2022 04 Digitale Resto Mondo Rid

haematologica
Journal of the Ferrata Storti Foundation
Editor-in-Chief
Jacob M. Rowe (Jerusalem)
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Carlo Balduini (Pavia), Jerry Radich (Seattle)
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Hélène Cavé (Paris), Monika Engelhardt (Freiburg), Steve Lane (Brisbane), Pier Mannuccio Mannucci (Milan), Pavan
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(Vancouver), Aaron Schimmer (Toronto), Richard F. Schlenk (Heidelberg), Sonali Smith (Chicago)
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haematologica
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haematologica
Table of Contents
Volume 107, Issue 4: April 2022
About the Cover
781 Images from the Haematologica Atlas of Hematologic Cytology: precursor lymphoid neoplasms, cytochemistry and immunocytochemistry
Rosangela Invernizzi
https://doi.org/10.3324/haematol.2022.280759
Landmark Paper in Hematology

782 The first achievement of complete remission in childhood leukemia by treatment with the folic acid antagonist aminopterin
Shai Izraeli
Editorials
783 FLT3-ITD signals bad news for core binding factor acute myeloid leukemia unless trisomy 22 comes to the rescue
Sun Loo and Andrew H. Wei
785 Thrombotic thrombocytopenic purpura and other immune-mediated blood disorders following vaccination against SARS-CoV-2
Pier Mannuccio Mannucci
787 Not all mismatches are equal: importance of alloreactivity direction

Jacinta Perram and Nada Hamad
Review Article
790 The mitochondrial anti-apoptotic dependencies of hematologic malignancies: from disease biology to advances in precision medicine
Isacco Ferrarini et al.
Articles
Acute Lymphoblastic Leukemia
803 LAMP-5 is an essential inflammatory-signaling regulator and novel immunotherapy target for mixed lineage leukemia-rearranged
acute leukemia
Gabriel Gracia-Maldonado et al.
Acute Myeloid Leukemia

816 Interleukin 4 promotes phagocytosis of murine leukemia cells counteracted by CD47 upregulation
Pablo Peña-Martínez et al.
825 Pevonedistat and azacitidine upregulate NOXA (PMAIP1) to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia
Dan Cojocari et al.
836 Characteristics and outcome of patients with core-binding factor acute myeloid leukemia and FLT3-ITD: results from an international
collaborative study
Sabine Kayser et al.
Haematologica 2022; vol. 107 no. 4 - April 2022

http://www.haematologica.org/
haematologica
Bone Marrow Transplantation

844 Refined HLA-DPB1 mismatch with molecular algorithms predicts outcomes in hematopoietic stem cell transplantation
Jun Zou et al.
Cell Therapy and Immunotherapy

857 Comparison of immune reconstitution between anti-T-lymphocyte globulin and posttransplant cyclophosphamide as acute graft-versus-host
disease prophylaxis in allogeneic myeloablative peripheral blood stem cell transplantation
Radwan Massoud et al.
Chronic Lymphocytic Leukemia

868 The complex karyotype landscape in chronic lymphocytic leukemia allows the refinement of the risk of Richter syndrome transformation
Andrea Visentin et al.
877 IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV
mutation load
Dianna Hussmann et al.
Hematopoiesis
887 Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome
Ayala Tovy et al.
Hodgkin Lymphoma
899 Improved outcomes of high-risk relapsed Hodgkin lymphoma patients after high-dose chemotherapy: a 15-year analysis
Yago Nieto et al.
909 Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect
Roberta Pece et al.
Plasma Cell Disorders

921 DIS3 mutations in multiple myeloma impact the transcriptional signature and clinical outcome
Katia Todoerti et al.
Platelet Biology & its Disorders

933 The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling
Adela Constantinescu-Bercu et al.
947 Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines
Stephan Michalik et al.
Red Cell Biology & its Disorders

958 Brain injury pathophysiology study by a multimodal approach in children with sickle cell anemia with no intra or extra cranial arteriopathy
Valentine Brousse et al.

haematologica
Letters to the Editor

966 Loss of 5-hydroxymethylcytosine expression is near-universal in B-cell lymphomas with variable mutations in epigenetic regulators
Kevin S. Tanager et al.
970 Immunophenotypic changes in leukemic blasts in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia after
treatment with CD19-directed chimeric antigen receptor (CAR)-expressing T cells
Ekaterina Mikhailova et al.
975 Early testicular maturation is sensitive to depletion of spermatogonial pool in sickle cell disease
Klara M. Benninghoven-Frey et al.
980 Targeting B-cell maturation antigen increases sensitivity of multiple myeloma cells to MCL-1 inhibition
Marta Cuenca et al.
984 Phase Ib dose-escalation study of the selective, non-covalent, reversible Bruton’s tyrosine kinase inhibitor vecabrutinib in B-cell malignancies
John N. Allan et al.
988 In vitro and in vivo effects of short-term cold storage of platelets in PAS-C
S. Lawrence Bailey et al.
991 Conventional interferon-α 2b versus hydroxyurea for newly-diagnosed patients with polycythemia vera in a real world setting:
a retrospective study based on 286 patients from a single center
Dan Liu et al.
996 Daratumumab with or without chemotherapy in relapsed and refractory acute lymphoblastic leukemia. A retrospective observational
Campus ALL study
Marco Cerrano et al.
1000 T-cell immune responses following vaccination with mRNA BNT162b2 against SARS-CoV-2 in patients with chronic lymphocytic leukemia:
results from a prospective open-label clinical trial
Lisa Blixt et al.
1004 Acute lymphoblastic leukemia cells are able to infiltrate the brain subventricular zone stem cell niche and impair neurogenesis
Lidia M. Fernández-Sevilla et al.
Case Reports
1008 Immune-mediated thrombotic thrombocytopenic purpura following administration of Pfizer-BioNTech COVID-19 vaccine
Gaetano Giuffrida et al.
1011 VEXAS syndrome in a female patient with constitutional 45,X (Turner syndrome)
Ryan J. Stubbins et al.
1014 A case series of primary cutaneous B-cell lymphomas with atypical presentations: diagnostic and therapeutic challenges
Emily Correia et al.

haematologica
The origin of a name that reflects Europe’s cultural roots.
Ancient Greek
Scientific Latin haematologicus (adjective) = related to blood
Scientific Latin haematologica (adjective, plural and neuter,

used as a noun) = hematological subjects
Modern English The oldest hematology journal,

publishing the newest research results.
2020 JCR impact factor = 9.94
ABOUT THE COVER
Images from the Haematologica Atlas of Hematologic Cytology: precursor lymphoid neoplasms,
cytochemistry and immunocytochemistry
Rosangela Invernizzi
University of Pavia, Pavia, Italy
E-mail: ROSANGELA INVERNIZZI - rosangela.invernizzi@unipv.it
doi:10.3324/haematol.2022.280759
I n the absence of signs of morphological differentiation, the lineage of acute leukemia blasts can be assessed by cytochem-
istry, flow-cytometry or immunocytochemistry. The cytochemical features of acute lymphoblastic leukemia are shown
in the Figure. Most lymphoblasts reveal strong periodic acid Schiff (PAS) staining with granules arranged in perinuclear rings
(A) or large, sometimes single, cytoplasmic blocks of glycogen (B). Differently from myeloblasts, lymphoblasts are negative
for the peroxidase (C, left) and Sudan black (C, right) reactions; note also the strong cytoplasmic positivity of neutrophils for
both reactions in panel (C). T-lymphoblasts, differently from B-lymphoblasts, are characterized by focal acid phosphatase
reactivity, due to the enzyme localization in the Golgi zone (D), and also by strong, localized, paranuclear dipeptidyl
aminopeptidase IV (DAP IV) activity (E). Nuclear terminal deoxynucleotidyl transferase (TdT) may be detected by immuno-
cytochemistry in both B-lineage (F) and T-lineage lymphoblasts.1
Disclosures
No conflicts of interest to disclose.
Reference
1. Invernizzi R. Precursor lymphoid neoplasms. Haematologica. 2020;105(Suppl. 1):127-138.
haematologica | 2022; 107(4) 781

LANDMARK PAPER IN HEMATOLOGY
The first achievement of complete remission in childhood leukemia by
treatment with the folic acid antagonist aminopterin
Shai Izraeli
Schneider Children’s Medical Center of Israel, Tel Aviv University, Tel Aviv, Israel
E-mail: sizraeli@gmail.com
doi:10.3324/haematol.2022.280670
TITLE Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-aminopteroyl-glutamic
acid (aminopterin).
AUTHORS Farber S, Diamond LK. Mercer RD, Slyvester RF Jr, Wolff JA.
JOURNAL New England Journal of Medicine. 1948;238(23):787-793.
T
hese days almost 90% of children with acute lym- one case, regression of subcutaneous nodules that were pre-
phoblastic leukemia and 70% of those with acute sumed to be leukemic. The major toxicity was “severe stomati-
myelogenous leukemia are cured. The first significant tis” and, in one case, pancytopenia and empty bone marrow,
step towards these results was published by Dr. Sidney Farber for which therapy with liver extracts was attempted. The
and colleagues 74 years ago.1 Their achievement was preceded longest complete remission off therapy was 47 days.
by a devastating failure. Following the great success of treat- Fast-forwarding to our times, methotrexate is one of the
ment of folate deficiency by conjugates of folic acid, and given cornerstones of treatment of acute lymphoblastic leukemia.
the morphological similarity between megaloblastic anemia Severe mucositis is indeed one of the major toxicities and
and leukemia, Dr. Farber attempted to treat children with folate, in the form of leucovorin, is the main rescue treatment
leukemia with folate conjugates. Remarkably, he was percep- after therapy with high-dose methotrexate. The remarkable
tive enough to note that treatment with folates had the oppo- observation by Dr. Farber that folate conjugates accelerate
site outcome from that desired: it markedly accelerated the leukemia growth serves as an important reminder that vita-
leukemias, as he observed both clinically and in postmortem mins and nutritional supplements, given with the intention
examinations.2 These observations led him to conduct a clinical to “strengthen the patient”, may fuel a tumor. Cancer and
trial with the newly synthetised anti-folate, amiopterin;3 the normal cells compete for the same resources with the former
results were published on June 3, 1948 in the New England being more dependent on the essential fuel. Treatment with
Journal of Medicine.1 Sixteen children with leukemia were treat- L-asparaginase followed the introduction of anti-folates as an
ed with aminopterin, ten entered transient remissions charac- effective means to starve tumor cells. Interestingly, con-
terized by a reduction or even complete disappearance of blasts trolled calorie restriction has recently been shown to further
from peripheral blood and bone marrow with recovery of nor- improve the rate of molecular remission of children with
mal hematopoiesis (Figure 1, from the original article). These acute lymphoblastic leukemia and is now being tested in a
hematologic findings were accompanied by resolution of the large clinical trial funded by the National Institutes of
clinical symptoms, regression of hepatosplenomegaly and, in Health.4
A B Figure 1. A reproduction of
the original figure from the
paper in the New England
Journal of Medicine1 demon-
strating the morphology of the
bone marrow before (A) and
after 1 month of treatment
with aminopterin and crude
liver extract (B).
References
1. Farber S, Diamond LK. Mercer RD, Slyvester RF Jr, Wolff JA.Temporary remissions in acute leukemia in children produced by folic acid antagonist, 4-
aminopteroyl-glutamic acid (aminopterin). N Engl J Med. 1948;238(23):787-793.
2. Farber S, Cutler EC, Hawkins JW, Harrison JH, Peirce EC 2nd, Lenz GG. The action of pteroylglutamic conjugates on man. Science. 1947;106(2764):619-
621.
3. Seeger DR, Smith JM Jr, Hultquist ME. Antagonist for pteroylglutamic acid. J Am Chem Soc. 1947;69(10):2567.
4. Orgel E, Framson C, Buxton R, et al. Caloric and nutrient restriction to augment chemotherapy efficacy for acute lymphoblastic leukemia: the IDEAL trial.
Blood Adv. 2021;5(7):1853-1861.
782 haematologica | 2022; 107(4)

EDITORIALS
FLT3-ITD signals bad news for core binding factor acute myeloid leukemia unless trisomy 22
comes to the rescue
Sun Loo1 and Andrew H. Wei1,2
1
Department of Clinical Haematology, Alfred Hospital and 2Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria,
Australia
E-mail: ANDREW H. WEI - andrew.wei@monash.edu
doi:10.3324/haematol.2021.279409
S
tructural rearrangements resulting in either also absent for the small group of patients treated non-inten-
t(8;21)(q22;q22) [RUNX1-RUNX1T1] or sively. These results prompted the authors to conclude that
inv(16)(p13q22)/t(16;16)(p13.1;q22) [CBFB-MYH11] are patients with FLT3-ITD CBF AML should be given intensive
pathognomonic for core binding factor (CBF) acute myeloid induction and consolidation therapy, when possible, and to
leukemia (AML). Prognostic classifications have consistently reserve allogeneic HCT as a strategy in second complete
positioned CBF AML as a favorable entity, particularly if the remission in the event of relapse after first-line therapy. A
patient can tolerate conventional induction and consolida- major caveat is the retrospective nature of the study, which
tion chemotherapy. Optimal outcomes for patients with CBF introduces the risk of potential bias. Only 39% of relapsing
disease are achieved through incorporation of gemtuzumab patients were transplanted, suggesting that the opportunity
ozogamicin into 7+3 based induction and high-dose cytara- for cure was lost for the majority of those in whom primary
bine into the consolidation phase of therapy.1,2 therapy failed. The failure to observe enhanced outcomes for
Recent molecular studies have highlighted striking differ- those treated in first complete remission, however, suggests
ences in the genomic landscape between the two forms of that not all patients with FLT3-ITD CBF AML have a poor
CBF AML. Although kinase activating mutations are prognosis and that heterogeneity in survival must exist.
observed frequently in both groups, RUNX1-RUNX1T1 In search of genetic factors differentiating prognosis in CBF
more commonly harbors mutations in ASXL1 (14%), ASXL2 AML, Kayser et al. identified an association between inv(16)
(14%), TET2 (11%), RAD21 (11%) and ZBTB7A (19%), and trisomy 22 in 23% of cases. Although prior studies have
whereas CBFB-MYH11 AML is more frequently associated already reported favorable outcome for this chromosomal
with WT1 mutation (10%). At the cytogenetic level, t(8;21) is duet,10 the current study extends this finding to patients with
more closely linked to del(9q) or loss of a sex chromosome, trisomy 22, inv(16) and FLT3-ITD mutation. For patients
whereas inv(16) may occur in the company of del(7q) and tri- with this molecular triad, relapse-free survival at 4 years was
somy 22 abnormalities.3-5 80%, compared to only 38% for other patients. The authors
In terms of prognosis, although there is general agreement conclude that patients with CBF and FLT3-ITD with inv(16)
that additional cytogenetic abnormalities do not consistently and trisomy 22 should be classified as favorable risk, the
increase the risk of relapse in CBF AML, the role of kinase remainder as poor risk. It remains uncertain, however,
activating mutations has been more controversial.6 The pre- whether outcomes would be improved by upfront allogeneic
dominant kinase activating mutations in CBF AML involve HCT in first complete remission or whether transplant at
RAS (27%), KIT (26%) and FLT3 (17%).5 The presence of relapse would suffice for this poor-risk CBF subgroup with
mutant RAS is generally associated with a favorable progno- FLT3-ITD. Another intriguing question is what candidate
sis in CBF AML.5 In contrast, several series suggest that KIT genes are carried on chromosome 22, which when amplified
mutations, in particular exon 17 mutations, are associated by just one copy, can result in dramatic enhancement of
with increased relapse risk among patients with RUNX1- prognosis in patients with FLT3-ITD CBF AML.
RUNX1T1, whereas prognostic concordance is lacking for A major limitation of the study was the absence of flow or
CBFB-MYH11 AML.7,8 molecular measurable residual disease (MRD) correlation
The paper published by Kayser and colleagues9 in this issue with these prognostic observations. Favorable prognosis in
of Haematologica is a multi-institutional retrospective cohort CBF AML is strengthened by multi-log reduction or eradica-
analysis addressing the role of FLT3-internal tandem duplication of MRD after commencing treatment. Despite an
tion (ITD) co-mutation in CBF AML. The study included 97 admirable effort to refine prognostic outcomes in FLT3-ITD
patients with similar proportions of t(8;21)(q22;q22) and CBF AML, a recurring question is whether the importance of
inv(16)(p13q22)/t(16;16)(p13.1;q22). Most were treated baseline prognostic risk stratification is diminished by
intensively, resulting in a very high complete remission rate dynamic assessment of post-treatment MRD. Although cur-
of 98%, despite the presence of FLT3-ITD, with only three rent European LeukemiaNET guidance recommends post-
patients receiving concomitant FLT3 inhibitor. Allogeneic treatment MRD monitoring every 3 months, several studies
hematopoietic cell transplant (HCT) was performed in 14% suggest that the window of opportunity to intervene
of the patient population in first complete remission. Among between initial detection of MRD progression by reverse-
patients not transplanted in first complete remission, almost transcriptase quantitative polymerase chain reaction (RT-
40% relapsed with subsequent allogeneic HCT performed in qPCR ) and clinical relapse is too narrow, making it logistical-
~39% of this group. In this analysis of patients with FLT3- ly difficult to orchestrate a meaningful therapeutic interven-
ITD CBF AML, the authors found that allogeneic HCT was tion.12-14 Increasing the intensity of MRD monitoring with
only beneficial for patients at relapse, whereas outcomes more frequent peripheral blood surveillance e.g., monthly for
were not improved by allogeneic HCT in first complete the first 12 months when relapse risk is highest, could enable
remission. If allogeneic HCT was not performed at relapse, earlier detection of rising MRD. It remains to be proven
there were no long-term survivors. Long-term survival was whether overall survival would be enhanced by earlier, pre-

Editorials
emptive intervention, as opposed to salvage at the time of Disclosures

morphological progression. With allogeneic HCT in sec- AW has served on advisory boards for Novartis, Janssen,
ond complete remission the main priority for patients Amgen, Roche, Pfizer, Abbvie, Servier, Celgene-BMS,
with relapsing disease, it is likely that early detection and Macrogenics, Agios, and Gilead; receives research funding to the
treatment to suppress rising MRD could increase the pro- Institution from Novartis, Abbvie, Servier, Celgene-BMS, Astra
portion of patients bridged to transplant in remission and Zeneca, and Amgen; serves on speakers bureaus for Abbvie,
negative for MRD. Alternatively, it remains an open ques- Novartis, Celgene; and receives royalty payments from the
tion whether outcomes will be improved by a pre-trans- Walter and Eliza Hall Institute of Medical Research related to
plant MRD reduction strategy, or whether equivalent out- venetoclax
comes could be achieved by proceeding directly to trans-
plantation, especially if myeloablative conditioning is Contributions
planned. The median time to relapse from detection of Both authors wrote and reviewed the paper.
MRD failure to clinical relapse is only about 3-4 months.12
Therefore, a pre-emptive MRD suppression strategy
could buy the treating team more time, keeping the References
patient in remission and free from relapse until the allo-
1. Magina KN, Pregartner G, Zebisch A, et al. Cytarabine dose in the
geneic HCT can be organized and carried out. consolidation treatment of AML: a systematic review and meta-
In terms of targeting FLT3 to improve clinical outcome in analysis. Blood. 2017;130(7):946-948.
FLT3-ITD CBF AML, treatment could be introduced at the 2. Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzum-
ab ozogamicin to induction chemotherapy in adult patients with
induction/consolidation stage, during maintenance, pre- acute myeloid leukaemia: a meta-analysis of individual patient data
emptively at the time of MRD progression, at morpholog- from randomised controlled trials. Lancet Oncol. 2014;15(9):986-
ical relapse, or as maintenance therapy in the post-allo- 996.
geneic HCT setting. Unfortunately, robust data to answer 3. Opatz S, Bamopoulos SA, Metzeler KH, et al. The clinical mutatome
of core binding factor leukemia. Leukemia. 2020;34(6):1553-1562.
any of these questions are lacking, with patients harboring 4. Faber ZJ, Chen X, Gedman AL, et al. The genomic landscape of core-
FLT3-ITD CBF accounting for only ~2% of the AML pop- binding factor acute myeloid leukemias. Nat Genet. 2016;48(12):
ulation, making randomized trial data with any new or 1551-1556.
5. Jahn N, Terzer T, Strang E, et al. Genomic heterogeneity in core-
future agent or combinations within this orphan sub-pop- binding factor acute myeloid leukemia and its clinical implication.
ulation an unlikely prospect. The RATIFY trial, which Blood Adv. 2020;4(24):6342-6352.
examined the role of midostaurin during induction, consol- 6. Han SY, Mrozek K, Voutsinas J, et al. Secondary cytogenetic abnor-
idation and maintenance in patients with FLT3-mutant malities in core-binding factor AML harboring inv(16) vs t(8;21).
Blood Adv. 2021;5(10):2481-2489.
AML, only enrolled 16 patients (4%) with CBF AML to the 7. Ishikawa Y, Kawashima N, Atsuta Y, et al. Prospective evaluation of
midostaurin arm.15 In the SORAML trial, the FLT3 inhibitor prognostic impact of KIT mutations on acute myeloid leukemia with
sorafenib was combined with standard induction and con- RUNX1-RUNX1T1 and CBFB-MYH11. Blood Adv. 2020;4(1):66-75.
8. Paschka P, Marcucci G, Ruppert AS, et al. Adverse prognostic signif-
solidation therapy and as maintenance for 12 months.16 In icance of KIT mutations in adult acute myeloid leukemia with
the favorable cytogenetic risk group, which formed only inv(16) and t(8;21): a Cancer and Leukemia Group B study. J Clin
10% of the study population, sorafenib was associated Oncol. 2006;24(24):3904-3911.
9. Kayser S, Kramer M, Martínez-Cuadrón D, et al. Characteristics and
with improved event-free, relapse-free and overall survival outcome of patients with core binding factor acute myeloid
in a post-hoc subgroup analysis. The outcomes of patients leukemia and FLT3-ITD: results from an international collaborative
with FLT3-ITD within this CBF subgroup were, however, study. Haematologica. 2022;107(4):836-843.
not defined. 10. Marcucci G, Mrozek K, Ruppert AS, et al. Prognostic factors and out-
come of core binding factor acute myeloid leukemia patients with
In summary, as the genomic age continues to reveal fur- t(8;21) differ from those of patients with inv(16): a Cancer and
ther prognostic heterogeneity within conventional AML Leukemia Group B study. J Clin Oncol. 2005;23(24):5705-5717.
subgroups, we will increasingly be challenged with when 11. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management
of AML in adults: 2017 ELN recommendations from an international
to pull the trigger on the use of allogeneic HCT and when expert panel. Blood. 2017;129(4):424-447.
to use a growing number of newly approved AML drugs, 12. Willekens C, Blanchet O, Renneville A, et al. Prospective long-term
such as FLT3 inhibitors and so forth, for uncommon clin- minimal residual disease monitoring using RQ-PCR in RUNX1-
ical scenarios for which definitive randomized evidence RUNX1T1-positive acute myeloid leukemia: results of the French
CBF-2006 trial. Haematologica. 2016;101(3):328-335.
may never become available. The current work by Kayser 13. Yin JA, O'Brien MA, Hills RK, et al. Minimal residual disease moni-
et al.9 adds to the growing list of AML scenarios in which toring by quantitative RT-PCR in core binding factor AML allows
the presence of FLT3-ITD represents bad news, including risk stratification and predicts relapse: results of the United Kingdom
MRC AML-15 trial. Blood. 2012;120(14):2826-2835.
among patients with CBF AML. Physicians are likely to 14. Puckrin R, Atenafu EG, Claudio JO, et al. Measurable residual dis-
formulate a logic circuit that suggests that: (i) it makes ease monitoring provides insufficient lead-time to prevent morpho-
sense to use an FLT3 inhibitor to target FLT3-ITD when logic relapse in the majority of patients with core-binding factor
acute myeloid leukemia. Haematologica. 2021;106(1):56-63.
detected in CBF AML; (ii) patients with concurrent tri- 15. Stone RM, Mandrekar SJ, Sanford BL, et al. Midostaurin plus
somy 22 should not be candidates for allogeneic HCT in chemotherapy for acute myeloid leukemia with a FLT3 mutation. N
first complete remission; (iii) close monitoring of MRD, Engl J Med. 2017;377(5):454-464.
potentially with RT-qPCR performed monthly on blood 16. Rollig C, Serve H, Huttmann A, et al. Addition of sorafenib versus
placebo to standard therapy in patients aged 60 years or younger
for at least the first 12 months, is warranted; and (iv) allo- with newly diagnosed acute myeloid leukaemia (SORAML): a mul-
geneic HCT should be ready to action early if MRD pro- ticentre, phase 2, randomised controlled trial. Lancet Oncol.
gression is confirmed. 2015;16(16):1691-1699.

Editorials
Thrombotic thrombocytopenic purpura and other immune-mediated blood disorders follow-

ing vaccination against SARS-CoV-2
Pier Mannuccio Mannucci
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy
E-mail: PIER MANNUCCIO MANNUCCI - piermannuccio.mannucci@policlinico.mi.it
doi:10.3324/haematol.2021.279649
I
n this issue of Haematologica, Giuffrida et al.1 report & Johnson vaccine, which is based on a human aden-
two cases of new-onset, immune-mediated thrombot- ovirus vector,3 and a relapse of recurrent TTP which
ic thrombocytopenic purpura (TTP) in 81-year-old and occurred 6 days after the second dose of the Pfizer-
30-year-old women diagnosed with this very rare disease BioNTech vaccine.4 The new-onset cases described by
14 and 18 days after the first dose of the mRNA-based Giuffrida et al.1 of such a rare immune-mediated blood
vaccine against severe acute respiratory syndrome coron- disease associated with a bleeding tendency follow the
avirus 2 (SARS-CoV-2) manufactured by Pfizer- report of a mRNA-vaccine (Pfizer-BioNTech)-associated
BioNTech. The older woman (case 1) had a history of dia- case of autoimmune hemophilia due to anti-factor VIII
betes and connective tissue disease positive for antinucle- antibodies5 and multiple cases of immune thrombocy-
ar antibodies, whereas the younger (case 2) was negative topenic purpura (ITP) due to platelet autoantibodies
regarding clinical history and laboratory markers of occurring after either of the two mRNA-based vaccines
potential triggers of TTP such as autoimmune disorders, produced by Pfizer and Moderna.6 Common features of
tumors and infections. Both women were promptly treat- these cases are that the majority of them occurred in
ed with glucocorticoids and daily sessions of plasma women, at young but also at older ages, thus reproducing
exchange, each followed by the nanobody caplacizumab. the two typical age peaks of occurrence of autoimmune
This state-of-the-art therapeutic approach based upon diseases. At variance with the recent reports of vaccine-
plasma therapy, immunomodulation and anti-von induced immune thrombotic thrombocytopenia (VITT),7
Willebrand factor medicines was successful in the these cases were not associated with thrombosis in cere-
younger woman, who had rapid normalization of a very bral or abdominal veins but only with hemorrhagic
low platelet count, even though plasma ADAMTS13 was symptoms compatible with the degree of thrombocy-
still unmeasurable on days 14 and 30 after eight plasma topenia in ITP and TTP and of factor VIII deficiency in
exchanges and anti-ADAMTS13 were still present. The autoimmune acquired hemophilia. Another feature that
older woman with comorbidities had only a modest distinguishes these cases from VITT is that they were not
improvement of platelet count and she died suddenly accompanied by serological positivity for autoantibodies
after the second plasma exchange as the result of an ill- directed against platelet factor 4. Table 1 summarizes the
defined cardiac event, thus once again emphasizing that main clinical symptoms and laboratory findings in the
TTP is still associated with a significant mortality toll different thrombocytopenias that did occur after vaccina-
notwithstanding prompt and impeccable management. tion against COVID-19.
The main interest of these two cases lies in the fact that What are the general messages that may be drawn from
new-onset autoimmune TTP occurred within 2 to 3 these reports of immune-mediated hematological dis-
weeks after the first dose of a vaccine to protect against eases associated with a bleeding tendency in persons
coronavirus disease 2019 (COVID-19). Administration of recently vaccinated to prevent COVID-19? It is well
the vaccine within this short time window prior to the established that a number of diseases due to the forma-
TTP episode as well as no evidence for other causes (at tion of autoantibodies against autologous cells and/or
least in the younger woman) are consistent with causality proteins may occur after vaccination against various
according to the World Health Organization criteria for infectious agents:8-10 common examples are measles-
post-vaccination adverse events.2 Until now, new-onset mumps-rubella and diphtheria-tetanus-pertussis vac-
TTP had been reported as a single case after the Johnson cines, but also vaccines against polio, rabies, influenza
Table 1. Main features of vaccine-induced, immune mediated thrombocytopenias.

Disease (and acronym) Severe Mucocutaneous Intracerebral Associated Thrombosis Laboratory
thrombocytopenia bleeding hemorrhage thrombosis sites diagnosis
(<10x109/L) symptoms
Immune thrombocytopenic Frequent Frequent Rare Rare - Anti-platelet antibodies
purpura (ITP)
Thrombotic thrombocytopenic Frequent Rare Rare Frequent, Microcirculation ADAMTS-13
purpura (TTP) microvascular of heart, brain deficiency and
and GI tract ADAMTS-13 antibody
Vaccine-induced immune Frequent Rare Frequent Frequent, Cerebral and Anti-PF4 ELISA
thrombotic thrombocytopenia macrovascular abdominal veins positivity
(VITT)
GI: gastrointestinal; ADAMTS13: a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; PF4: platelet factor 4; ELISA: enzyme-linked immunosor-
bent assay.

Editorials
and bacterial pneumonia, especially in children but also in and in the specific instance of COVID-19, the effective-
adults. There is no evidence that the innovative technolo- ness of vaccines, which are the only weapon currently
gies recently developed for anti-COVID vaccine produc- available to control this pandemic. The majority of ITP
tion have a particular role in the dysregulation of the and TTP cases seem to be less severe than VITT and are
immune system that led to the production of antibodies usually not life-threatening, except in older individuals
other than those towards the spike SARS-CoV-2 protein, with multiple comorbid conditions, such as case 2. In
because autoimmune diseases have occurred after all addition, it appears that, albeit with the limited amount
types of vaccines, spanning from those traditionally of available knowledge given the recent onset and short
based upon inactivated virions to those newly employing follow-up of these complications, responses to state-of-
viral DNA vectors or mRNA technology.8-10 Only VITT the-art therapies, as well as tendencies to recur or
appears to be peculiar, because this complication has so become chronic, are not overtly different from those of
far been described with convincing documentation only cases that occur irrespective of vaccination. By the same
in patients receiving the vaccines based on adenoviral token, no prophylactic measure is warranted before or
vectors, such as the AstraZeneca and the Johnson & after vaccination, because useless and potentially danger-
Johnson products. In VITT the very rare but catastrophic ous.
thrombohemorrhagic complications are due to the forma-
tion of highly pathogenic autoantibodies against a com- Disclosures
plex between platelet factor 4 and a still poorly defined No conflicts of interest to disclose.
polyanion that triggers platelet activation, consumptive
thrombocytopenia and a hypercoagulable state perhaps
amplified by antibody-induced NETosis.7 However, it is References
not yet fully understood why venous thrombi occur in
1. Giuffrida G, Condorelli A, Di Giorgio MA, et al. Immune-mediated
unusual sites, and the source and composition of the thrombotic thrombocytopenic purpura following administration of
polyanion are still unlcear. Moreover, it remains uncertain the Pfizer-BioNTech COVID-19 vaccine. Haematologica. 2022;
whether or not these rare post-vaccination disorders are 107(4):1008-1010.
2. World Health Organization. Global manual on surveillance of
more frequent than expected in the population at large, adverse effect following immunization. 2015 Update. World Health
because epidemiologically-based studies evaluating their Organization. 2014.
incidences in vaccinated versus non-vaccinated persons 3. Yocum A, Simon EL. Thrombotic thrombocytopenic purpura after
are scanty or absent. The reported prevalences in vacci- Ad26.COV2-S vaccination. Am J Emerg Med. 2021 Nov;49:441.e3-
441.e4
nated people, usually affected by limited sample size, 4. Sissa C, Al-Khaffaf A, Frattini F, et al. Relapse of thrombotic throm-
range from 1 in 50.000-100.000 for VITT depending on bocytopenic purpura after COVID-19 vaccine. Transfus Apher Sci.
the age and gender of the vaccine recipients to a lower 2021;60(4):103145.
5. Radwi M, Farsi S. A case report of acquired hemophilia following
prevalence (1 in 1,000,000) for ITP.6,11,12 COVID-19 vaccine. J Thromb Haemost. 2021;19(6):1515-1518.
An array of innate or adaptive immunological mecha- 6. Lee EJ, Cines DB, Gernsheimer T, et al. Thrombocytopenia follow-
nisms may be responsible for these adverse events, but ing Pfizer and Moderna SARS-CoV-2 vaccination. Am J Hematol.
vaccine-induced danger signals accompanied by inflam- 2021;96(5):534-537.
7. Cines DB, Bussel JB. SARS-CoV-2 vaccine-induced immune throm-
mation, as well as antigenic mimicry with activation of botic thrombocytopenia. N Engl J Med. 2021;384(23):2254-2256.
quiescent autoreacting B and T cells, are the most plausi- 8. Guimarães LE, Baker B, Perricone C, Shoenfeld Y. Vaccines, adju-
ble.8,10 It is unlikely that adjuvants, frequently employed vants and autoimmunity. Pharmacol Res. 2015;100:190-209.
9. Perricone C, Ceccarelli F, Nesher G, et al. Immune thrombocytopenic
in some vaccines in order to boost antibody production purpura (ITP) associated with vaccinations: a review of reported
towards the target antigen, play a mechanistic role, cases. Immunol Res. 2014;60(2-3):226-235.
because the currently licensed anti-COVID vaccines do 10. Watad A, De Marco G, Mahajna H, et al. Immune-mediated disease
flares or new-onset disease in 27 subjects following mRNA/DNA
not need nor contain typical adjuvants such as squalene SARS-CoV-2 vaccination. Vaccines (Basel). 2021;9(5):435.
and aluminum, because their RNA and DNA components 11. Simpson CR, Shi T, Vasileiou E, et al. First-dose ChAdOx1 and
offer intrinsic ‘adjuvanticity’. BNT162b2 COVID-19 vaccines and thrombocytopenic, throm-
On the whole, these exceptional cases of immune- boembolic and hemorrhagic events in Scotland. Nat Med.
2021;27(7):1290-1297.
mediated hematological diseases associated with bleed- 12. Pottegård A, Lund LC, Karlstad Ø, et al. Arterial events, venous
ing and/or thrombosis that have occurred in the current thromboembolism, thrombocytopenia, and bleeding after vaccina-
frame of global vaccination of more than 400 million tion with Oxford-AstraZeneca ChAdOx1-S in Denmark and
Norway: population based cohort study. BMJ. 20215;373:n1114.
people should not put in doubt nor jeopardize, in general

Editorials
Not all mismatches are equal: importance of alloreactivity direction

Jacinta Perram1,2 and Nada Hamad1,2,3
1
Department of Haematology, St Vincent's Hospital Sydney; 2St Vincent's Clinical School Sydney, University of New South Wales and
3
School of Medicine Sydney, University of Notre Dame Australia, Sydney, Australia
E-mail: NADA HAMAD - nada.hamad@svha.org.au

doi:10.3324/haematol.2021.279587
A
llogeneic hematopoietic stem cell transplantation epitope (TCE) analysis.6,7 While these methods assess the
(HSCT) remains the only curative therapy for qualitative character of a mismatch, they do not evaluate
numerous hematologic malignant and benign con- direction or anticipate immunogenicity of a given mis-
ditions. Transplantation does, however, come with signif- match. This poorly characterized potential risk represents
icant risks of morbidity and mortality related to the trans- a limitation of current donor selection algorithms.
plant, graft-versus-host disease (GVHD) and relapse. The By contrast, molecular matching techniques assess
art of medicine in allogeneic HSCT lies in walking the structural components of epitopes, called eplets, allowing
tightrope between relapse risk and GVHD. We have come quantification of donor and recipient mismatched eplets
a long way in minimizing the risks of GVHD with rates of (ME). This quantification, when combined with the
clinically significant (grade 2-4) acute GVHD and moder- PIRCHE score (PS), a predictor of TCE alloreactivity, has
ate to severe chronic GVHD reported to be as low as 50% been shown to predict immunogenicity and clinical out-
and 30%, respectively.1,2 This is, no doubt, related to more comes in haploidentical transplant recipients.8 Zou et al.5
widespread use of T-cell depletion methods and better present novel data on the use of molecular algorithms for
HLA typing.3 Optimal donor selection in the absence of a HLA-DPB1 mismatch in a cohort of more than 1,500
matched relative relies on an assessment of the relative patients who received an unrelated donor transplant
risk, and selection of the donor most genetically suitable between 2005-2018 at The University of Texas MD
based on HLA matching at HLA-A, -B, -C, DR and DQ. Anderson Cancer Center. The primary question in this
Human-leukocyte-antigen DPB1 (HLA-DPB1) mismatch study is whether molecular matching offers superior prog-
is known to be broadly associated with decreased relapse nostic guidance than the traditional TCE model. The
at the cost of increased rates of acute GVHD.4 HLA-DPB1 group reports concordance testing of bidirectional ME and
mismatch in otherwise matched donors is common, yet PS, as well as the TCE model, with acute GVHD out-
our ability to predict GVHD severity based on this mis- comes. The central finding is that high levels of ME in the
match is limited. In this issue, Zou et al.5 present data sup- graft-versus-host (GVH) direction are the strongest single
porting HLA-DPB1 mismatch associated risks of acute predictor of acute GVHD. The authors propose use of
GVHD while using clinical correlation to investigate the molecular algorithms to guide the choice of or augmenta-
clinical impact of HLA-DPB1 molecular mismatch. tion of acute GVHD prophylaxis.
In the last two decades, donor selection algorithms have Another crucial finding is the importance of direction of
classified HLA-DPB1 mismatches as permissive or non- alloreactivity. Bidirectional high ME or PS mismatch is
permissive, based on functional toxicity assays and T-cell universally associated with high rates of acute GVHD and
Figure 1. Competing risks

regression for acute graft-
versus-host disease grade
2-4. GVHD: graft-versus-host
disease; HR: hazard ratio;
ME: mismatched eplets;
GVH: graft-versus-host; HVG:
host-versus-graft.

Editorials
Figure 2. Competing risks regres-

sion for non-relapse mortality.
HR: hazard ratio; ME: mis-
matched eplets; GVH: graft-ver-
sus-host.
Figure 3. Competing risks regres-

sion for relapse. HR: hazard ratio;
ME: mismatched eplets; GVH:
graft-versus-host; HVG: host-ver-
sus-graft.
relapse, suggesting a synergistic effect. The reduced cases, an isolated high HVG ME is associated with low
relapse risk purported to arise from HLA-DPB1 permissive GVHD risk. The empirical inconsistency of these results
mismatch was only observed among those with high ME highlights the persisting incomplete understanding of
or PS in the GVH direction, and not those with isolated HVG pathophysiology.
high PE or MS in the host-versus-graft (HVG) direction, While the outcomes reported here have potential to
who in fact had an increased rate of relapse. This is a clin- alter practice in the future, the authors acknowledge the
ically important outcome as it forces re-evaluation of the need for significant further study. Involvement of only a
rationale for tolerating increased GVHD in recipients of single center is a significant limitation. The retrospective
transplants with HLA-DPB1 permissive mismatch. nature of this research is not problematic in itself given the
Building on the existing TCE model for HLA-DPB1 mis- correlative nature of the analyses performed. However,
match classification, there are several findings offering one of the great challenges in allogeneic HSCT is the rap-
refined stratification. Among the permissive mismatch idly evolving landscape across which research is per-
group, high HVG ME and PS are associated with a high formed. Since the commencement of this study, the adop-
risk of GVHD, yet among non-permissive mismatched tion of T-cell-depleting therapies has rapidly expanded.

Editorials
Across the trial period, the use of anti-thymocyte globulin Disclosures

increased significantly (23.7% to 41.2%). It is unknown No conflicts of interest to disclose.
whether, and if so how, this has augmented results.
Confirmatory investigations will be important to validate Contributions
current findings and confirm them as enduring in the set- JP and NH wrote and edited the editorial.
ting of routine T-cell depletion.
Another important development has been the use of References
post-transplant cyclophosphamide in haploidentical allo-
1. Jagasia M, Perales MA, Schroeder M, et al. Ruxolitinib for the treat-
geneic HSCT. The associated rapid improvement in clini- ment of steroid-refractory acute GVHD (REACH1): a multicenter,
cal outcomes has shifted the donor selection paradigm. If open-label phase 2 trial. Blood. 2020;135(20):1739-1749.
anything, the uptake of haploidentical transplants rein- 2. Zeiser R, Polverelli N, Ram R, et al. Ruxolitinib for glucocorticoid-
refractory chronic graft-versus-host disease. N Engl J Med.
forces the importance of this trial. With increased avail- 2021;385(3):228-238.
ability of alternative donors, the imperative to refine out- 3. Bacigalupo A. ATG in allogeneic stem cell transplantation: standard of
come prediction in HLA-DPB1 mismatch is all the more care in 2017? Point. Blood Adv. 2017;1(9):569-572.
relevant. 4. Fleischhauer K, Shaw BE, Gooley T, et al. Effect of T-cell-epitope
matching at HLA-DPB1 in recipients of unrelated-donor haemopoietic-
A further potentially significant development is related cell transplantation: a retrospective study. Lancet Oncol.
to the evolution of therapies for GVHD.2,9 More effica- 2012;13(4):366-374.
cious treatment and prevention strategies for GVHD may 5. Zou J, Kongtim P, Oran B, et al. Refined HLA-DPB1 mismatch with
molecular algorithms predicts outcomes in hematopoietic stem cell
redefine donor selection algorithms, permitting mismatch- transplantation. Haematologica. 2022;107(4):844-866.
es that were previously prohibited. 6. Zino E, Frumento G, Marktel S, et al. A T-cell epitope encoded by a
Zou et al.5 present an important novel approach to assess- subset of HLA-DPB1 alleles determines nonpermissive mismatches for
ment of HLA-DPB1 mismatch permissibility. The authors hematologic stem cell transplantation. Blood, 2004;103(4):1417-1424.
7. Crivello P, Zito L, Sizzano F, et al. The impact of amino acid variability
acknowledge that confirmation of their findings with on alloreactivity defines a functional distance predictive of permissive
multi-center data is needed before refinement of algorithms HLA-DPB1 mismatches in hematopoietic stem cell transplantation.
can be considered. One of the great challenges for the field Biol Blood Marrow Transplant. 2015;21(2):233-241.
8. Zou J, Ciurea S, Kongtim P, et al. Molecular dispartiy in human leuko-
of HSCT moving forward is access to progressively more cyte antigens is associated with outcomes in haploidentical stem cell
specialized molecular testing. There is also the need to transplantation. Blood Adv. 2020;4(15):3474-3485.
embrace international research collaborations to allow real- 9. Zeiser R, von Bubnoff N, Butler J, et al. Ruxolitinib for glucocorticoir-
refractory acute graft-versus-host disease. N Engl J Med.
time outcome reporting in a rapidly progressing field. 2020;382(19):1800-1810.

REVIEW ARTICLE
Ferrata Storti Foundation The mitochondrial anti-apoptotic dependencies

of hematologic malignancies: from disease
biology to advances in precision medicine
Isacco Ferrarini, Antonella Rigo and Carlo Visco
Department of Medicine, Section of Hematology, University of Verona, Verona, Italy
ABSTRACT
Haematologica 2022
M
Volume 107(4):790-802 itochondria are critical organelles in the regulation of intrinsic
apoptosis. As a general feature of blood cancers, different anti-
apoptotic members of the BCL-2 protein family localize at the
outer mitochondrial membrane to sequester variable amounts of pro-
apoptotic activators, and hence protect cancer cells from death induc-
tion. However, the impact of distinct anti-apoptotic members on apop-
tosis prevention, a concept termed anti-apoptotic dependence, differs
remarkably across disease entities. Over the last two decades, several
genetic and functional methodologies have been established to uncover
the anti-apoptotic dependencies of the majority of blood cancers,
inspiring the development of a new class of small molecules called BH3
mimetics. In this review, we highlight the rationale of targeting mito-
chondrial apoptosis in hematology, and provide a comprehensive map
of the anti-apoptotic dependencies that are currently guiding novel
therapeutic strategies. Cell-extrinsic and -intrinsic mechanisms confer-
ring resistance to BH3 mimetics are also examined, with insights on
potential strategies to overcome them. Finally, we discuss how the field
of mitochondrial apoptosis might be complemented with other dimen-
sions of precision medicine for more successful treatment of ‘highly
complex’ hematologic malignancies.
Correspondence:
ISACCO FERRARINI
isacco.ferrarini@univr.it
Introduction
CARLO VISCO
carlo.visco@univr.it Prevention of programmed cell death is a hallmark of cancer cells and efforts to
re-establish pro-death pathways have been the mainstay of research in the field of
anti-cancer therapeutics.1 Among modalities of programmed cell death, apoptosis
Received: October 18, 2021.
is the best characterized in terms of triggering stimuli, sequencing of biochemical
Accepted: January 7, 2022. events, intracellular organelles involved, and morphological changes.2 Two inter-
Prepublished: January 20, 2022. connected forms of apoptosis have been described: the extrinsic and the intrinsic
(i.e., mitochondrial) pathways. The former is triggered on the cell surface by the
engagement of death receptors, such as tumor necrosis factor receptor and tumor
https://doi.org/10.3324/haematol.2021.280201 necrosis factor-related apoptosis-inducing ligand receptor, and proceeds through
caspase-8/10 activation and BID cleavage.3 The latter is induced by oncogenic sig-
naling, nutrient deprivation, genotoxic drugs and other cellular stressors, and is
©2022 Ferrata Storti Foundation regulated at the level of the outer mitochondrial membrane by pro- and anti-apop-
Material published in Haematologica is covered by copyright. totic BCL-2 family members.4
All rights are reserved to the Ferrata Storti Foundation. Use of
published material is allowed under the following terms and
conditions:
https://creativecommons.org/licenses/by-nc/4.0/legalcode. Overview of the BCL-2 family
Copies of published material are allowed for personal or inter-
nal use. Sharing published material for non-commercial pur- The BCL-2 family members are distinguished into three main categories: anti-
poses is subject to the following conditions:
apoptotic, pro-apoptotic BH3-only, and pro-apoptotic effectors4 (Figure 1). BCL-2,
https://creativecommons.org/licenses/by-nc/4.0/legalcode,
sect. 3. Reproducing and sharing published material for com- MCL-1, BCL-w, BCL-B, BCL-xL, and BFL-1 are the main pro-survival relatives and
mercial purposes is not allowed without permission in writing contain all four BH domains.4,5 BID, BIM, PUMA, NOXA, BAD, HRK, and BMF are
from the publisher. the pro-apoptotic BH3-only proteins, and only share the BH3 domain with the rest
of the family. Among these, BID and BIM can directly activate the pro-apoptotic
effectors, while the others act as sensitizers by antagonizing the pro-survival

The anti-apoptotic landscape of blood cancers
members.6 BAX, BAK, and BOK contain three out of four nately with all of them. BAD binds BCL-2, BCL-w and
BH domains and represent the pro-apoptotic effector pro- BCL-xL, HRK binds BCL-xL, while NOXA preferentially
teins that form homodimers and heterodimers through binds MCL-1 and BFL-1.6
the outer mitochondrial membrane.7 Overall, the mito- In hematologic malignancies, there is selective pressure
chondrial cascade of apoptosis starts when a death stim- for upregulating the pro-survival members via genetic
ulus triggers the translocation of BH3-only proteins to the and non-genetic mechanisms. For example, in both follic-
mitochondrial surface. This leads to subsequent displace- ular lymphoma (FL) and double-hit diffuse large-B cell
ment of further pro-apoptotic activators and effectors lymphoma (DLBCL), t(14;18) juxtaposes BCL2 to the
from the pro-survival members, promoting BAX/BAK immunoglobulin heavy chain (IGH) locus, increasing
assembly through the outer mitochondrial membrane. BCL-2 protein levels.9,10 In chronic lymphocytic leukemia
These pore-like structures provoke the cytoplasmic leak- (CLL) with del(13q), the lack of microRNA 15/16 de-
age of cytochrome c and other apoptogenic factors, represses BCL-2 expression.11 Multiple myeloma (MM)
which in turn activate the executioner caspases, and dis- and selected subtypes of DLBCL harbor the 1q21 ampli-
rupt the mitochondrial transmembrane potential that is fication, which leads to MCL-1 overexpression.12,13 On
necessary for oxidative phosphorylation (OxPHOS).8 the other hand, pro-apoptotic BH3-only members are
BH3-only proteins have different interaction modalities sometimes downregulated. BIM is epigenetically
for each anti-apoptotic member. BAD, HRK and NOXA silenced in a subset of patients with acute lymphoblastic
bind preferentially to selected anti-apoptotic members, leukemia (ALL), leading to glucocorticoid resistance and
whereas BIM, BID, PUMA and BMF interact indiscrimi- poorer outcomes.14 Similarly, a subgroup of patients with
Figure 1. The BCL-2 protein family. The three subgroups of the BCL-2 protein family and their recurrent genetic alterations are shown in the colored boxes. A schemat-
ic of the interactions occurring among the BCL-2 family members is represented in the lower right box, based on the “indirect activation” model. Overall, the anti-
apoptotic proteins sequester the pro-apoptotic BH3-only activators (A) and effectors, preventing the initiation of the apoptotic cascade. By contrast, the pro-apoptotic
sensitizers (S) antagonize the anti-apoptotic members thus freeing the activators, which in turn trigger the polymerization of the effectors. This creates pore-like struc-
tures at the outer mitochondrial membrane which favor the release of cytochrome c (cyt c) and other apoptogenic factors. While the model shown here has been
widely adopted to define the concept of apoptotic priming, recent evidence suggests that, at least in selected cancer types, BAX and BAK activation may only require
that these members are freed from the anti-apoptotics, with no need of direct interaction with pro-apoptotic members. DLBCL: diffuse large B-cell lymphoma; FL: fol-
licular lymphoma; ABC: activated B-cell; T-NHL: T-cell non-Hodgkin lymphoma; MM: multiple myeloma; HL: Hodgkin lymphoma; MCL: mantle cell lymphoma; GC-R: ALL
glucocorticoid-resistant acute lymphoblastic leukemia; BL: Burkitt lymphoma.

I. Ferrarini et al.
mantle cell lymphoma expresses low levels of BIM and is Genetic and functional approaches to detect
less likely to achieve complete response to standard anti-apoptotic dependencies
treatments.15 All of these mechanisms converge towards
apoptosis evasion, a common denominator for cancer Over the last 15 years, several genetic and functional
cells. methodologies have been set up to derive anti-apoptotic
dependencies in cancer. Genetic knock-out of selected
anti-apoptotic proteins using CRISPR-Cas9 or related
Evasion from mitochondrial apoptosis gene-editing techniques have pointed out the anti-apop-
totic role of MCL-1 and BCL-w in myc-driven lym-
Solid tumors and hematologic malignancies evade phomas.21-23 Moreover, doxycycline-inducible silencing
mitochondrial apoptosis in markedly different ways RNA (siRNA) targeting specific pro-survival proteins has
(Figure 2), perhaps reminiscent of the biology of the nor- been successfully transfected in human lymphoma cell
mal tissue counterparts.16 Diseases such as kidney, col- lines to evaluate which member has the greatest impact
orectal, and cervical cancer show an extremely poor pro- on in vitro cell survival.24 While the strength of these
clivity to activate intrinsic apoptosis, even when strong approaches lies in their ability to accurately inform about
pro-apoptotic stressors are directly applied on cancer cell the role of a selected BCL-2 family gene, they are poorly
mitochondria. Such low apoptotic priming, defined by applicable to primary cells from cancer patients. As their
the near absence of BH3-only activators on the mitochon- use is mostly limited to cancer cell lines, genetic
drial surface, is a major factor contributing to the approaches are precious to infer general principles of
chemoresistance of solid tumors, and suggests targeting apoptotic regulation in a given cancer type, but lack scal-
solely this pathway might not be successful against these ability to large numbers of patient-derived samples. This
diseases.16 By contrast, mitochondria of blood cancer cells is of relevance because some of the most common hema-
struggle to maintain the integrity of the outer mitochon- tologic malignancies such as acute myeloid leukemia
drial membrane due to its occupation by several pro- (AML) and DLBCL show heterogeneity of anti-apoptotic
apoptotic activators (i.e., high mitochondrial priming). In dependencies across patients, and perhaps even within
this type of cancers, apoptotic evasion is based on the patients over the course of their disease.19,25,26
activity of several anti-apoptotic proteins aiming at Functional approaches provide higher scalability but
buffering large amounts of pro-apoptotics that dynami- less molecular insight. They are mainly based on in vitro
cally shuttle between the cytoplasm and the mitochon- and ex vivo pharmacological targeting of pro-survival BH3
dria.16 The need for an efficient anti-apoptotic arsenal cre- proteins, and on BH3 profiling.27-30 Side-by-side compar-
ates a specific vulnerability that is being successfully tar- isons of different BH3 mimetics targeting distinct anti-
geted by venetoclax and other BH3 mimetics.17 This class apoptotic proteins have been performed on cancer cell
of small molecules targets the interaction interface lines and primary samples, and several readouts such as
between the anti- and pro-apoptotic members thus intracellular ATP content, annexin V externalization, and
allowing the latter to initiate the apoptotic cascade. While caspase 3/7 activation have been utilized to detect differ-
in some cases a single anti-apoptotic protein is the only ences in cell viability.27,28 These pharmacological assays
barrier to apoptotic triggering, in others multiple anti- inform about the anti-apoptotic dependencies of tumor
apoptotic members act in concert to oppose outer mito- samples, working at the same time as apoptosis-tailored
chondrial membrane permeabilization18-20 (Figure 2). drug sensitivity screens with potential impact on preci-
Figure 2. Different forms of evasion from mitochondrial apoptosis. Three distinct scenarios are depicted. In cancers such as chronic lymphocytic leukemia, large
amounts of pro-apoptotic activators are sequestered (i.e., high priming) by a single anti-apoptotic relative. In other hematologic cancers (e.g., B-cell acute lymphoblas-
tic leukemia), apoptotic priming is high, but the pro-apoptotics are concurrently sequestered by multiple anti-apoptotic relatives (e.g., BCL-2, MCL-1 and BCL-xL). In
solid tumors, pro-apoptotic members are mostly not bound to the anti-apoptotic proteins, and hence sensitivity to BH3 mimetics is generally low. CLL: chronic lym-
phocytic leukemia; B-ALL: B-cell acute lymphoblastic leukemia.

sion strategies. Long BH3-mimetic incubation times (i.e., ily dynamics, without off-target effects that might
more than 18-24 hours), which may be needed to detect instead be encountered using small molecules. A potential
many of the cell-death readouts, is a common shortcom- downside is that BH3 profiling is an organelle-centered
ing of these assays as primary cells do not often survive method that does not take into account how other cellu-
long-term in ex vivo cultures.31 Moreover, it has been lar components might react to peptide-induced
reported that in vitro-cultured primary cancer cells lose cytochrome c release. For instance, defective activation of
similarities with the original tumor over time,32 thus downstream cytosolic caspases, which sometimes occurs
weakening the reliability of the results. Off-target effects in solid tumors due to somatic mutations,35 may blunt the
of some BH3 mimetics, for example BCL-2-independent apoptotic response triggered by cytochrome c leakage.
inhibition of OxPHOS by venetoclax,33 might be an addi- BH3 profiling also functions as a platform to set up an
tional limitation of these assays when the primary aim is additional assay, named dynamic BH3 profiling, which
to study the biology of the disease rather than the drug measures changes in apoptotic priming and anti-apoptot-
efficacy. A clinically applicable declination of such ic dependencies triggered by drug candidates. In this case,
approaches, called the BH3-mimetic toolkit, has been the incubation with pro-apoptotic peptides is preceded
employed on MM samples using CD138 loss as a flow by ex vivo treatment with a panel of drugs, with the aim
cytometry readout of cancer cell death. Venetoclax (a of identifying those that most efficiently lower the
BCL-2 inhibitor), A1155463, A1331852 (both BCL-xL threshold for cytochrome c release.36,37 Dynamic BH3 pro-
inhibitors), and A1210477 (a MCL-1 inhibitor) were test- filing has proven useful to assess the impact of Bruton
ed at multiple concentrations, and three dependency tyrosine kinase (BTK) inhibitors on BCL-2 dependence of
groups were derived in an unbiased way using analytical CLL cells,38 and as a functional precision medicine strate-
tools.30 BH3 profiling is a different functional technique gy for T-cell prolymphocytic leukemia.39
based on exposing cancer cell mitochondria to an array of
pro-apoptotic peptides with distinct binding modalities
for different anti-apoptotic members. In this assay, the The anti-apoptotic map of hematologic
anti-apoptotic addiction of cancer cells can be inferred by malignancies
the pattern of cytochrome c release upon peptide incuba-
tion.34 Due to the short incubation time, BH3 profiling Extensive preclinical research and clinical observations
averts the risk of artifactual genetic selections or function- are building our knowledge on how cancer cells evade
al modifications that could potentially occur during pro- apoptosis. The anti-apoptotic map of hematologic malig-
longed ex vivo culture. In addition, the specificity of treat- nancies (Figure 3) has recently guided highly effective
ing peptides that act directly on the mitochondrial surface treatments for CLL and AML,40,41 and is currently inspiring
renders this assay particularly focused on the BCL-2 fam- novel antineoplastic regimens for other types of blood
Figure 3. The anti-apoptotic map of hematologic

malignancies. The colored map shows the anti-
apoptotic dependencies of several hematologic
malignancies, based on preclinical data and clini-
cal results (see references). With the exception of
follicular lymphoma, in which the BCL-xL depen-
dence is driven by microenvironmental stimuli, all
the other anti-apoptotic dependencies depicted
here refer to cell-intrinsic dependencies. AML:
acute myeloid leukemia; MLL-r B-ALL: MLL-rear-
ranged B-cell acute lymphoblastic leukemia; ETP
T-ALL: early T-cell acute lymphoblastic leukemia;
CLL: chronic lymphocytic leukemia; DLBCL: dif-
fuse large B-cell lymphoma; FL: follicular lym-
phoma; MM multiple myeloma; ALCL: anaplastic
large T-cell lymphoma; T-NHL NOS: T-cell non-
Hodgkin lymphoma not otherwise specified; CTCL:
cutaneous T-cell lymphoma; T-PLL: T-cell prolym-
phocytic leukemia; HL: Hodgkin lymphoma;
BPDCN: blastic plasmacytoid dendritic cell neo-
plasia.

I. Ferrarini et al.
disorders. This section focuses on key preclinical findings BCL-xL, with MCL-1 being identified as a further anti-
relevant to apoptosis avoidance. Clinical results of BH3 apoptotic member able to confer resistance to single or
mimetics in hematology will only be touched on, as they dual BCL-2/BCL-xL antagonism.48-50 Indeed, sensitivity to
have been recently reviewed by Roberts and colleagues.42 venetoclax, which was predicted by BCL2 gene expres-
sion level as well as BH3 profiling, was highly heteroge-
Acute myeloid leukemia and myelodysplastic syndromes neous in a panel of B-ALL cell lines and patient-derived
Regulation of intrinsic apoptosis in AML shows both xenograft, with EC50 values ranging from 1.8 nM to 5.5
intratumor and interpatient heterogeneity. In 2014, Pan mM.49 Accordingly, venetoclax was effective in vivo in only
and co-workers identified a BCL-2 dependence for rough- a minority of B-ALL xenografts, whereas combined inhi-
ly 80% of primary AML cases, with rapid apoptotic trig- bition of BCL-2 and BCL-xL resulted in synergistic killing
gering upon ex vivo exposure to venetoclax. BCL-2 protein of most B-ALL in vivo models.51 A recently reported phase
expression correlated with sensitivity to venetoclax, I trial of the combination of venetoclax with low-dose
whereas BCL-xL and, to a lesser extent MCL-1, showed navitoclax (dual BCL-2/BCL-xL inhibitor) plus
anti-correlation with susceptibility to BCL-2 inhibition.43 chemotherapy in relapsed/refractory (R/R) B-ALL has
A phase II trial evaluating the activity of venetoclax as a shown encouraging results, with a complete remission
single agent in high-risk AML demonstrated an overall rate of 60%.52 Despite the heterogeneity and the high
response rate of 19%, with particularly favorable degree of anti-apoptotic co-dependencies in B-ALL, the
responses among patients carrying isocitrate dehydroge- subgroup carrying the t(4;11) translocation proved uni-
nase 1/2 (IDH1/2) mutations.41 BH3 profiling identified formly BCL-2-dependent.51,53 The fusion protein MLL/AF4
patients who were more likely to stay on venetoclax ther- activates BCL2 transcription via DOT1L-mediated
apy longer than 30 days, working as a predictive func- H3K79me2/3, without altering the expression level of
tional assay with potential clinical applications.41 Bone other anti-apoptotic members. This renders MLL-rear-
marrow cells from patients with high-risk myelodysplas- ranged cells susceptible to venetoclax-induced apoptosis
tic syndromes are also sensitive to BCL-2 antagonism. In in vitro, and sensitive to venetoclax-based combinations in
vitro treatment with ABT-737 or venetoclax depletes the vivo.53
myelodysplastic syndrome progenitor compartment, and The anti-apoptotic dependency of T-ALL reflects the
decreases the colony-forming capacity and the percentage maturation stage of lymphoblasts. Early T-cell progenitor
of CD34+ cells.44 The combination of venetoclax plus (ETP) ALL cells most closely resemble early thymic, CD4–
intensive chemotherapy led to complete remission in /CD8– T cells, and are dependent upon BCL-2.54 Instead,
82% of patients with newly-diagnosed AML and high- non-ETP T-ALL cells have a gene expression and pheno-
risk myelodysplastic syndromes.45 Despite the meaning- typic profile resembling that of more mature, CD4+/CD8+
ful clinical activity of venetoclax-based regimens in these T cells, expressing abundant BCL-xL protein levels, and
settings, about 20% of patients are primarily refractory. having functional dependency upon BCL-xL.54 While
Among AML patients achieving complete remission with ETP-ALL patient-derived xenograft models are very sen-
venetoclax plus azacytidine, the median duration of sitive to venetoclax in vivo, the non-ETP counterpart is rel-
response was only 11.3 months.46 This suggests that a atively resistant.54 Moreover, sensitivity to long-term
subset of AML cases is not BCL-2 dependent, and that BCL-2 inhibition in ETP-ALL might be compromised by
additional groups of patients may harbor subclonal microenvironment-derived signals. More specifically, the
dependencies to different anti-apoptotic proteins. A spleen has been identified as a sanctuary site for residual
recent study addressed this point and found that BCL-2 ETP lymphoblasts following venetoclax treatment. Such
dependence decreases through stages of AML morpho- surviving cells display decreased BCL-2 expression and
logical maturation.47 Indeed, lower BCL-2 expression, at reduced BCL-2 dependence, with requirement of con-
both mRNA and protein levels, was observed in French- comitant MCL-1 inhibition to evoke robust cell death.55
American-British (FAB) M5 AML compared to FAB-
M0/M1/M2 leukemia. A combination of venetoclax plus Chronic lymphocytic leukemia
azacitidine failed to inhibit OxPHOS in monocytic AML, In the light of its broad heterogeneity in terms of chro-
with a modest impact on cell viability.47 By contrast, the mosomal aberrations, gene mutations, clinical character-
MCL-1 inhibitor VU661013 combined with azacitidine istics, and drug response profiles,56-58 CLL shows surpris-
significantly suppressed OxPHOS in monocytic cases and ingly homogeneous anti-apoptotic regulation.18 More
was more effective than venetoclax-based treatments in than three decades of basic discoveries have pointed out
inducing cell death. Genetic knockdown of MCL-1 was that BCL-2 is overexpressed in CLL cells compared to nor-
sufficient to trigger apoptosis in primary monocytic AML mal B lymphocytes due to gene promoter hypomethyla-
specimens, further indicating their reliance on MCL-1 to tion,59 miR15/16 downregulation,11 and, more rarely, BCL-
maintain survival.47 Accordingly, 62% of patients with 2 translocation.60 Such genetic bases for BCL-2 addiction
FAB-M5 AML were refractory to venetoclax-azacitidine, have been functionally confirmed by BH3 profiling,
whereas only 8% of non-FAB-M5 cases did not respond which revealed a clear-cut BCL-2 dependence of CLL cells
to this regimen.47 A side-by-side comparison of BH3 regardless of TP53 status and previous lines of therapy.18,61
mimetics in mediating AML cell killing confirmed that a This set the stage for the clinical introduction of veneto-
subset of immortalized and primary AML cells is highly clax, the first approved selective BCL-2 inhibitor, which
sensitive (low micromolar/nanomolar range) to the MCL- achieved an overall response rate of 79% with 20% of
1 inhibitor S63845, but not to BCL-xL or BCL-2 complete remissions in the R/R CLL setting.40
inhibitors.27 While cell-intrinsic genetic programs seem to be
responsible for the exceedingly high BCL-2 dependency
Acute lymphoblastic leukemia of CLL cells, signals from the microenvironment can add
B-cell ALL shows concurrent dependence on BCL-2 and further layers of anti-apoptotic protection. Indeed, anti-

gen stimulation and CXCL12, both converging on BTK, Multiple myeloma

decrease apoptotic priming and BCL-2 dependence.62 As a At least one-third of MM cases are predominantly
consequence, ibrutinib and other inhibitors of the B-cell MCL-1 dependent, whereas the others are either BCL-2
receptor signaling pathway increase BCL-2 dependence dependent or characterized by BCL-2/MCL-1 co-depen-
by impeding extrinsic signals to upmodulate MCL-1 and dence.12,20,30,72 Moreover, a minority of MM cell lines and
BCL-xL which would ultimately favor additional pro-sur- primary samples show some degree of BCL-xL depen-
vival forces.38 The CLARITY study, exploring the combi- dence.30,72 MCL-1 addiction can be driven by genetic alter-
nation of ibrutinib and venetoclax in R/R CLL, found ation and microenvironmental cues. The MCL1 gene is
51% complete remissions with a high rate of minimal located on 1q21, which is amplified in 43-72% of MM
residual disease eradication, which enabled treatment patients depending on disease status.73 1q21 copy gain
cessation in a subset of patients.63 directly correlates with MCL1 transcript abundance and
MCL-1 protein expression.12 Importantly, plasma cells
B-cell non-Hodgkin lymphoma from 1q21-amplified cases are especially sensitive to
DLBCL, the most common type of B-cell non- MCL-1 inhibition, while proving relatively resistant to
Hodgkin lymphoma (NHL), is remarkably heteroge- venetoclax.12 Interleukin-6 released by bone marrow stro-
neous in terms of mutational landscape and clinical pic- mal cells can further amplify MCL-1 dependence through
tures.64 Anti-apoptotic dependencies show heterogene- MCL-1 transcriptional upregulation and BIM phosphory-
ity as well, and do not correlate with cell of origin.19,26,65 lation. Such events switch the BIM binding partner from
Loss of the anti-apoptotic BCL-w was reported to delay BCL-2 and BCL-xL to MCL-1.74,75 Blocking the interleukin-
MYC-driven lymphoma development in Em-Myc trans- 6 signaling pathway decreases MCL-1 dependence and
genic mice by augmenting MYC-induced apoptosis.22 enhances sensitivity to venetoclax.76,77 On the other hand,
Moreover, BCL-w is overexpressed in a subset of BCL-2-dependent cases are enriched among MM with the
DLBCL characterized by shorter overall survival, sug- t(11;14)(q13;q32) translocation, which is detected in 15%
gesting a primary role for apoptosis evasion.22 Despite to 20% of cases.78 In the phase I study of venetoclax
this, a recent study found that CRISPR/Cas9-mediated monotherapy in R/R MM, the overall response rate was
loss of BCL-w did not trigger apoptosis in DLBCL cell 21% in the whole population, but reached up to 40% in
lines, nor did it increase the sensitivity to BH3 mimetics the subgroup harboring t(11;14).79 High BCL2:MCL1 and
targeting additional pro-survival proteins, casting BCL2:BCL2L1 mRNA expression ratios correlated with
doubts about the role of BCL-w in human DLBCL.28 venetoclax sensitivity.79 Given the anti-apoptotic hetero-
MYC-driven lymphomagenesis is also sustained by geneity and the frequent co-dependencies of MM cells,
MCL-1, which is highly expressed in activated B-cell dual targeting of BCL-2 and MCL-1 has been assessed in
DLBCL. 66 MCL1 copy number abnormalities were preclinical studies, showing a profound synergism poten-
detectable in 25.7% of activated B-cell -DLBCL versus tially translatable to the clinic.77 Moreover, the BH3-
12.5% of germinal-center DLBCL, and constitutive mimetic toolkit revealed an increase in MCL-1 addiction
STAT3 signaling further contributes to MCL-1 upregu- from 33% at diagnosis to 69% at relapse, indicating tem-
lation in selected cases. 66 The MCL-1 antagonist poral remodeling of cellular dependencies.30 This assay
AZD5991, currently in clinical testing, curtails tumor also identified a subset of newly diagnosed MM patients
growth and disrupts mitochondrial metabolism in not sensitive to any of the BH3 mimetics, highlighting
MCL-1-addicted DLBCL cells via TP53- and BAX- that apoptosis targeting might not always be a suitable
dependent mechanisms. 13 BCL2 is also frequently therapeutic option.30
deregulated in DLBCL due to the t(14;18) chromosomal
translocation, gene mutations, copy number alterations T-cell non-Hodgkin lymphoma
and amplifications.67 Such genetic events confer poor Protein expression analyses identified MCL-1 as the
prognosis especially when combined with MYC ampli- major anti-apoptotic member in cell lines and primary
fication.10 While in some cases BCL2 dysregulation is samples of systemic T-cell NHL.80,81 Copy number gains
associated with functional dependency on BCL-2 and involving the MCL1 locus were found in ten out of 21 T-
high sensitivity to venetoclax, in others targeting addi- NHL cell lines.80 Accordingly, loss of a single MCL1 allele
tional pro-survival members is needed to achieve apop- delayed tumor formation in T-NHL mouse models and
tosis. Indeed, several B-cell receptor-dependent DLBCL compromised the viability of neoplastic T cells,81 BH3
lines with genetic bases for BCL-2 dysregulation require profiling confirmed that most T-NHL cell lines, especially
dual targeting of PI3Ka/d, which in turns decreases those from anaplastic T-cell lymphomas and other
MCL-1 abundance, and BCL-2 to commit cell death.68 peripheral T-cell lymphomas, are MCL-1 dependent.80
Anti-apoptotic heterogeneity and frequent co-depen- The development of several MCL-1 antagonists has
dencies account for the disappointing results of single- recently allowed the translation of these biological find-
agent venetoclax against DLBCL, with an overall ings into pharmacological approaches. Indeed, AZD5991
response rate of only 18% in R/R cases.69 Less common reduced tumor volumes in vivo, and synergized with
subtypes of B-NHL, including follicular lymphoma and cyclophosphamide, vincristine, doxorubicin and pred-
mantle cell lymphoma, display better clinical responses nisone to improve survival of mice with T-NHL.80 By con-
to BCL-2 inhibition.69,70 However, durable remissions trast, cutaneous T-NHL cases are primarily BCL-xL
are not frequent and may require combination thera- dependent, and some of them carry copy number alter-
pies. At least in follicular lymphoma, in which high ation of the BCL2L1 gene.80 A proteolysis targeting
BCL-2 expression caused by the t(14;18) translocation chimera that targets BCL-xL for degradation has been
has ever been considered the pathogenic hallmark, con- developed to effectively kill cutaneous T-NHL cells in vitro
current antagonism of microenvironment-induced BCL- and in vivo, without causing significant thrombocytopenia
xL is needed to efficiently trigger apoptosis.71 as previously reported for navitoclax.82

I. Ferrarini et al.
T-cell prolymphocytic leukemia anti-apoptotic proteins in sustaining Hodgkin lymphoma

As compared to CLL, T-cell prolymphocytic leukemia is growth.85
less primed for apoptosis, less BCL-2 dependent, and
often BCL-2/MCL-1 co-dependent.39,83 BCL-xL depen- Blastic plasmacytoid dendritic cell neoplasm
dence is usually more pronounced in T-cell prolympho- Preclinical data indicate that blastic plasmacytoid den-
cytic leukemia than in CLL, but remains of secondary dritic cell neoplasm is primarily BCL-2 dependent. Blastic
importance when compared with BCL-2 and MCL-1 plasmacytoid dendritic cell neoplasm cells collected from
dependence.39 Clinical reports suggest that venetoclax patient-derived xenografts or directly from patients’ skin
monotherapy is often inadequate to get durable disease and bone marrow showed higher BCL-2 dependency
control.84 As demonstrated by dynamic BH3 profiling, compared with randomly selected AML cases.86 In vivo
inhibitors of histone deacetylase and the JAK/STAT path- experiments confirmed these findings and, to date, sever-
way increase apoptotic priming and BCL-2 dependence, al reports have been published about patients with
strengthening the pro-apoptotic effect of venetoclax in relapsed blastic plasmacytoid dendritic cell neoplasm suc-
vitro and in vivo.39 cessfully treated with single-agent venetoclax.87,88 Such a
therapeutic approach is currently being investigated in a
Hodgkin lymphoma phase I clinical trial (NCT03485547).
As compared to normal B lymphocytes, Hodgkin lym-
phoma cells express higher levels of BCL-w and BCL-xL
mRNA.85 BCL-w expression further increases in Resistance to BH3 mimetics: a tale of cellular
advanced stages and in the R/R setting.85 Fluorescence in interactions, mitochondrial biology and
situ hybridization analyses on Hodgkin lymphoma sam- mutational pressure
ples showed that copy number gains of chromosome 14
and amplifications of the BCL-w containing region were With the growing use of venetoclax in clinical practice,
common events in the pathogenesis of Hodgkin lym- several resistance mechanisms have been outlined, espe-
phoma and led to almost invariably high BCL-w protein cially in the context of CLL and AML which currently rep-
expression as detected by immunohistochemistry. resent the major indications for BCL-2 inhibition. Three
Genetic knockdown of BCL-w or pharmacological antag- broad concepts are emerging. First, clinical progressions
onism of BCL-xL significantly reduced Hodgkin lym- on venetoclax are mostly underpinned by “polyclonal pat-
phoma cell viability, pointing to a primary role for these terns”, whereby different leukemic clones within the same
Figure 4. Mechanisms of venetoclax resistance. The figure depicts the four major modalities of resistance to venetoclax: cell-extrinsic mechanisms, outer and inner
mitochondrial adaptation, genomic alterations, and expansion of intrinsically resistant subclones. Mechanisms highlighted in purple have been described in acute
myeloid leukemia. Those highlighted in blue have been reported in chronic lymphocytic leukemia. See text for pathway details. SCF: stem cell factors; OxPHOS: oxida-
tive phosphorylation; FAO: fatty acid oxidation.

Table 1. Treatment strategies that may prevent/overcome escape from venetoclax based on preclinical mechanistic predictions.
Treatment strategy Indication Mode of action of partner drug Study phase NCT number
Escape mechanism: extrinsic interactions and downstream signaling pathways
cirmtuzumab + venetoclax CLL anti-ROR1 I NCT03797261
duvelisib + venetoclax CLL PI3Kgd inhibitor I/II NCT03534323
copanlisib + venetoclax DLBCL PI3Kad inhibitor I/II NCT04572763
ruxolitinib + venetoclax AML JAK inhibitor I NCT03874052
BP1001 + venetoclax AML L-Grb2 antisense oligonucleotide II NCT02781883
ibrutinib + venetoclax CLL covalent BTK inhibitor II NCT02756897
loxo305 + venetoclax + rituximab CLL non-covalent BTK inhibitor III NCT04965493
plerixafor + venetoclax AML anti-CXCR4 / /
Escape mechanism: anti-apoptotic and metabolic mitochondrial adaptation
AMG 176 + venetoclax R/R heme malignancies MCL-1 inhibitor I NCT03797261
S64315 + venetoclax AML MCL-1 inhibitor I NCT03672695
AZD5991 + venetoclax AML MCL-1 inhibitor I/II NCT03218683
Navitoclax + venetoclax + chemo B-ALL BCL-xL inhibitor I NCT03181126
adi-peg 20 + venetoclax + azacytidine AML arginine depleting enzyme I NCT05001828
Omacetaxine + venetoclax AML protein translation inhibitor I NCT04874194
IACS-010759 + venetoclax AML OxPHOS inhibitor / /
ONC201 + venetoclax AML OxPHOS inhibitor / /
Escape mechanism: gene alterations
BGB-11417 B-cell malignancies BCL-2 inhibitor (active against G101V) I NCT04883957
eprenetapopt + venetoclax MCL p53 reactivator II NCT04990778
NCT: National Clinical Trials; CLL: chronic lymphocytic leukemia; DLBCL: diffuse large B-cell lymphoma; AML: acute myeloid leukemia; R/R: relapsed or refractory; ALL: acute
lymphoblastic leukemia; MCL: mantle cell lymphoma.
patient take distinct paths to survive BCL-2 inhibition lymph nodes, where most of the cell-to-cell and paracrine
independent of each other.89 Secondly, the general biolog- interactions take place.40
ical principles driving resistance to one specific BH3
mimetic are shared with other BCL-2 family antagonists. Mitochondrial adaptations
Although this section is mainly focused on the mecha- Mitochondria are active players in the initiation of vene-
nisms of resistance to venetoclax, the only Food and Drug toclax resistance due to anti-apoptotic remodeling, occur-
Administration-approved BH3 mimetic so far, early data ring at the outer mitochondrial membrane, and metabolic
are piling up about similar escape trajectories occurring in reprogramming, occurring at the inner mitochondrial
cells treated with MCL-1 or BCL-xL antagonists.25 Thirdly, membrane. Functional shifts towards alternative anti-
resistance to BH3 mimetics is highly “multimodal”, as apoptotic defenses render leukemic mitochondria progres-
multiple cellular components can be rewired to undermine sively less vulnerable to BCL-2 antagonism, with dual
the efficacy of BCL-2 family antagonism. The four major BCL-2 and MCL-1 antagonism outperforming the individ-
modalities of acquisition of resistance to venetoclax are ual targeting.25,92,94 Moreover, a global reduction of mito-
based on cell-extrinsic interactions,90,91 mitochondrial chondrial apoptotic priming is quite common in AML
adaptation,92 genomic alterations,89 and, as previously acquiring resistance to different types of BH3 mimetics.25
highlighted for the monocytic escape of AML, the emer- As apoptotic regulation is tightly connected with bioener-
gence of intrinsically resistant clones47 (Figure 4). While getic processes, the upmodulation of mitochondrial
such variety of resistance mechanisms contrasts with the metabolic pathways confers resistance to BH3 mimetics.
simpler mutational evolution often observed in patients While in venetoclax-sensitive AML stem cells OxPHOS is
treated with kinase inhibitors,93 it offers several clues to suppressed through the inhibition of amino acid
plan at best subsequent therapies, and to design strategic metabolism, in resistant clones it is restored through the
drug combinations as well (Table 1). enhancement of fatty acid oxidation.95 In this context, the
role of BCL-2 remains unclear because venetoclax is
Cell-extrinsic interactions reported to inhibit OxPHOS independently of BCL-2
In AML, CXCL12 released by surrounding stromal cells expression.96 Thus, resistant cells might have evolved the
protects the leukemic clone from BCL-2 inhibition by ability to restore OxPHOS, bypassing the inhibitory effect
activating the CD44/CXCR4 complex, which in turn of venetoclax. In addition, resistant AML stem cells show
induces the transcription of several pro-survival embry- elevated nicotinamide metabolism which promotes the
onic stem-cell core transcription factors.90 Likewise, in uptake and catabolism of amino acids, as well as the con-
CLL CD40 ligation enhances non-BCL-2 anti-apoptotic version of fatty acids into the tricarboxylic acid cycle inter-
dependencies by inducing BCL-xL transcription via the mediates 2-oxoglutarate and malate. Indeed, inhibitors of
canonical as well as the non-canonical NF-kB pathway.91 nicotinamide phosphoribosyltransferase specifically target
Induction of pro-survival members by cell-extrinsic fac- leukemic cells with acquired resistance to venetoclax.97
tors may account for the relatively less durable response The modulation of mitochondrial cristae ultrastructure
to venetoclax observed among CLL patients with >5 cm also impacts on bioenergetics and BH3 mimetic sensitivi-

I. Ferrarini et al.
ty. The mitochondrial chaperonin CLPB has been recently Genomic alterations
identified as a crucial interactor of the cristae-shaping pro- Emergence of subclones harboring BCL2 mutations is
tein OPA1, and its downregulation alters cristae architec- increasingly described in CLL patients progressing on
ture and renders cytochrome c more prone for cytosolic venetoclax, with the most frequent being Gly101Val and
release.98 An independent genome-wide CRISPR screen substitutions at Asp103.89 These BCL-2 structural variants
identified genes involved in mitochondrial translation as decrease venetoclax binding affinity by several folds.
an additional circuit to bypass BCL-2 antagonism, and Interestingly, BCL-2 mutants maintain the ability to bind
antibiotics targeting the mitochondrial ribosomes effec- and sequester BIM, thus preserving their fundamental
tively overcome venetoclax resistance.99 role in the regulation of apoptotic balance.89 In other
Figure 5. Integrated precision medicine for ‘highly complex’ hematologic malignancies. (A) Classification of hematologic malignancies based on their
molecular/functional complexity and treatment requirements. In chronic myeloid leukemia, the prototype of ‘purely genomic’ malignancies, a single genomic aber-
ration drives disease biology and treatment modalities. In chronic lymphocytic leukemia, a model for ‘mostly functional’ malignancies, a few functional pathways (B-
cell receptor signaling, BCL-2 dependence) sustain leukemic growth and have proven to be the best drug targets so far. In acute myeloid leukemia and other ‘highly
complex’ malignancies, multiple driver mutations and functional oncogenic pathways co-occur to promote cancer growth and escape treatments. This category might
be approached with an integrated precision medicine strategy. (B) Integrated precision medicine is based on interrogation of different static (i.e., microenvironment,
surfaceome, genomics) and functional (i.e., anti-apoptotic and metabolic dependencies, signaling pathways, drug sensitivity) domains through dedicated assays (ital-
ics). Each of these assays will provide information about different tumor-specific vulnerabilities (e.g., BH3 profiling might highlight BCL-2 dependence, extracellular
flux analysis might indicate oxidative phosphorylation utilization, microenvironment analysis might demonstrate CTLA-4-based interactions). Eventually, a combina-
tion of drugs targeting vulnerabilities from different domains is suggested, with potential benefits against ‘highly complex’ malignancies. CML: chronic myeloid
leukemia; APL: acute promyelocytic leukemia; HCL: hairy cell leukemia; CNL: chronic neutrophilic leukemia; AML: acute myeloid leukemia; DLBCL: diffuse large B-cell
lymphoma; ALL: acute lymphoblastic leukemia; MM: multiple myeloma; T-NHL: T-cell non-Hodgkin lymphoma; OxPHOS: oxidative phosphorylation.

cases, focal amplification of MCL1 or larger chromosomal in predicting clinical endpoints is still under investigation
gains at 1q drive venetoclax resistance. In this context, (e.g., NCT03943342, NCT03214562, NCT03709758), it
MCL-1 overexpression takes control of the anti-apoptotic looks clear that targeting functional cell biology domains
dependencies at the expense of BCL-2.92 A recent work (e.g., mitochondrial apoptosis, B-cell receptor signaling,
indicates that also BAX variants, including missense, non- immune interactions), in addition to the static mutational
sense, frameshift, and splice site mutations, emerge in repertoire, provides therapeutic benefits in hematology.
AML progressing on venetoclax.100 In AML cell lines, BAX Nevertheless, there should be awareness that mitochon-
but not BAK1 loss is associated with resistance to BCL-2 drial apoptosis is only one of the functional domains of
and MCL-1 antagonists.100 A mutation at BAX c.370 was cancer cell biology and, for most hematologic malignan-
detected in a case of mantle cell lymphoma that relapsed cies, mitochondrial targeting alone does not provide deep
on venetoclax.101 In contrast to BCL2 mutations, BAX and durable remissions.
alterations function as a more downstream resistance Blood cancers might be currently classified into three
mechanism potentially affecting sensitivity to a wider categories based on their underpinnings and treatment
range of BH3 mimetics, with important implications requirements. The rare ‘purely genomic’ malignancies
when MCL-1 and BCL-xL inhibitors become available for have a single genetic abnormality that almost entirely
clinical practice. Mutations in genes that do not encode sustains the neoplastic growth. In this case, genomic-driv-
BCL-2 family members have also been implicated in en precision medicine is a highly effective therapeutic
resistance mechanisms. Although TP53-disrupted AML strategy and has already gained success. CML is the pro-
and CLL cells are sensitive to acute venetoclax treatment, totype of these diseases that are effectively treated with
they are capable of escaping chronic BCL-2 inhibition, molecules targeting their genetic hallmarks.105 The ‘most-
partly because of an increased threshold for BAK/BAX ly functional’ malignancies, such as CLL, are particularly
activation.102 Moreover, lack of TP53 activity reduces the vulnerable to the targeting of selected oncogenic path-
transcription of the pro-apoptotic genes PUMA and ways that have been discovered through cell biology
NOXA, potentially heightening the apoptotic threshold experiments rather than mutational analyses.106 While
and impairing the efficacy of individual BH3 mimetics CLL cells have several mutations across their genome, the
when used at suboptimal doses or over long periods of targets of the most effective drugs, such as ibrutinib and
time.102 In AML, KRAS and PTPN11 mutations also venetoclax, are never mutated.106 In this category, knowl-
decrease sensitivity to venetoclax. Mutant KRAS down- edge built on perturbation of live cells proved perhaps
regulates BCL-2 and BAX, while it upregulates MCL-1 more useful than DNA sequencing in terms of therapeutic
and BCL2A1, possibly through the activation of the NF- applications, and targeting only one functional domain at
kB pathway. Similarly, mutant PTPN11 increases the a time yields considerable results.40 The ‘highly complex’
expression of BCL-xL, MCL-1 and its phosphorylated malignancies, such as AML and DLBCL, are those in
form, potentially targetable by the correspondent which different genetic drivers frequently co-occur, and
inhibitors.103 multiple functional domains simultaneously sustain can-
cer cell survival.107 In this context, single-agent treatments
rarely provide impressive results due to intrinsic and
Anti-apoptotic profiles at the forefront adaptive resistance. An integrated precision medicine
of integrated precision medicine approach encompassing genetic and functional testing
might be needed to improve the outcome of this catego-
Until very recently, the paradigm of precision medicine ry, especially in the R/R setting in which tumor hetero-
in clinical oncology consisted in matching the right drug geneity is further amplified.107 As illustrated in Figure 5,
with the right patient based on a tumor’s genomic signa- analysis on an individual basis of anti-apoptotic depen-
ture. Results from the NCI-MATCH trial indicated, how- dencies together with complementary static and func-
ever, that only 37.6% of enrolled patients had actionable tional measurements might help to reach this goal in the
alterations and only 17.8% were assigned to a treatment future. A prerequisite for this approach will be to design
arm.104 Furthermore, resistance-conferring tumor muta- clinically applicable ex vivo assays, each with the ability to
tions were found in 71.3% of specimens, thus lowering interrogate one specific domain of cancer cell biology.
the chance of meaningful responses.104 Overall, the co- Because cancer cells usually maintain BAX and BAK
occurrence of multiple driver mutations, the poor ability expression, and hence are susceptible to the restoration of
to predict which mutation is a driver and which is a mere mitochondrial apoptosis, rational targeting of extra-mito-
bystander, the complexity of resistance mechanisms, and chondrial vulnerabilities through integrated precision
the paucity of available drugs compared to the variety of medicine might eventually increase apoptotic priming
genomic alterations limit the success rate of genomic- and enhance the effectiveness of concomitant BCL-2 fam-
driven precision medicine. The emergence of mitochon- ily antagonism.
drial apoptosis as a cancer vulnerability highlights that
successful targets can be found outside genomic alter-
ations, and that genomic alterations not always predict Concluding remarks
response to mitochondrial targeting. Indeed, BCL-2 rear-
rangements or expression level do not always correlate The clinical success of venetoclax in CLL and AML gen-
with venetoclax response. By contrast, functional assays erated considerable enthusiasm on targeting apoptosis in
were able to identify tumors that were more likely to hematology. Basic discoveries in the field will be further
respond to BCL-2 inhibition in the clinic.20,49 Moreover, in rewarded with the possible clinical introduction of MCL-
the context of CLL, results of BH3 profiling correlated 1 and BCL-xL inhibitors, which are currently under inves-
with lymphocyte count reduction upon venetoclax initia- tigation. Different methods have been established to
tion in vivo.61 Although the reliability of these approaches derive tumor-specific anti-apoptotic dependencies, with

I. Ferrarini et al.
potential clinical applications. In particular, functional ing for some hematologic malignancies, it is clear that a
precision medicine platforms such as BH3 profiling and multitude of other genetic and functional dependencies
the BH3-mimetic toolkit have proven promising to prior- exists in complex cancers. In such cases, different angles
itize apoptosis-targeting agents in a clinically appropriate of cell biology need to be explored simultaneously to
timeframe. In several studies, they predicted clinical instruct more effective combination strategies.
results more accurately than the expression of the BCL-2
family proteins or other static measurements. Moreover, Disclosures
these approaches are somehow breaking the paradigm of No conflicts of interest to disclose.
precision medicine as an omics-based concept, and pave
the way for novel companion diagnostic assays based on Contributions
ex vivo perturbation of live cells. To pursue this path, clin- IF, AR, and CV conceived and wrote the manuscript, and
ical and technical efforts will be needed to obtain ade- reviewed the literature.
quate amounts of live cancer cells from patients, and to
standardize ex vivo protocols aiming at minimizing inter- Acknowledgments
laboratory variability. Despite the undoubted advantages This research received no external funding. The figures were
that BH3 domain-related pharmacology has been provid- created with Biorender.com.
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Acute Lymphoblastic Leukemia ARTICLE
LAMP-5 is an essential inflammatory-signaling Ferrata Storti Foundation

regulator and novel immunotherapy target for
mixed lineage leukemia-rearranged acute
leukemia
Gabriel Gracia-Maldonado,1,2 Jason Clark,1,2 Matthew Burwinkel,1,2
Brenay Greenslade,1,3 Mark Wunderlich,1,4 Nathan Salomonis,5,7 Dario Leone,6
Evelina Gatti,6 Philippe Pierre,6 Ashish R. Kumar1,2,7 and Lynn H. Lee1,3,7
1
Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Haematologica 2022
Cincinnati, OH, USA; 2Division of Bone Marrow Transplantation and Immune Deficiency, Volume 107(3):803-815
Cincinnati, Children’s Hospital Medical Center, Cincinnati, OH, USA; 3Division of
Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; 4Division
of Experimental Hematology and Cancer Biology, Cincinnati, Children’s Hospital Medical
Center, Cincinnati, OH, USA; 5Division of Biomedical Informatics, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH, USA; 6Aix Marseille Université, CNRS, INSERM,
Centre d'Immunologie de Marseille-Luminy (CIML), Marseille, France and 7Department
of Pediatrics, University of Cincinnati School of Medicine, Cincinnati, OH, USA
ABSTRACT
A
lthough great advances have been made in understanding the
pathobiology of mixed lineage leukemia-rearranged (MLL-r)
leukemias, therapies for this leukemia have remained limited, and
clinical outcomes remain bleak. In order to identify novel targets for
immunotherapy treatments, we compiled a lineage-independent MLL-r
leukemia gene signature using publicly available data sets. Data from
large leukemia repositories were filtered through the in silico human sur-
faceome, providing a list of highly predicted cell surface proteins overex-
pressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane
protein, is expressed highly and specifically in MLL-r leukemia. We
found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. Correspondence:
LAMP5 depletion significantly inhibited leukemia cell growth in vitro and
in vivo. Functional studies showed that LAMP-5 is a novel modulator of LYNN H. LEE
lynn.lee@cchmc.org
innate-immune pathways in MLL-r leukemias. Downregulation of
LAMP5 led to inhibition of NF-kB signaling and increased activation of ASHISH R. KUMAR
type-1 interferon signaling downstream of Toll-like receptor/interleukin ashish.kumar@cchmc.org
1 receptor activation. These effects were attributable to the critical role
of LAMP-5 in transferring the signal flux from interferon signaling endo- Received: April 30, 2020.
somes to pro-inflammatory signaling endosomes. Depletion of IRF7 was
able to partially rescue the cell growth inhibition upon LAMP5 downreg- Accepted: April 2, 2021.
ulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r Pre-published: April 29, 2021.
leukemia cells. Targeting surface LAMP-5 using an antibody-drug conju-
gate leads to significant cell viability decrease specifically in MLL-r
leukemias. Overall, based on the limited expression throughout human
tissues, we postulate that LAMP-5 could potentially serve as an
immunotherapeutic target with a wide therapeutic window to treat ©2022 Ferrata Storti Foundation
MLL-r leukemias. Material published in Haematologica is covered by copyright.
All rights are reserved to the Ferrata Storti Foundation. Use of
conditions:
https://creativecommons.org/licenses/by-nc/4.0/legalcode.
Introduction Copies of published material are allowed for personal or inter-
nal use. Sharing published material for non-commercial pur-
Translocations in the mixed lineage leukemia (MLL) gene account for 10% of all https://creativecommons.org/licenses/by-nc/4.0/legalcode,
human leukemias and are associated with pediatric, adult, and therapy-related cases. sect. 3. Reproducing and sharing published material for com-
In infants, around 80% of acute lymphoid leukemia (ALL) and 35%-50% of acute mercial purposes is not allowed without permission in writing
myeloid leukemia (AML) cases carry a translocation in the MLL gene.1 However, from the publisher.
despite improvements in conventional chemotherapy treatments for leukemia,
patients with MLL-rearranged leukemia (MLL-r) have a poor response to treatment

G. Gracia-Maldonado et al.
and poor prognosis.2,3 Immunotherapy strategies have Animal experiments

proven effective in multiple blood cancers, mainly targeting All animal experiments were carried out in accordance with the
lineage-specific proteins like CD19 (blinatumomab, tis- guidelines of the Institutional Animal Care and Use Committee
agenlecleucel) and CD33 (gemtuzumab), abundantly (IACUC). For xenograft experiments with MV4;11 and MLL-AF10
expressed in ALL and AML patients, respectively.4 primary patient cells, immunocompromised NOD-Rag1null IL2rgnull
However, mounting evidence in recent clinical trials and (NRG) (Jackson Laboratories, stock no. 007799) recipient mice
case reports have shown that patients with MLL-rearrange- were conditioned with busulfan and transplanted 24 hours later.
ments frequently relapse after treatment with CD19 In xenograft experiments bone marrow samples were collected 4
immunotherapies, arising as AML or mixed phenotype weeks after transplantation as well as when signs of leukemia
acute leukemia (MPAL).5-12 The exact mechanism of lineage were present; bone marrow aspirates were analyzed via flow
switch induced by CD19 immunotherapies is still unclear. cytometry for the presence of human CD45+ cells and the pres-
One approach to overcome the lineage switching is to ence of short hairpin RNA (shRNA)-transduced Venus+ cells
develop MLL-r specific immunotherapies targeting cell
surface proteins essential for the survival of MLL-r Retroviral and lentiviral transductions
leukemias. Recently, NG2/CSPG4 and CD133/PROM1 Retroviral and lentiviral supernatants were generated by trans-
have been shown to be promising MLL-r specific fection of HEK293T cells using the FuGENE 6 reagent (Promega)
immunotherapy targets, however, these targets are or Lipofectamine 3000 (Thermo Fisher Scientific) according to the
restricted to lymphoid lineage, increasing the potential manufacturer’s recommendations.
for lineage switching within the leukemic population.13-15 All lentiviral shRNA constructs were purchased from Millipore
Gene-expression profiling based on underlying cytoge- Sigma. Cells transduced with constructs containing a fluorescent
netic mutations is one way to identify proteins that are marker (Venus) were sorted 4-5 days after transduction by using
overexpressed and thus might be essential for the propa- MoFlo XDP (Beckman Coulter), FACSAria (BD Biosciences), or a
gation of the specific leukemia.16,17 Both AML and ALL SONY SH800S (Sony Biotechnology).
with MLL-rearrangements share a common gene signa-
ture that is distinct from that of MLL-germline (MLL-G) Flow cytometry
leukemias.18 Most of the well-studied and validated MLL- For apoptosis assays, cells were incubated with allophyco-
r gene targets however are DNA binding proteins like the cyanin (APC)-conjugated annexin V (BD Biosciences) for 15 min-
HOXA gene cluster and its co-factor MEIS1,19,20 which are utes at room temperature in 1X annexin V binding buffer (BD
not suitable targets for immunotherapy. Biosciences) followed by staining with 7-aminoactinomycin D (7-
In several of the published gene-expression studies, we AAD) (eBioscience). For surface LAMP-5 detection, cells were
found LAMP5 significantly and specifically overexpressed incubated with 3 µg anti-human LAMP-5 therapeutic antibody
in MLL-r leukemias.18,21–23 LAMP-5 is a member of the overnight and then stained with anti-mouse IgG1-PE. Data were
lysosome-associated membrane protein (LAMP) family. acquired on a BD FACSCanto analyzer and results were analyzed
In contrast to other LAMP proteins which show using FlowJo Version 10 (BD Biosciences).
widespread expression, Lamp5 expression in mice is con-
fined to several regions of the postnatal brain. In neurons, Colony-forming unit assays
the protein was found to recycle between the plasma Transduced human cells were sorted 4-5 days after transduction
membrane and a non-classical endosomal vesicle.24–26 In and were cultured in methylcellulose-containing media (StemCell
humans, aside from its conserved expression in the brain, Technologies, H4434). Colonies were scored 10-14 days after plat-
LAMP5 is specifically expressed in plasmacytoid dendritic ing.
cells (pDC).27 Upon activation of pDC, LAMP-5 aids in
the transport of Toll-like receptor 9 (TLR9) from early Real-time- and quantitative real-time polymerase chain
endosomal to lysosomal signaling vesicles, thereby regu- reaction
lating type 1 interferon (IFN-1) and pro-inflammatory sig- Total RNA was extracted from human puromycin-selected or
naling respectively, downstream of TLR9 activation.28 sorted Venus+ cells using the RNeasy Mini kit (QIAGEN). For
Importantly, results of in silico modeling predict LAMP-5 quantitative real-time polymerase chain reaction (RT-PCR) 5-10 ng
as a cell surface protein.29 In this report, we demonstrate of cDNA was analyzed using iTaq Universal SYBR Green
LAMP5 as being highly expressed and essential for MLL- Supermix (Bio-Rad) or PowerUP SYBR Green (Thermo Fisher
r leukemias through the regulation of innate immune sig- Scientific) in a StepOnePlus RT-PCR machine (Applied
naling and describe its potential as a target for MLL-r spe- Biosystems).
cific immunotherapy.
Cell viability
MOLM-13, RS4;11, THP-1, and Kasumi-1 cells were plated at
Methods 10,000 cells per well in a 96-well plate. Cells were incubated with
LAMP-5 therapeutic antibody (Creative Biolabs) and anti-mouse
Cell lines and primary patient-derived xenograft cells immunoglobulin G (IgG) Fc-DM1 antibody with non-cleavable
Human leukemia cell lines were maintained in Iscove’s linker (Moradec) at 5 ng/uL and 1 ng/uL final concentrations,
Modified Dulbecco Medium (IMDM) or Roswell Park Memorial respectively. In order to measure cell viability CellTiter-Glo® 2.0
Institute (RPMI) 1640 medium supplemented with 10% fetal Cell Viability Assay (Promega) was used following the manufac-
bovine serum (FBS), 1% penicillin, and 1% streptomycin. turer’s protocol.
Fully de-identified primary cells were obtained from the
Cincinnati Children’s Hospital Medical Center Biorepository. Immunofluorescence
Cells were cultured in IMDM supplemented with 20% FBS and Cells seeded on alcian blue-treated coverslips were fixed with
10 ng/mL human cytokines including SCF, FLT3-ligand, throm- 3.5% paraformaldehyde and permeabilized with 0.05% saponin.
bopoietin, IL-3, and IL-6. Cells were stained overnight with primary antibodies against

LAMP5 as an essential target of MLL-r leukemias
LAMP-5, MYD88, and LAMP-1. Immunofluorescence and confo- locus generate MLL fusion proteins (MLL-FP) which acti-
cal microscopy were performed with a Zeiss LSM580 63x objec- vate transcription of downstream target genes.32,33 In
tive and accompanying imaging software. order to determine if LAMP5 expression was dependent
on the MLL-FP, we transformed CB-CD34+ cells with a
Statistics retrovirus carrying a tetracycline-repressible MLL-AF9
The statistical methodology used, and sample sizes are construct. Treatment of transformed cells with doxycy-
described in the individual Figure legends. t-tests were two-tailed cline led to a simultaneous reduction in the levels of both
unless otherwise stated. Results are presented as mean ± standard MLL-AF9 and LAMP5 (Figure 1G). in order to determine
error of the mean (SEM) unless otherwise stated. A two-sided if the MLL-FP directly activates the LAMP5 gene locus,
time-stratified Cochran-Mantel-Haenszel was used for the we interrogated previously published MLL-FP chromatin
Kaplan-Meier Survival analysis. ROC curves were used to deter- immunoprecipitation sequencing (ChIP-seq) datasets
mine the diagnostic utility of LAMP5 mRNA. The sensitivity and derived from the SEM, RS4;11, MV4;11, THP-1, and ML-
specificity were identified at the optimal cutoff point that was 2 cell lines, CD34+ cells transformed with FLAG-MLL-Af4,
chosen at which Youden's index was maximal. A significance level and primary patient sample.32–36 Almost all cell lines exhib-
cutoff of 0.05 was used unless otherwise stated. Statistical analysis ited peaks within the LAMP5 promoter region, suggesting
was performed using GraphPad Prism. direct binding of the MLL-FP (as evidenced by coincident
More detailed information on the materials and methods used signal in both MLL and fusion partner ChIP-seq tracks).
can be found in the Online Supplementary Appendix. Additionally, there was accompanying significant enrich-
ment of H3K79me2 and H3K79me3 along the gene body,
further supporting our hypothesis that LAMP5 undergoes
Results transcriptional activation in MLL-r leukemia via direct tar-
geting by the MLL-FP complex (Figure 1H). In mice,
LAMP5 is highly expressed in mixed lineage Lamp5 does not show any expression in blood, as it does
leukemia-rearranged leukemias and is a direct target in humans (Online Supplemental Figure S3A and B).
of the mixed lineage leukemia-fusion protein Furthermore, we did not detect upregulation of Lamp5 in
In order to determine genes that are highly expressed in mouse models of MLL-AF9, E2A-HLF, and AML1-ETO
AML and ALL with MLL-rearrangements, we compared leukemia, hence we focused our studies exclusively on
recently published RNA sequencing (RNA-seq) studies human cells (Online Supplemental Figure S3C).
that identified differentially expressed genes between
MLL-r and MLL-G leukemias in both AML and B-ALL LAMP-5 is required for in vitro and in vivo leukemia
samples30,31(Figure 1A). Twenty-seven genes were com- cell survival
monly overexpressed in MLL-r ALL and AML (Online The ideal immunotherapy target should be essential for
Supplementary Table S1). Using the in silico human sur- the survival of MLL-r leukemias. In order to test the func-
faceome tool (http://wlab.ethz.ch/surfaceome/) five of these tional role of LAMP-5 in MLL-r leukemia, we transduced
27 genes were predicted to be expressed on the cell sur- both MLL-r leukemia (MOLM-13, MV4;11, RS4;11, THP-1)
face29 (Online Supplementary Table S1). Of the five predict- and MLL-G leukemia cells (Kasumi-1 and REH) with
ed proteins, LAMP-5 stood out for being present in mul- lentiviral shRNA vectors targeting LAMP5. We obtained
tiple previous MLL-r leukemia gene expression stud- efficient knockdown of LAMP5 with two independent hair-
ies17,18,21–23 (Online Supplementary Figure S1A and B). We pins as compared to non-targeting control (NT) (Online
further validated the specificity of LAMP5 expression in Supplementary Figure S4A). Upon LAMP5 depletion, we
MLL-r leukemias by analyzing the 1,109 pediatric observed a significant reduction of cell growth in MLL-r
leukemia patient samples from the St.Jude PeCan Portal leukemia cell lines (Figure 2A), while no effect was seen in
which revealed LAMP5 as significantly overexpressed in Kasumi-1 and REH (Figure 2B). Additionally, LAMP5
92% of ALL and 72% of AML with MLL-rearrange- knockdown led to a significant decrease in colony-forming
ments16 (Figure 1B). In order to determine if LAMP5 units (CFU) in the MLL-r leukemia cell lines (Figure 2C) sug-
expression could discriminate between MLL-r leukemia gesting an effect on the clonogenicity of these cells.
and MLL-G leukemia patients, we performed a receiving Furthermore, LAMP5 knockdown led to apoptosis in
operating curve (ROC) analysis. LAMP5 achieved a statis- MLL-r leukemia cells, as evident by a significant increase in
tically significant area under the curve (AUc) score in both annexin V and 7-AAD double-positive staining (Figure 2D;
the microarray innovations in both the (MILE) Online Supplementalry Figure S4B). We next sought to deter-
(GSE13159) and the St. Jude PeCan datasets, with high mine the role of LAMP-5 in leukemia propagation in vivo.
sensitivity and specificity at the optimal cutoff points MV4;11 cells were transduced with shLAMP5-2 or NT con-
(Figure 1C). Further, a Kaplan-Meier survival analysis of trol followed by transplantation into immunocompromised
B-ALL and AML patients correlated higher expression of NOD-Rag1null IL2rgnull (NRG) mice (Figure 2E). In the bone
LAMP5 with poor survival (Online Supplementary Figure marrow, both groups showed similar human cell engraft-
S2A and B). At the protein level, patient-derived ment based on human CD45 expression. On the other
xenograft (PDX) pediatric AML and ALL samples show hand, the transduced Venus+ fraction was significantly
high expression of LAMP-5 only in the MLL-r samples as reduced in shLAMP5-2 compared to shNT mice 4 weeks
compared to MLL-G (Figure 1D). Similar results were after transplantation (Figure 2F, left panels). We repeated
seen in human MLL-r AML and ALL cells lines (MOLM- this experiment using cells from an AML PDX with MLL-r
13, MV4;11, THP-1, and RS4;11) at the mRNA (Figure 1E) (MLL-AF10) leukemia. We again observed a significant
and protein (Figure 1F) levels as compared to MLL-G reduction in the proportion of Venus+ cells with LAMP5
leukemia cell lines (HL-60, Kasumi-1, K562, REH, and knockdown compared to NT control (Figure 2F, right pan-
RCH-ACV) and normal human CD34-enriched cord els). Overall, these data underscore a critical role for LAMP-
blood cells (CB-CD34+ cells). Translocations of the MLL 5 in the growth of MLL-r leukemia cells.

LAMP-5 is required for activation of Toll-like et al. showed that LAMP-5 plays an important role in con-
receptor/interleukin 1 receptor signaling in leukemia trolling the subcellular location of TLR9 after activation in
Acute leukemias exhibiting constitutive activation of human pDC. Upon activation of TLR9, LAMP-5 shuttles
innate immune signaling pathways have been character- TLR9 from the VAMP3+-interferon response factor signal-
ized as having a pro-inflammatory profile which is ing endosome (IRF-SE), to the LAMP-1+ pro-inflammato-
required for their survival.37 These physiologic cellular ry-signaling endosome (PI-SE). This transition of TLR
systems involve TLR/IL-1R signaling and culminate in the localization in turn acts as a negative regulator of IFN-1
release of pro-inflammatory cytokines via NF-kB and/or signaling.28 Based on the known role of LAMP-5 in TLR9
of type I interferons (IFN-1).38 Recent studies reveal localization in pDC, we first examined the localization of
heightened activation of NF-kB signaling in MLL-r intracellular LAMP-5 in MOLM-13 cells. We performed
leukemia compared to other leukemias.39 Furthermore, co-staining of MOLM-13 cells with antibodies against
MLL-r leukemias have been shown to require the TLR/IL- LAMP-5, LAMP-1, and myeloid differentiation primary
1R signaling pathway to survive, through degradation of response 88 (MYD88), a scaffold protein that is required
the wild-type MLL protein, allowing the MLL-FP to bind for TLR and IL-1R signaling. Confocal microscopy
to its target genes without restriction.40 Recently, Combes showed that in MOLM-13 leukemia cells, LAMP-5 local-
A B C
D E
F G
Figure 1. Continued on following page.

Figure 1. LAMP5 is highly expressed in mixed lineage leukemia-rearranged leukemias and is a direct target of the mixed lineage leukemia-fusion protein. (A) The
intersection of published gene expression signatures composed of genes overexpressed in mixed lineage leukemia-rearranged (MLL-r) acute myeloid leukemia (AML)
and acute lymphoid leukemia (ALL) when compared to MLL-germline (MLL-G) leukemias. (B) Log2 FPKM expression of LAMP5 in AML and ALL pediatric patients with
MLL-rearrangement (AML MLL-r, n=36 and ALL MLL-r, n=76) compared to MLL-G (AML MLL-G, n=270) (ALL MLL-G, n=727) patients. Data obtained from the St. Jude
PeCan Portal and presented as median value with quartiles (t-test, ***P<0.0001). (C) Receiving operating curve (ROC) analysis showing the capacity of LAMP5 to
discriminate acute leukemia patients with MLL-G or MLL-r leukemias. Data obtained from GSE13159 and St. Jude Pecan Portal. (D) Western blot analysis of LAMP-
5 expression in pediatric primary ALL and AML samples. Actin or vinculin was used as a loading control. (E) Relative expression of LAMP5 in MLL-r leukemia (MV4;11,
MOLM-13, THP-1, and RS4;11) and MLL-G leukemia (K562, HL-60, Kasumi-1, REH, RCH-ACV) cell lines. The graph represents the relative expression of LAMP5 nor-
malized to b-ACTIN. Data are from three biological replicates. LAMP5 expression in cord blood cells was set as 1.0. Bars show mean ± standard error of the mean
(SEM). (F) Western blot analysis of the LAMP-5 levels in MLL-r leukemia and MLL-G leukemia human cell lines. CD34+ cord blood cells were used as control. Actin
was used as a loading control. (G) Quantitative real-time polymerase chain reaction (RT-PCR) analysis of LAMP5 and MLL-AF9 gene expression in CD34+ cord blood
cells transformed with a tetracycline-repressible MLL-AF9 construct. Gene expression was analyzed 24 hours after doxycycline incubation. Relative expression of
LAMP5 and MLL-AF9 was normalized to b-ACTIN. (H) Representative chromatin immunoprecipitation sequencing (ChIP-seq) tracks at the LAMP5 locus from different
MLL-r cell lines. ChIP-seq data were obtained from GSE95511 for ML-2, GSE79899 for MV4;11 and THP-1, GSE38403 for RS4;11, GSE38338 for SEM, GSE84116
for CB CD34+ MLL-Af4, and GSE83671 for primary patient MLL-AF4.
ized to LAMP-1+ vesicles. As suspected, we found reporter. Robust activation of NF-kB was evident in con-
MYD88 accumulating highly in the periphery of LAMP- trol cells upon incubation with PAM3CSK4 (TLR2 agonist)
1+ vesicles in MLL-r leukemia, suggestive of TLR/IL-1R or LPS (TLR4 agonist). Knockdown of LAMP5 led to a
activation (Figure 3A). Conversely, in Kasumi-1 cells, near-complete blockade of this activation, suggesting that
MYD88 does not co-localize with LAMP-1+ vesicles. TLR-induced NF-kB signaling is disrupted upon LAMP5
However, overexpression of wild-type LAMP-5 in this depletion (Figure 3D). Correspondingly, in Kasumi-1 cells,
cell line led to the relocation of MYD88 around LAMP-1+ overexpression of LAMP5 led to increased phosphoryla-
vesicles (Figure 3B). tion of p38, JNK, and NF-kB along with increased cell
We subsequently hypothesized that LAMP-5 loss may growth (Figure 3E and F).
dampen TLR/IL-1R signaling in MLL-r leukemias. We thus A previous study showed that NF-kB plays a critical
analyzed known effector proteins downstream of TLR/IL- role in MLL-r leukemias.39 We thus hypothesized that NF-
1R activation by western blot. Upon LAMP5 knockdown, kB activation would rescue the cell growth defect seen by
we observed a reduction in phosphorylated IRAK1, NF- LAMP5 depletion. We induced persistent activation of
kB, p38, and JNK, key players in the signal transduction NF-kB in leukemia cells by overexpressing a constitutive-
downstream of TLR/IL-1R (Figure 3C). In order to further ly active version of inhibitor of nuclear factor kB kinase
determine the impact of LAMP5 depletion in TLR-mediat- subunit b (IKBKB-EE) in these cells.41 Despite sustained
ed NF-kB activation, we measured NF-kB activity using NF-kB activation, knockdown of LAMP5 in MOLM-13
the THP-1 NF-kB-SEAP cell line, which contains an NF-kB and RS4;11 cells still led to growth inhibition, suggesting
inducible secreted embryonic alkaline phosphatase (SEAP) that loss of NF-kB is not the only signaling event being

affected by LAMP5 depletion (Online Supplementary Figure levels of the MLL-FP or LC3 A/B in THP-1 and MOLM-13
S5A and B). A potential mechanism underlying this essen- cells upon LAMP5 depletion (Online Supplemental Figure
tiality was proposed by Wang et al., where they suggested 6A and B). Overall, these results underscore a critical role
that loss of LAMP5 in MLL-r leukemia led to degradation for LAMP-5 in the activation of TLR/IL-1R signaling in
of the MLL-FP due to increased autophagy.42 However, in MLL-r leukemia, while also indicating the presence of
our experiments, we did not observe any change in the additional attributes that are also essential.
A B
C E
Figure 2. LAMP5 expression is required for mixed lineage leukemia-rearranged leukemia survival in vitro and in vivo. (A and B) In vitro growth of MLL-r and MLL-G
leukemia cell lines (A) (MOLM-13, MV4;11, RS4;11 and THP-1) and (B) (Kasumi-1 and REH) respectively upon short hairpin RNA (shRNA) knockdown of LAMP5. Data
are from three independent experiments, t-test, **P<0.01, ***P<0.001. (C) Colony-forming units (CFU) of MV4;11, MOLM-13, THP-1, RS4;11 cells upon LAMP5
shRNA knockdown. Data are from three biological replicates, represented as mean and SEM, t-test, **P<0.01, ***P<0.001. (D) Percentage of annexin V+/7-AAD+
cells after transduction with shNT or shLAMP5-2. Data are from three biological replicates, represented as mean and standard error of the mean (SEM) of at least
three experiments. t-test, *P<0.05, **P<0.01 (E) Schematic of in vivo xenograft transplantation. MV4;11 or MLL-AF10 PDX cells were transduced with short hairpin
non-targeting control (shNT) or shLAMP5-2 (shLAMP5) lentivirus and the mixed population of Venus+ and Venus- cells were transplanted into mice. (F) Plots show the
percentage of human CD45+ (upper) and Venus+ cells in the CD45+ fraction (lower) in MV4;11 (left) and MLL-AF10 patient-derived xenograft (PDX) sample (right).
Data are from eight biological replicates for MV4;11 and five biological replicates for the MLL-AF10 PDX, represented as mean and SEM, t-test, **P<0.01,
***P<0.001.

LAMP-5 is a negative regulator of interferon-1 LAMP-5 on IFN-1 signaling in MLL-r leukemia, we turned
signaling in mixed lineage leukemia-rearranged to THP-1-ISG-SEAP cells containing an interferon-stimu-
leukemias lated gene (ISG) inducible-SEAP reporter. Upon TLR acti-
Since activation of NF-kB was not sufficient to rescue vation by PAM3CSK4, IFN-1 signaling activation was evi-
the cell growth inhibition seen upon LAMP5 depletion, dent only in the LAMP5-depleted cells but not in the con-
we next sought to understand the mechanistic signifi- trol condition (Figure 4C). Furthermore, gene set enrich-
cance of the inflammatory-signal-regulation function of ment analysis of RNA-seq from MOLM-13 cells trans-
LAMP-5 in MLL-r leukemia. In pDC, the carboxy-termi- duced with shNT or shLAMP5-2 showed enrichment of
nal YKHM domain of LAMP-5 was found to be required IFN gene signatures (Online Supplementary Figure S8).
for normal localization of LAMP-5 and transportation of Additionally, we validated the increase in IFN-1 signaling
TLR9 from the early endosome vesicle to the pro-inflam- in several MLL-r cell lines by demonstrably increased
matory vesicle.24,27,28 We thus overexpressed wild-type expression of interferon a2 (IFNA2) and interferon b
LAMP5 (LAMP5-WT), a Y276A mutant LAMP5 (LAMP5- (IFNB) upon depletion of LAMP5 (Figure 4D). In order to
mut), or control vector (EV) in MV4;11 and THP-1 cells, assess the role of LAMP5-depletion mediated IFN-1 acti-
followed by selective knockdown of endogenous LAMP5 vation on cell growth, we performed knockdown of inter-
using an shRNA targeting the 3’UTR region of LAMP5 feron regulatory factor 7 (IRF7), a known regulator of IFN
(Online Supplementary Figure S7A). Overexpression of signaling downstream of TLR/IL1R activation, along with
LAMP5-WT completely prevented cell growth inhibition LAMP5 in MV4;11 and THP-1 cells. We found that loss of
and apoptosis upon knockdown of endogenous LAMP5, IRF7 alone had no significant effect on MLL-r leukemia
validating LAMP5 as the main target of the shRNA. In cell growth but importantly, its depletion prevented the
contrast, LAMP5-mut was unable to rescue cell growth or growth inhibition observed upon LAMP5 knockdown
apoptosis in MV4;11 (Figure 4A and B). In pDC, LAMP5 (Figures 4E and F; Online Supplementary Figure S9A and B).
knockdown or overexpression of LAMP5-mut induced Collectively, these results demonstrate that a critical func-
IFN-1 activation upon TLR9-stimulation, due to retention tion of LAMP-5 in MLL-r leukemias is to promote the
of TLR9 in the IRF-SE. In order to determine the effect of transfer of TLR/IL-1R from the IFN-1–activating signaling
A C D
B E F
Figure 3. LAMP-5 is required for activation of Toll-like receptor/interleukin 1 receptor signaling. (A) Representative confocal microscopy images showing MOLM-13
cells stained with LAMP-5 (red), LAMP-1 (blue), and MYD88 (green); scale bar =1 mm. (B) Confocal microscopy image showing Kasumi-1 cells overexpressing empty
vector (EV) or wild-type LAMP5 (LAMP5-WT) stained with antibodies against LAMP-5, LAMP-1, and MYD88; scale bar =1 mm. (C) Western blot analysis showing that
LAMP5 depletion (shL5) led to a decrease of p-IRAK1, p-p38, p-JNK, and p-NF-kB, known downstream targets of Toll-like receptor (TLR) signaling. (D) THP-1-Blue-NF-
κB reporter cell line was treated with PAM3CSK4 10 ng/mL or LPS 100 ng/mL in the presence or absence of LAMP-5. Data are from three independent experiments.
t-test, ***, P<0.001. (E) Western blot analysis of Kasumi-1 cells with overexpression of empty vector (EV) or LAMP5 showing increased activation of p-NF-kB, p-p38,
and p-JNK. (F) In vitro cell growth of Kasumi-1 cells overexpressing EV or LAMP5-WT. Data are from three individual experiments. t-test, ***P<0.001.

B C
Figure 4. Continued on following page.

E F
Figure 4. LAMP-5 is a negative regulator of interferon-1 signaling in mixed lineage leukemia-rearranged leukemias. (A) In vitro growth of MV4;11 and THP-1 cells
overexpressing empty vector control (EV), wildtype LAMP5 (LAMP5-WT), or mutated LAMP5 (LAMP5-mut) upon shRNA knockdown of LAMP5. Data are from three
independent experiments. t-test ***, P<0.001. (B) Fold change of % apoptotic cells in MV4;11 cell line overexpressing empty vector (EV), wild-type LAMP5 (LAMP5-
WT), or mutant LAMP5 (LAMP5-mut) upon short hairpin RNA (shRNA) knockdown of LAMP5. Data are from three independent experiments. t-test, *P<0.05,
**P<0.01. (C) THP-1 ISG blue reporter cell line was untreated (UT) or treated with 10 ng/mL PAM3CSK4 in the presence or absence of LAMP-5. Data are from three
independent experiments. Bars show mean ± standard error of the mean (SEM). t-test, ***P<0.001. (D) Relative expression of LAMP5, IFNA2, and IFNB upon knock-
down of LAMP5 in MV4;11, MOLM-13, THP-1 and RS4;11 cells. The graph represents the relative expression of LAMP5, IFNA2, and IFNB normalized to b-actin. Data
are from three biological replicates. Bars show mean ± SEM. t-test *, P<0.05. (E) In vitro growth of MV4;11 after LAMP5, or IRF7 or LAMP5+IRF7 shRNA knockdown.
Data are from three independent experiments, represented as mean and ± standard deviation. ***P<0.001. (F) Colony-forming units (CFU) of THP-1 cells upon
shRNA knockdown of LAMP5, IRF7, or LAMP5+IRF7 together. Data are from three independent experiments, represented as mean and ± SEM. t-test **, P<0.01.
cascade to the pro-inflammatory signaling cascade. immune signaling in MLL-r leukemias, specifically direct-
Depletion of LAMP5 thus leads not only to loss of NF-kB ing the flux of activity away from IRF-SE towards the PI-
activation but also to activation of IFN-1-signaling, the SE, leading to constant activation of NF-kB (Figure 6).
latter inducing cell death. Recent discoveries have highlighted how the specific
subcellular location and timing of TLR activation affect
Surface LAMP-5 can be detected and targeted with signaling outcomes in normal immune cells.43 Combes et
antibody drug conjugate therapy al. showed that LAMP-5 is a negative regulator of IFN-1
LAMP-5 has been found to briefly localize in the plas- signaling in pDC wherein it transports activated TLR9
ma membrane of cortical neurons in mice and is highly from the IRF-SE to the PI-SE. Although dispensable for
predicted to reach the cell membrane based on the pDC cell survival, LAMP5 depletion led to unrestricted
human surfaceome.24,29 We thus sought to confirm if activation of IFN-1 signaling. Furthermore, aberrant
LAMP-5 was expressed on the surface of MLL-r leukemia expression of LAMP-5 can lead to diminished activation
cells. Using an antibody targeting the N-terminus of of pDC in tumors and contribute to their immunomodu-
LAMP-5, we were able to detect LAMP-5 on the surface latory phenotype by decreasing the IFN-1 production
of MLL-r leukemia cell lines, while none was detected in capacity.28 However, how these mechanisms function in
the MLL-G leukemias (Figure 5A and B). In order to vali- leukemia is still poorly understood. Innate immune sig-
date the specificity of the antibody, we overexpressed naling and inflammation have been shown to play a cru-
LAMP5 or control empty vector (EV) in Kasumi-1 cells. cial role in acute leukemias.37 MLL-r leukemias rely on
We detected surface LAMP-5 only in the cells that express activation of NF-kB downstream of TLR/IL-1R to main-
high levels of LAMP5 (Figure 5C). tain the MLL-FP gene signature and block cell differentia-
As a proof-of-concept for potential therapeutic use, we tion.39,40 Furthermore, it has been shown that treatment
used a secondary antibody conjugated to the tubulin-toxin with IFN-1 or activation of IFN-1 signaling is deleterious
Mertansine, targeting the surface-LAMP-5 antibody. We for MLL-r leukemias.44 In our study, we describe a novel
observed that a 72-hour treatment with this antibody-sand- role for LAMP-5 in maintaining NF-kB activation and
wich comprised of the surface LAMP-5 antibody along blocking IFN-1 signaling downstream of TLR/IL-1R in
with the secondary antibody drug conjugate (ADC) anti- MLL-r leukemias. We show that LAMP-5 acts as a molec-
body is sufficient to reduce cell viability in MLL-r leukemia ular switch to maintain active TLR/IL-1R signaling in the
cell lines MOLM-13, RS4;11 and THP-1, while no effect pro-inflammatory endosome leading to NF-kB activation,
was seen in Kasumi-1 cells (Figure 5D). These results sug- whereas LAMP5 depletion leads to activation of IFN-1
gest that LAMP-5 could be exploited as an MLL-r specific signaling and cell death. This suggests that both the
biomarker and could potentially be used as a target for LAMP-5-mediated induction of pro-inflammatory signal-
immunotherapy. ing and inhibition of IFN-1 signaling contribute to the
pathogenesis of MLL-r leukemias. We confirmed that
activation of IFN-1 signaling upon LAMP-5 depletion was
Discussion deleterious for leukemia propagation, and that by deplet-
ing IRF7, cell growth and clonogenicity were rescued in
Our findings further reaffirm LAMP5 as a novel and LAMP5-depleted cells. This suggests that increased IFN-1
essential core gene in MLL-r leukemias, directly upregu- signaling is at least partly responsible for inducing cell
lated by the MLL-FP. Additionally, we found that one of death upon LAMP5 depletion. Additionally, overexpres-
the critical functions of LAMP-5 is to regulate innate- sion of LAMP5 in MLL-G leukemia led to increased acti-

vation of NF-kB, p38, and JNK, and increased cell growth, tion of LAMP-5 on the surface of MLL leukemias provides
which suggests that this signaling pathway might be con- the opportunity to potentially use it as a target for
tributing to the therapy-resistant phenotype of MLL-r immunotherapy in this treatment-refractory malignancy.
leukemias. Furthermore, LAMP-5 is highly expressed in other can-
In humans, LAMP5 expression is generally restricted to cers such as multiple myeloma (MM) and blastic plasma-
the brain and blood. In blood, LAMP5 is exclusively cytoid dendritic cell neoplasm (BPDCN).45,46 Therefore,
expressed in nonactivated pDC,27 wherein LAMP-5 LAMP-5 immunotherapies could benefit other blood dis-
resides in the ERGIC compartment and is transported to eases. Finally, total loss of Lamp5 had no major effects on
endo-lysosomal vesicles upon TLR9 activation.27,28 We the health or lifespan of mice, only causing minor behav-
found that the aberrant increased expression of LAMP-5 ioral effects like deficits in olfactory discrimination and
in MLL-r leukemia leads to its accumulation in the plasma increased startle response to auditory and tactile stim-
membrane, as demonstrated by a novel LAMP-5 anti- uli,25,26 suggesting that there could be a wide therapeutic
body targeting the N-terminus of the protein. The detec- window for LAMP-5-directed therapies in humans.
B C D
Figure 5. Surface LAMP-5 can be detected and targeted with antibody drug conjugate therapy. (A) Representative histogram plots showing LAMP-5 surface expres-
sion in mixed lineage leukemia-rearranged (MLL-r) leukemia (MOLM-13, RS4;11, MV4;11, and THP-1) and MLL-germline (MLL-G) leukemia (Kasumi-1, HL-60, and
NB4) cell lines. (B) Graph showing mean fluorescence intensity (MFI) of LAMP-5 surface staining in MLL-r leukemias vs. MLL-G leukemias represented as mean and
± standard deviation (SD). t-test,**P<0.01. (C) Representative histogram of LAMP-5 staining in Kasumi-1 expressing empty vector (EV) or LAMP5, confirming the
specificity of the antibody. (D) MOLM-13, RS4;11, THP-1, and Kasumi-1 cells were incubated with surface LAMP-5 antibody clone D1 and aMFc-NC-DM1 antibody
drug conjugate (ADC) antibody for 72 hours. Bar graph represents cell viability from three biological replicates presented as mean and ± SEM. t-test, *P<0.05.

Figure 6. Proposed model to illustrate the mechanism of action of LAMP-5 in mixed lineage leukemia-rearranged leukemias and potential immunotherapy usage.
Left panel: 1. The mixed lineage leukemia (MLL)-FP induces expression of LAMP-5. 2. LAMP-5 gets internalized from the cell surface to the interferon signaling endo-
some (IFN-SE), 3. and 4. LAMP-5 is quickly shuttled to the LAMP-1+ pro-inflammatory signaling endosome (PI-SE), activating NF-kΒ signaling. 5. NF-kΒ activates pro-
inflammatory signaling. Right panel: 6. depletion of LAMP-5 leads to blockage of transport of TLR to PI-SE, with retention in and activation of the IFN-SE and 7. induc-
tion of interferon related genes and cell death. 8. Surface-LAMP-5 can be targeted in mixed lineage leukemia-rearranged (MLL-r) leukemias with immunotherapies.
Similar to our observations, Wang et al. recently altogether.49,50 Overall, our results show that LAMP-5
showed that LAMP-5 is essential for the survival of MLL- localizes both on the surface and in LAMP-1+ endosomes
r leukemias in vitro and in vivo using shRNA knockdown. in leukemia, leading to constitutive activation of pro-
However, they propose that LAMP-5 is a negative regula- inflammatory signaling, and dampening of interferon-sig-
tor of autophagy leading to MLL-FP stabilization. They naling and that it can be used as a target for immunother-
show that LAMP-5 and ATG5 co-localize in MLL-r apy.
leukemia cells and that blockade of autophagy is suffi-
cient to rescue the increased levels of apoptosis after Disclosures
LAMP5 knockdown.42 We were unable to detect any sig- No conflicts of interest to disclose.
nificant change in the levels of the MLL-FP or LC3A/B
upon LAMP5 knockdown. Since TLR-mediated innate Contributions
immune signaling can regulate autophagy, the function of GGM, LHL and ARK contributed to study conception and
LAMP-5 in regulating autophagy as described by Wang et design; GGM, JC, MB and BG acquired data; GGM, JC,
al. may be downstream of its impact on endosome-lyso- MW, DL, PP, EG and LHL analyzed and interpreted data; NS
some trafficking.47 On the other hand, it is also possible and LHL analyzed and interpreted RNA-seq data; GGM, JC,
that these effects are not directly linked, and that LAMP- LHL and ARK wrote and revised the manuscript; GGM, JC,
5 might exert its growth-promoting effects in MLL-r DL, PPE and ARK reviewed the manuscript; MW, DL and JC
leukemia by multiple mechanisms. It is notable; however, provided administrative, technical, or material support.
that the role of autophagy in leukemia is controversial. In
murine MLL leukemia models, heterozygous loss of Atg5 Acknowledgments
leads to increased leukemia cell proliferation in vitro and We would like to thank Daniel Starczcynowski, Ph.D., for his
more aggressive leukemia in vivo, while homozygous loss intellectual input. We thank J. Bailey and V. Summey for assis-
is lethal to these cells.48 Additionally, while some studies tance with transplantations (Comprehensive Mouse and Cancer
suggest that Atg5-dependent autophagy may contribute Core at CCHMC). We would like to acknowledge the assis-
to the development of MLL-AF9 driven leukemia but distance of the Research Flow Cytometry Core in the Division of
pensable for propagation and chemosensitivity, others Rheumatology at Cincinnati Children’s Hospital Medical
suggest that Atg5-dependent autophagy is dispensable Center. All flow cytometric data were acquired using equipment

maintained by the Research Flow Cytometry Core in the NIH grant (R01 CA226802). PP and EV were supported by the
Division of Rheumatology at Cincinnati Children’s Hospital Institut National du Cancer (INCA) (PLBIO17-187),
Medical Center. Canceropole Paca GEFLUC (RAK18024AAA), and Fondation
ARC PJA (20131200330). GGM was supported by the
Funding Chateaubriand Fellowship. ARK was supported by a Hyundai
The SH800S is supported by an NIH Shared Hope on Wheels grant. LHL is a St. Baldrick’s Foundation
Instrumentation Grant (S10OD023410). MW was supported Scholar and is supported by grants from CancerFree KIDS and
by an NIH grant (R50 CA211404). NS was supported by an the NIH (L40 HL143713-01).
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ARTICLE Acute Myeloid Leukemia
Ferrata Storti Foundation Interleukin 4 promotes phagocytosis of

murine leukemia cells counteracted by CD47
upregulation
Pablo Peña-Martínez, Ramprasad Ramakrishnan, Carl Högberg,
Caroline Jansson, David Gisselsson Nord and Marcus Järås
Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund,
Sweden
Haematologica 2022
Volume 107(4):816-824 ABSTRACT
C
ytokines are key regulators of tumor immune surveillance by con-
trolling immune cell activity. Here, we investigated whether
interleukin 4 (IL4) has antileukemic activity via immune-mediat-
ed mechanisms in an in vivo murine model of acute myeloid leukemia
driven by the MLL–AF9 fusion gene. Although IL4 strongly inhibited
leukemia development in immunocompetent mice, the effect was
diminished in immune-deficient recipient mice, demonstrating that the
antileukemic effect of IL4 in vivo is dependent on the host immune sys-
tem. Using flow cytometric analysis and immunohistochemistry, we
revealed that the antileukemic effect of IL4 coincided with an expansion
of F4/80+ macrophages in the bone marrow and spleen. To elucidate
whether this macrophage expansion was responsible for the
antileukemic effect, we depleted macrophages in vivo with clodronate
liposomes. Macrophage depletion eliminated the antileukemic effect of
IL4, showing that macrophages mediated the IL4-induced killing of
leukemia cells. In addition, IL4 enhanced murine macrophage-mediated
phagocytosis of leukemia cells in vitro. Global transcriptomic analysis of
macrophages revealed an enrichment of signatures associated with alter-
natively activated macrophages and increased phagocytosis upon IL4
stimulation. Notably, IL4 concurrently induced Stat6-dependent upregu-
lation of CD47 on leukemia cells, which suppressed macrophage activi-
ty. Consistent with this finding, combining CD47 blockade with IL4
Correspondence: stimulation enhanced macrophage-mediated phagocytosis of leukemia
MARCUS JÄRÅS cells. Thus, IL4 has two counteracting roles in regulating phagocytosis in
marcus.jaras@med.lu.se mice; enhancing macrophage-mediated killing of leukemia cells, but also
inducing CD47 expression that protects target cells from excessive
phagocytosis. Taken together, our data suggest that combined strategies
Received: August 25, 2020.
that activate macrophages and block CD47 have therapeutic potential in
Accepted: April 27, 2021. acute myeloid leukemia.
Pre-published: May 6, 2021.
Introduction
Acute myeloid leukemia (AML) is a fatal disease characterized by an accumula-
tion of myeloid blasts in the bone marrow. For AML to develop, the malignant cells
©2022 Ferrata Storti Foundation must escape tumor immune surveillance. Several evasion mechanisms have been
Material published in Haematologica is covered by copyright. described in AML, mainly associated with suppression of natural killer (NK) cells
All rights are reserved to the Ferrata Storti Foundation. Use of and macrophages.1-3 Suppression of NK cells is mediated by secretion of ligands
conditions:
from the leukemic blasts and through direct cell–cell interactions with leukemic
https://creativecommons.org/licenses/by-nc/4.0/legalcode. cells.4 An absence of NKG2D ligands on leukemia stem cells mediates their
Copies of published material are allowed for personal or inter- immune evasion.5 The main inhibitory signal to macrophages is CD47, which is
nal use. Sharing published material for non-commercial pur- upregulated on AML cells and protects them from phagocytosis.2 Paradoxically,
tumor-associated macrophages in AML also contribute to immune suppression.6,7
sect. 3. Reproducing and sharing published material for com- Whereas interleukin (IL)2 and IL15 promote restoration of NK cell function in
mercial purposes is not allowed without permission in writing AML,8 anti-CD47 blocking antibodies can rescue macrophage function.9 Whether
from the publisher. cytokine treatment can restore and boost macrophage-mediated antileukemic
activity is currently unclear.
In a syngeneic murine AML model, we previously found that IL4 exerts

IL4 enhances phagocytosis of murine leukemia cells
antileukemic activity by inducing Stat6-dependent apop- For human phagocytosis assays, we labeled human leukemia
tosis of AML cells.10 Elevated IL4 levels in mice eradicate cell lines with the PKH67 green fluorescent cell dye according to
AML cells in both the spleen and bone marrow, resulting the manufacturer’s instructions (Sigma-Aldrich, Darmstadt,
in increased survival. Under physiological conditions, IL4 Germany) and stained macrophages with the PKH26 red fluores-
is a pleiotropic cytokine that regulates several immunolog- cent cell dye (Sigma-Aldrich). AML cells were mixed with human
ical processes, such as B-cell class switching, T helper cell macrophages in a 2:1 ratio and incubated for either 2 h (Mono
maturation, alternative activation of macrophages, and Mac 6 cells) or 18 h (MA9:16 cells). The percentage of PKH26+
activation of NK cells.11,12 IL4 can bind to the IL4 receptor PKH67+ macrophages was determined by FACS.
(IL4R) type I receptor complex, a heterodimer of the IL4R
alpha (IL4RA) and IL2 receptor subunit gamma (IL2RG) RNA sequencing analysis
chains, or to the IL4R type II receptor complex, a dimer of Global gene expression profiling was performed on sorted
IL4RA and IL13RA1.13 Whether immune cells also mediate F4/80+ spleen cells from mice transplanted with IL4-overexpress-
the antileukemic activity of IL4 has not been previously ing leukemia cells and non-transplanted irradiated controls. Cells
explored. were collected 12 days after irradiation. In addition, RNA sequenc-
In this study, we show that IL4 regulates phagocytosis ing was performed on macrophages produced in vitro by stimulat-
by enhancing macrophage-mediated killing of AML cells ing murine monocytes for 7 days with murine (m)CSF1 (25
and increasing CD47 expression on leukemia cells, which ng/mL) and mIL4 (20 ng/mL) or only mCSF1.
inhibits macrophages. Combined blockade of CD47 and Raw data and normalized gene expression data are available in
IL4 stimulation enhanced macrophage-mediated killing of the Gene Expression Omnibus database under accession number
AML cells. Hence, our data suggest that combined strate- GSE155048.
gies that activate macrophages and block CD47 have ther-
apeutic potential in AML.
Results
Methods The antileukemic activity of interleukin 4 in vivo
is predominantly mediated via immune cells
The murine leukemia model To determine whether immune cells contribute to the
All animal experiments were conducted according to the proto- previously described antileukemic effects of IL4 in vivo,10
col approved by the Animal Care and Use Committee of the we used a murine AML model driven by the MLL–AF9
Lund/Malmö Ethical Committee. MLL–AF9 leukemias were gen- (KMT2A-MLLT3) fusion gene.14 The leukemia cells were
erated in a dsRed C57BL/6 transgenic background (6051; Jackson generated in a dsRed+ transgenic background, allowing
Laboratory, Bar Harbour, NY, USA), as previously described.14 The for convenient tracking of leukemia cells upon serial
MLL–AF9 leukemia was serially propagated in sublethally irradi- transplantations.16,17 Serial passaging of leukemia cells in
ated (600 cGy) C57BL/6 recipient mice and leukemia stem cells mice did not alter IL4RA expression on AML blasts
were enriched as previously described.15 All experiments involving (Online Supplementary Figure S1A). Consistent with previ-
murine leukemia cells were performed using tertiary or quaternary ous results,10 we confirmed that elevated IL4 levels medi-
transplanted leukemia cells. As immunodeficient murine recipi- ated by retroviral expression in c-Kit+ AML cells trans-
ents, sublethally irradiated (250 cGy) NOD/SCID and NOD.Cg- planted into mice (IL4 group) resulted in strong in vivo
PrkdcscidIl2rgtm1Wjl/SzJl (NSG) mice were used (in-house breeding). antileukemic activity. The IL4 group showed prolonged
All mice used in experiments were age- and sex-matched. survival compared to controls and had almost no
leukemia cells in the bone marrow or spleen at the time
In vivo depletion of macrophages of sacrifice (Figure 1A, B, Online Supplementary Figure
To deplete macrophages in mice transplanted with retrovirally S1B).
transduced leukemia cells, we used intraperitoneal (i.p.) injection To address whether the antileukemic activity of IL4 in
of 200 mL of clodronate liposomes (5 mg/mL; Liposoma B.V., vivo was immune-mediated, we used two strains of
Amsterdam, the Netherlands). Controls were injected with phos- immunodeficient recipient mice: NOD/SCID mice, which
phate-buffered saline. We administered the first injection of clo- lack T and B cells and have decreased activity of both NK
dronate liposomes 1 day before injections of leukemia cells and cells and macrophages,18 and NSG mice, which addition-
repeated the procedure every tenth day. All mice in the survival ally lack NK cells.19 In NOD/SCID animals, the
experiments were sacrificed based on at least one of the following antileukemic effect of IL4 was reduced, and we observed
criteria: immobility, hunched back, hind leg paralysis, or dehydra- increased levels of leukemia cells in the bone marrow and
tion. spleens compared to the levels in immunocompetent mice
(Figure 1C, Online Supplementary Figure S1C). These find-
Phagocytosis assay ings suggest that immune cells at least partially mediate
For mouse phagocytosis assays, c-Kit+ dsRed+ murine MLL–AF9 the antileukemic effect of IL4. To further characterize the
leukemia cells were added to macrophage cultures in a 2:1 ratio. antileukemic effect of IL4, we used the NSG mouse strain,
After 18 h, the cells were stained with a BV421–F4/80 antibody which lacks a functional IL4 receptor type I complex
(BioLegend, San Diego, CA, USA), and the percentage of because of deficiency in the Il2rg gene. Of note, in NSG
F4/80+dsRed+ cells was determined by FACS analysis. mice, the antileukemic effect of IL4 was abolished, and
For the CD47 blocking experiments, we incubated c-Kit+ dsRed+ survival was even shorter than in controls, with high lev-
murine MLL–AF9 leukemia cells for 30 min with an anti-CD47 els of leukemia cells in both the bone marrow and spleens
antibody or rat IgG2a isotype control (30 mg/mL; BioXCell, at the time of sacrifice (Figure 1D, Online Supplementary
Lebanon, NH, USA), before co-culture with macrophages for 1 h Figure S1D). These findings suggest that the antileukemic
at 37°C. The percentage of F4/80+dsRed+ cells was determined by effect of IL4 in vivo depends on immune cells expressing
flow cytometry as described above. the IL4 receptor type I complex.

P. Peña-Martinez et al.
A B
C D
Figure 1. Interleukin-4 has antileukemic activity in a microenvironment-dependent manner. (A) dsRed+ c-Kit+ MLL–AF9 acute myeloid leukemia (AML) cells were
transduced with retroviral vectors coexpressing green fluorescent protein (GFP) and a murine interleukin 4 cDNA (MIG–IL4) or an empty control vector (MIG). Two
days later, sorted GFP+ AML cells were transplanted into sublethally irradiated mice. (B) Transplantation of 10,000 leukemia cells into C57BL/6 mice. Kaplan-Meier
survival curves (9 mice per group, pooled from 2 independent experiments), and percentage of leukemia (dsRed+) cells in the bone marrow (BM) of mice at the time
of sacrifice. (C) Transplantation of 30,000 leukemia cells into NOD/SCID mice. Kaplan-Meier survival curves (6 mice per group) and percentage of leukemia cells in
the BM of mice at the time of sacrifice. (D) Transplantation of 30,000 leukemia cells into NSG mice. Kaplan-Meier survival curves (14 mice per group, pooled from
2 independent experiments), and percentage of leukemia cells in the BM of mice at the time of sacrifice. ***P<0.001; ****P<0.0001.
Interleukin 4 expands macrophages in vivo that the elevated IL4 levels resulted in bone marrow fail-
To identify the type of immune cell that mediates the ure in these animals. By contrast, in NSG mice, the IL4
IL4-induced antileukemic effects, we analyzed the group exhibited high levels of leukemia cells in the bone
hematopoietic compartment in mice receiving IL4-secret- marrow, similar to the levels in the MIG control group
ing AML cells. At day 19 after transplantation, we detect- (Online Supplementary Figure S2H).
ed no IL4-induced alterations in blood cell lineages by
flow cytometry (Figure 2A). Moreover, at this time-point, Interleukin 4 stimulation increases murine
we detected no circulating leukemia cells in the blood of macrophage-mediated phagocytosis of leukemia cells
mice in the IL4 group (Figure 2B). In contrast, at day 27 To assess whether the IL4-induced expansion of
after transplantation, the white blood cell, red blood cell, macrophages in vivo was responsible for the antileukemic
and platelet counts in the IL4 group were reduced com- activity of IL4, we depleted macrophages by intraperi-
pared to those in controls that had not been injected with toneal injections of clodronate liposomes,20,21 followed by
leukemia cells (Figure 2C, Online Supplementary Figure S2A, injection of IL4-secreting AML cells (Figure 3A). Efficient
B). Of note, at the time of sacrifice, when the mice had depletion of macrophages was observed in the spleen but
succumbed to disease (Figure 1B), there was significant not in the bone marrow (Figure 3B). Consistent with the
expansion of F4/80+ macrophages in the bone marrow (on macrophage depletion, we found a proportional increase
average, 2.4% vs. 1%; P<0.001) and spleens (on average, of leukemia cells in the spleen of these mice (on average,
7.9% vs. 1.3%; P<0.0001) of IL4 mice (Figure 2D, Online 33% vs. 6%; P<0.05), but not in the bone marrow (Figure
Supplementary Figure S2C, D). We confirmed this 3C). In contrast, depletion of macrophages had no effect
IL4-induced increase in the proportion of macrophages by on the level of leukemia cells in the MIG control group
immunohistochemistry (Figure 2E, Online Supplementary (Online Supplementary Figure S3A, B). These findings sug-
Figure S2E). We also confirmed IL4RA expression on the gest that macrophages mediate the IL4-induced killing of
F4/80+ cells from both groups of mice, supporting that IL4 leukemia cells.
receptor signaling may directly stimulate macrophages in Because macrophages kill cells by phagocytosis, we
this model (Online Supplementary Figure S2F). Hematoxylin next assessed whether IL4 stimulation results in increased
staining of sections revealed extramedullary macrophage-mediated phagocytosis of leukemia cells in
hematopoiesis in the spleens of the IL4 mice, as indicated culture. Murine monocytes isolated from bone marrow
by a marked increase in megakaryocytes and altered were differentiated into macrophages for 7 days by sup-
spleen architecture with increased red pulp and decreased plementation of the culture medium with CSF1 (Figure
white pulp (Online Supplementary Figure S2G). In addition 3D). The addition of IL4 to the medium resulted in
to a reduction in leukemia cells, the decrease in circulating increased phagocytosis of leukemia cells, as evident by
white blood cells, increased extramedullary macrophage acquisition of dsRed fluorescence (Figure 3E,
hematopoiesis, and hypocellular bone marrow indicated F). Consistent with a more activated state, the IL4-stimu-

A B
C D
Figure 2. Interleukin 4 stimulation increases the frequency of macrophages in vivo. C57BL/6 mice were transplanted with 30,000 sorted green fluorescent protein
(GFP)+ MLL-AF9 acute myeloid leukemia (AML) cells 2 days after transduction with retroviral vectors co-expressing GFP and a murine interleukin 4 cDNA (MIG–IL4)
or a control vector (MIG). (A) Percentages of blood cell populations within dsRed– cells 19 days after transplantation (n=3). (B) Percentage of leukemia (dsRed+ ) cells
in the peripheral blood on day 19 after transplantation (n=3). (C) White blood cell counts at days 12 and 27 for MIG–IL4 and non-transplanted irradiated control
mice (IL4 group, n=4; controls, n=3). (D) Percentage of F4/80+ cells within dsRed– cells in bone marrow and spleens of mice at the time of sacrifice (controls, n=4;
IL4 group, n=5). (E) Representative immunohistochemistry staining of F4/80+ cells in bone marrow (40×; scale bar, 20 mm) and spleens (10x; scale bar, 100 mm).
BM: bone marrow; N.D.: not detected; PB: peripheral blood; WBC: white blood cell; IHC: immunohistochemistry. **P<0.01; ***P<0.001; ****P<0.0001.

A B C
D E F
G H I
Figure 3. Interleukin 4 stimulation causes macrophage-mediated depletion of leukemia cells in vivo. (A) C57BL/6 mice were transplanted with 30,000 sorted green
fluorescent protein (GFP)+ MLL-AF9 acute myeloid leukemia (AML) cells transduced with retroviral vectors expressing a murine interleukin 4 cDNA (MIG–IL4) or GFP
only (MIG; data presented in Online Supplementary Figure S2). One day prior to transplantation, mice received intraperitoneal (i.p.) injections of clodronate liposomes
(MΦdep group; n=4) or phosphate-buffered saline as control (n=5). Every tenth day, new i.p. injections were performed. (B) Percentage of F4/80+ cells and (C)
leukemia cells in bone marrow (BM) and spleens at the time of sacrifice in the IL4 group. (D) Monocytes were isolated from mouse BM and differentiated into
macrophages in culture with mCSF1 (25 ng/mL) and mIL4 (20 ng/mL) for 7 days, and then MLL-AF9 dsRed+ AML cells were co-cultured with the macrophages. (E)
Representative flow cytometry contour plots showing dsRed+ cells within F4/80+ cells in freshly mixed cultures (0 h) and after 18 h of co-culture with macrophages
and dsRed+ leukemia cells. (F) Phagocytosis assay with dsRed+ AML cells and murine macrophages (n=3). The percentage of dsRed+ cells within F4/80+ cells is pre-
sented. (G) CD14+ cells were isolated from human blood and differentiated into macrophages in culture with human (h)CSF1 (25 ng/mL) and hIL4 (20 ng/mL) for 7
days and then co-cultured with membrane-stained AML cell lines. (H) Phagocytosis assay with PKH67+ MA9:16 cells and PKH26+ human macrophages (n=4). The
percentage of PKH67+ cells within PKH26+ cells is presented. (I) Phagocytosis assay with PKH67+ Mono Mac 6 cells and PKH26+ human macrophages (n=5). BM,
bone marrow; MM6, Mono Mac 6; MΦ, macrophage. **P<0.01; ***P<0.001; ****P<0.0001.
lated macrophages had an increased volume and were less polarization, IL4 induced the expression of several genes
irregular than unstimulated cells, as evaluated using phase associated with alternative activation of macrophages,
holograph imaging (Online Supplementary Figure S4). including Arg1, Chil3, and Retnla (Figure 4A, Online
In contrast to its effect on murine macrophages, human Supplementary Figure S5A, B),22,23 which were among the
IL4 is well known to differentiate human monocytes into most differentially upregulated genes (Online
anti-inflammatory macrophages.22 To assess how human Supplementary Tables S1 and S2). Of note, IL4 also induced
IL4 affects phagocytosis of leukemia cells, human strong upregulation in vivo of the chemokine Ccl24, a bio-
macrophages were stimulated with IL4 before mixing marker for macrophages that originate from monocytes
with AML cell lines. In line with a differential role of IL4 rather than tissue-resident macrophages (Figure 4A).24
in mice and humans, IL4 suppressed human macrophage- Moreover, the IL4-induced macrophages showed down-
mediated phagocytosis of the AML cells (Figure 3G-I). regulation of genes such as Cd68, which is associated with
tumor-associated macrophages (Figure 4B),25 indicating
Interleukin 4 induces polarization of macrophages that IL4 differentiates macrophages into a phenotype that
To investigate how IL4 affects the global gene expres- is distinct from tumor-associated macrophages.
sion of macrophages, we performed RNA sequencing of We next performed gene set enrichment analysis to iden-
murine macrophages generated in vitro with or without IL4 tify gene expression signatures enriched in the IL4-induced
stimulation. In addition, we performed RNA sequencing macrophages in vivo. In accordance with increased phagocy-
on sorted dsRed– F4/80+ macrophages from mice trans- tosis of macrophages stimulated with IL4 in vitro, we found
planted with IL4-expressing leukemia cells and an enrichment of phagocytosis signatures in macrophages
macrophages from leukemic control mice. In agreement harvested from mice in the IL4 group (Figure 4C).
with a described role for IL4 in promoting macrophage Moreover, IL4 stimulation resulted in enrichment of genes

A B
Figure 4. Interleukin 4 expands macrophages enriched for gene expression signatures associated with alternative activation of macrophages and phagocytosis.
RNA sequencing was performed on murine macrophages generated from monocytes in vitro, and on sorted dsRed-F4/80+ macrophages from mice in the interleukin
4 (IL4) and control groups. (A) Volcano plots displaying differential gene expression between IL4-stimulated macrophages and control macrophages in vitro (left plot),
and macrophages from mice in the IL4 or control group (right plot). The y-axis corresponds to the –log10(q-value) and the x-axis to the log2 of the gene expression
fold change. Green dots represent significantly differentially expressed genes with a q-value <0.05 and fold change >2.0. (B) Heatmap showing expression of genes
associated with upregulation in tumor-associated macrophages. IL4-stimulated macrophages and control macrophages were harvested from mice. (C) Gene set
enrichment analysis revealed enrichment of phagocytosis and MHC protein complex signatures in macrophages harvested from mice. FDR, false discovery rate; GO:
gene ontology; MΦ, macrophage; NES, normalized enrichment score; TAM: tumor-associated macrophage.
associated with major histocompatibility complex (MHC) was upregulated in both c-Kit+ AML cells and normal
proteins (Figure 4C). To determine the influence of the in c-Kit+ bone marrow cells stimulated with IL4 (Figure 5C).
vivo microenvironment, we compared the gene expression We next explored the mechanistic basis of the IL4-
profiles of IL4-stimulated macrophages generated in vitro induced upregulation of CD47. Because STAT6 is a critical
and those generated in vivo (Online Supplementary Table S3). downstream mediator of IL4R signaling, we used
Macrophages generated in vivo exhibited a preferential CRISPR/Cas9 genetic engineering to knock out Stat6 in
upregulation of several markers associated with inflamma- Cas9-expressing MLL–AF9 AML cells using Stat6 sgRNA
tion and immune activation (Online Supplementary Figure that we had previously characterized.10 Stat6 disruption
S5C, D). Altogether, the gene expression data suggest that hindered the IL4-induced upregulation of CD47 (Figure
IL4 stimulation leads to an expansion of monocyte-derived 5D), demonstrating that IL4 upregulates CD47 in a
macrophages with increased phagocytic activity. STAT6-dependent manner. Thus, in addition to activating
murine macrophages, we identified a previously
Interleukin 4 upregulates CD47 in a Stat6-dependent unknown role of IL4 in protecting cells from phagocytosis
manner via CD47 upregulation.
We next searched for IL4-induced mechanisms in
leukemia cells that might affect their interactions with Combined interleukin 4 treatment and CD47 blockade
macrophages. Interestingly, the macrophage-inhibitory results in enhanced macrophage-mediated
protein CD47 was upregulated on leukemia cells in the phagocytosis of acute myeloid leukemia cells
IL4 group compared to controls at the time of sacrifice Because CD47 protects cells from phagocytosis, we
(Figure 5A). Consistent with this finding, IL4 induced the next evaluated whether the IL4-induced upregulation of
expression of CD47 in leukemia cells in a dose-dependent CD47 on AML cells counteracts enhanced phagocytosis
manner, showing that IL4 activates signaling that induces by IL4-stimulated macrophages. Consistent with this
CD47 expression (Figure 5B). Moreover, according to RNA hypothesis, AML cells pre-treated for 24 h with IL4 and
sequencing data that we had previously generated,10 Cd47 washed before co-culture with macrophages were partial-

ly resistant to phagocytosis (Figure 5E). To overcome the Discussion

inhibitory signal provided by increased CD47 expression,
we used an a-CD47 blocking antibody. Combined block- Distinct types of macrophages control tumor develop-
ing of CD47 on AML cells and IL4 stimulation of ment. Whereas tumor-associated macrophages promote
macrophages resulted in enhanced phagocytosis of AML tumor development by suppressing the immune system,
cells (Figure 5F). These findings show that IL4 has a dual other types of macrophages achieve tumor immune sur-
role in murine phagocytosis by directly activating veillance through phagocytosis of malignant cells.26-29 We
macrophages and enhancing their phagocytic activity, found that IL4 has antileukemic effects in mice, predomi-
while also inducing CD47 expression that counteracts nantly mediated by alternatively activated macrophages
phagocytosis in target cells. that normally play a key role in tissue repair and immune
A B
C D
E F
Figure 5. Combined interleukin 4 stimulation and CD47 blockade result in enhanced macrophage-mediated phagocytosis of acute myeloid leukemia cells. (A)
Representative histograms showing CD47 expression on acute myeloid leukemia (AML) cells in bone marrow (BM) and spleens of mice transplanted with dsRed+
leukemia cells transduced with the MIG–interleukin 4 (MIG-IL4) or control (MIG) vectors. (B) CD47 expression on AML cells following IL4 stimulation for 24 h. (C)
Cd47 expression shown as FPKM values of normalized reads from RNA sequencing data of c-Kit+ dsRed+ leukemia cells and c-Kit+ normal BM cells stimulated with
IL4 for 18 h. Data are presented as box and whiskers diagrams; the line indicates median, box limits are first and third quartiles, and bars indicate maximum and
minimum values. (D) CD47 expression measured by flow cytometry after 24 h of stimulation with murine (m)IL4 (100 ng/mL) in cells transduced with lentiviral vectors
expressing Stat6 or control sgRNA. (E) Phagocytosis assay with macrophages derived from murine BM monocytes stimulated with mCSF1 (25 ng/mL) and mIL4 (20
ng/mL) for 7 days. The AML cells were treated with mIL4 (100 ng/mL) or no IL4 (control) for 24 h prior to co-culture (n=3). Phagocytosis is presented as the percent-
age of dsRed+ cells within F4/80+ cells. (F) Phagocytosis assay with mouse BM monocyte-derived macrophages stimulated for 7 days with mCSF1 (25 ng/mL) and
mIL4 (20 ng/mL) or mCSF1 only (n=3). AML cells were cultured for 1 h with a blocking anti-CD47 antibody or corresponding isotype control and then mixed with the
macrophages. FPKM, fragments per kilobase million; gMFI, geometric mean fluorescence intensity; NBM, normal bone marrow. *P<0.05; **P<0.01; ***P<0.001;
****P<0.0001.

regulation.30,31 The observed expansion of alternatively viously unrecognized mechanism that regulates CD47
activated macrophages is consistent with findings show- expression and thereby protects cells from phagocytosis.
ing that IL4, via the IL4 receptor type I complex, promotes This mechanism could possibly have evolved to protect
the outgrowth of macrophages beyond homeostatic levels endogenous cells from phagocytosis in areas in which
in the setting of nematode infections.32 However, nema- high IL4 levels activate macrophages to fight invading
tode infections trigger the expansion of tissue resident pathogens. Consistent with these findings, a super-
macrophages.32 In contrast, the IL4-induced macrophages enhancer region with binding sites for STAT6 has been
with antileukemic activity showed higher expression of shown to regulate CD47 expression,38 providing a puta-
Ccl24, Mrc1, and Pdcd1lg2, suggesting that they are of tive mechanistic basis for how CD47 is upregulated via
monocytic origin, from either the bone marrow or periph- the IL4/STAT6 pathway. Given that combined IL4 stimu-
eral blood.24 Among hematopoietic cells, only lation and CD47 inhibition enhanced macrophage-medi-
macrophages showed increased numbers following ated phagocytosis of AML cells, our data suggest thera-
enforced expression of IL4 in vivo. IL4 also boosted the peutic potential for strategies that combine direct activa-
phagocytic activity of murine monocyte-derived tion of macrophages with blocking of inhibitory signals to
macrophages in vitro, suggesting that IL4 acts directly on macrophages. Because IL4 has opposing effects in murine
the monocytes/macrophages that mediate the and human macrophages, we speculate that other
antileukemic effect. Moreover, consistent with their cytokines that activate human macrophages may also
increased phagocytic activity, the IL4-induced upregulate CD47 or other ‘don’t eat me’ signals on target
macrophages were functionally and molecularly distinct cells. Identifying these mechanisms may translate into
from tumor-associated macrophages, which are classically new therapeutic opportunities in AML and possibly other
associated with an alternatively activated phenotype.25 types of cancer.
Furthermore, the IL4-induced macrophages were func- In summary, here we show that IL4 has a potent in vivo
tionally distinct from AML-associated macrophages, antileukemic effect in mice by promoting macrophage-
which polarize into a leukemia-supportive state that mediated phagocytosis of AML cells. IL4 stimulation
accelerates disease development.3 The reason why IL4 induced CD47 upregulation in a STAT6–dependent man-
induced stronger macrophage activation in vivo than in vitro ner, and combined IL4 stimulation with CD47 blockade
could be related to interactions with other immune cells or further enhanced macrophage-mediated phagocytosis of
the AML blasts, resulting in enhanced phagocytic activity. AML cells. These findings deepen our understanding of
Of note, the macrophages were dependent on IL4 for their how IL4 regulates murine macrophages and suggest that
anti-leukemic activity as depletion of macrophages in the strategies to combine macrophage activation with CD47
MIG control group did not affect the leukemia burden. inhibition should be explored further as a therapeutic
Constitutive expression of IL4 in mice has not been approach in cancer.
linked previously to anti-cancer activity, but it has been
associated with excessive phagocytosis resulting in Disclosures
decreased blood cell counts, extramedullary No conflicts of interest to disclose.
hematopoiesis, and increased mortality.33,34 We found that
IL4 induced potent antileukemic activity, with some mice Contributions
surviving long-term without signs of disease or problems PPM, RR, CH and CJ performed research, PPM and MJ ana-
of tolerability, while other mice eventually had to be sac- lyzed data and wrote the manuscript, and all other authors con-
rificed despite very low levels of leukemia cells in their tributed with valuable comments.
bone marrow and spleen. The low blood cell counts and
expansion of megakaryocytes in the spleen indicated Acknowledgments
extramedullary hematopoiesis and suggests that elevated The authors thank Dr Benjamin Ebert (Brigham and Women’s
IL4 levels induced macrophage activation with excessive Hospital, Boston, MA, USA) for sharing the dsRed+ MLL-AF9
phagocytosis. This pattern resembles that of hemophago- leukemia cells. We also thank Dr James Mulloy, (University of
cytic lymphohistiocytosis (HLH), a disease characterized Cincinnati, Cincinnati, OH, USA) for sharing the MA9:16 cells.
by aberrantly activated macrophages.35 Hence, we specu-
late that the cause of death of non-leukemic mice in the Funding
IL4 group was due to the HLH-like symptoms. Of note, We thank the following granting agencies for their support: the
the leukemic cells were selectively depleted, indicating Swedish Cancer Society, the Swedish Childhood Cancer
that the IL4-induced macrophages preferentially attacked Foundation, the Swedish Research Council, the Crafoord
them. The reason is unclear but could be related to altered Foundation, the Royal Physiographic Society in Lund, and the
expression of genes by leukemia cells that regulate Medical Faculty of Lund University.
macrophages, such as MHC class I molecules or calretic-
ulin.36,37 Data-sharing statement
In addition to IL4 boosting macrophage-mediated Raw data and normalized gene expression data are available
phagocytosis, stimulation of AML cells with IL4 induced in the Gene Expression Omnibus database under accession num-
STAT6-dependent upregulation of CD47, revealing a pre- ber GSE155048.
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Ramakrishnan R, et al. Agonistic targeting 2019;10:2746. 38. Betancur PA, Abraham BJ, Yiu YY, et al. A
of TLR1/TLR2 induces p38 MAPK-depen- 26. Chen Y, Zhang X. Pivotal regulators of tis- CD47-associated super-enhancer links pro-
dent apoptosis and NFkB-dependent differ- sue homeostasis and cancer: macrophages. inflammatory signalling to CD47 upregula-
entiation of AML cells. Blood Adv. Exp Hematol Oncol. 2017;6:23. tion in breast cancer. Nat Commun. 2017;
2017;1(23):2046-2057. 27. Feng M, Chen JY, Weissman-Tsukamoto R, 8:14802.

Acute Myeloid Leukemia ARTICLE
Pevonedistat and azacitidine upregulate NOXA Ferrata Storti Foundation

(PMAIP1) to increase sensitivity to
venetoclax in preclinical models of acute
myeloid leukemia
Dan Cojocari,1* Brianna N Smith,2-4*Julie J. Purkal,1 Maria P. Arrate,3
Jason D. Huska,1 Yu Xiao,1 Agnieszka Gorska,3 Leah J. Hogdal,5
Haley E. Ramsey,3,4 Erwin R. Boghaert,1 Darren C. Phillips1#
and Michael R. Savona3,4,6,7#
1
Haematologica 2022
Oncology Discovery, AbbVie Inc., North Chicago, IL; 2Department of Pediatrics, Volume 107(4):825-835
3
Medicine, and 4Program in Cancer Biology, Vanderbilt University School of Medicine,
Nashville, TN; 5Precision Medicine, AbbVie Inc., North Chicago, IL; 6Vanderbilt-Ingram
Cancer Center, Nashville, TN and 7Center for Immunobiology, Vanderbilt University
School of Medicine, Nashville, TN, USA
*DC and BNS contributed equally as co-first authors.
#
DCP and MRS contributed equally as senior authors.
ABSTRACT
D
ysregulation of apoptotic machinery is one mechanism by which
acute myeloid leukemia (AML) acquires a clonal survival advan-
tage. B-cell lymphoma protein-2 (BCL2) overexpression is a com-
mon feature in hematologic malignancies. The selective BCL2 inhibitor,
venetoclax (VEN) is used in combination with azacitidine (AZA), a DNA-
methyltransferase inhibitor (DNMTi), to treat patients with AML. Despite
promising response rates to VEN/AZA, resistance to the agent is common.
One identified mechanism of resistance is the upregulation of myeloid cell
leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits
NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a
BCL2 family protein that competes with effector molecules at the BH3
binding site of MCL1. We demonstrate that PEV/AZA combination
induces NOXA to a greater degree than either PEV or AZA alone, which
enhances VEN-mediated apoptosis. Herein, using AML cell lines and pri-
mary AML patient samples ex vivo, including in cells with genetic alter- Correspondence:
ations linked to treatment resistance, we demonstrate robust activity of MICHAEL R. SAVONA
the PEV/VEN/AZA triplet. These findings were corroborated in preclinical michael.savona@vanderbilt.edu
systemic engrafted models of AML. Collectively, these results provide
rational for combining PEV/VEN/AZA as a novel therapeutic approach in
overcoming AML resistance in current therapies. Received: September 17, 2020.
Accepted: April 7, 2021.
Pre-published: April 15, 2021.
Introduction
Acute myeloid leukemia (AML) is a heterogeneous hematopoietic neoplasm https://doi.org/10.3324/haematol.2020.272609

characterized by the arrest of differentiation and clonal proliferation of myeloid
precursor cells. Despite recent advances, only 24% of patients survive their disease
beyond 5 years of diagnosis.1–6 One mechanism by which AML clones acquire a
©2022 Ferrata Storti Foundation
survival advantage is the dysregulation of apoptotic machinery that in normal cells Material published in Haematologica is covered by copyright.
regulates homeostasis in the bone marrow. Apoptosis is controlled at the mito- All rights are reserved to the Ferrata Storti Foundation. Use of
chondrial level via mechanisms regulated by the B-cell lymphoma protein-2 (BCL2) conditions:
family of proteins.7 This group of proteins is divided into three sub-groups which https://creativecommons.org/licenses/by-nc/4.0/legalcode.
include pro-apoptotic “BH3-only” proteins BIM, BID, PUMA, NOXA, BAD, BIK, Copies of published material are allowed for personal or inter-
BMF and HRK, and the effector proteins BAX and BAK that are induced by various nal use. Sharing published material for non-commercial pur-
cell death stimuli to trigger mitochondrial outer membrane permeabilization https://creativecommons.org/licenses/by-nc/4.0/legalcode,
(MOMP) and apoptosis.8,9 A separate group of anti-apoptotic family members, sect. 3. Reproducing and sharing published material for com-
including BCL2, BCL-XL, MCL1, BCL-W, and BCL2-A1, function to bind and inhibit mercial purposes is not allowed without permission in writing
the pro-apoptotic family members, preventing MOMP and subsequently from the publisher.
apoptosis.8,10,11 BCL2 overexpression in human hematologic malignancies is associ-
ated with poor responses to conventional chemotherapy.12,13 A deep understanding

D. Cojocari et al.
of mitochondrial protein control of apoptosis has fueled a line-derived and patient-derived xenograft models of
desire to discover small molecules specifically intended to AML. Our work provides important mechanistic insight
occupy the hydrophobic BH3 binding site of antiapoptotic to support the use of these agents in combination in ongo-
proteins and allow initiation of apoptosis by effector mol- ing clinical trials.
ecules.14,15
Venetoclax (VEN), a selective BCL2 inhibitor, binds BCL2
to directly inhibit sequestration of pro-apoptotic proteins Methods
such as the activator BIM. Free BIM can bind to and activate
BAX/BAK, inducing conformational changes that result in Cell culture and reagents
BAX/BAK homo-oligomerization and mitochondrial outer AML cell lines and patient cells were cultured as previously
membrane permeabilization (MOMP), initiating apopto- described, and were short tantem repeats (STR) validated and
sis.16,17 VEN has been effective in the clinic for patients with mycoplasma tested.34 See the Online Supplementary Materials and
chronic lymphocytic leukemia but has limited efficacy in Methods for reagent details.
relapsed-refractory AML as a single agent.18,19 In recent clin-
ical trials in newly-diagnosed AML patients ineligible for Patient samples
intensive chemotherapy, VEN demonstrated an overall Experiments were conducted on primary patient samples pro-
response rate of 67% or 48% in combination with DNA vided by the Vanderbilt-Ingram Cancer Center Hematopoietic
methyltransferase inhibitors (DNMTi; AZA or decitabine) Malignancies Repository, after obtaining informed consent, and
or low-dose cytosine arabinoside (LDAC), respectively.20,21 approval of the Vanderbilt University Medical Center Institutional
Despite this progress, many patients treated with Review Board.
VEN+DNMTi/LDAC ultimately relapse, and a subset of
patients never respond.20–23 One proposed mechanism of Knockout cell line generation
resistance to VEN is cellular upregulation of the anti-apop- Genomic deletion of BBC3 and PMAIP1 were previously
totic protein myeloid cell leukemia-1 protein (MCL1). VEN described.34 The BAK1/BAX-deficient OCI-AML5 cells were gen-
in combination with selective MCL1 inhibitors has demon- erated using the same protocol except by combining the two
strated enhanced cytotoxic activity over either agent alone CRISPR RNA (crRNA) targeting BAK1/BAX. ATF4-deficient cells
in AML cells in vitro and in xenograft models pre-clinically, were generated by combining the two crRNA (Online
but efficacy of this combination in the clinic has yet to be Supplementary Table S1).
reported.24 Both MCL1-dependent and MCL1-independent
mechanisms of VEN resistance are emerging and new Cell proliferation assay
approaches aimed at addressing these are moving toward For combinatorial studies, cells were treated for 24 hours as pre-
the clinic.25-29 viously described.34 For primary cells, compounds were dispensed
Pevonedistat (PEV) was developed as a targeted into a 384-well plate using the Echo 555 liquid handler (Labcyte)
inhibitor of NEDD-8 activating enzyme (NAE), which and 2,000-8,000 cells/well were incubated for 24 hours or 72 hours
activated the Cullin-RING E3 ubiquitin ligases (CRL) in a for VEN/AZA resistant patient samples and cell viability was
process called neddylation. Thus, PEV disrupts the protea- measured using the Cell TiterGlo (Promega). Percent viability was
somal-mediated degradation of proteins targeted by the defined as the relative luminescence units (RLU) of each well
CRL, leading to their accumulation.30 Neddylation is divided by the RLU of cells in dimethyl sulfoxide (DMSO) control
upregulated in AML, and pevonedistat has been tested as and the effective concentration (EC50) to induce 50% cell death
a single agent and in combination with the DNMTi, were determined by nonlinear regression algorithms using Prism
AZA.31-33 In a phase Ib clinical trial of untreated AML 8.0 (GraphPad).
patients ≥60 years of age, an intention to treat analysis
revealed PEV in combination with AZA induced an overall Western blot
response rate of 50%.31 Interestingly, PEV and AZA both Western blotting was performed as previously described.34
upregulate NOXA (PMAIP1), a pro-apoptotic BCL2 family Membranes were incubated with the respective primary antibod-
protein known to compete with effector molecules at the ies (Online Supplementary Table S2).
BH3 binding site of MCL1 to inhibit its anti-apoptotic
function.10,34,35 Given the emerging critical role of MCL1 in Patient-derived xenografts
VEN resistance, we postulated that the combination of 2×106 primary AML mononuclear cells were engrafted in 7-9-
PEV/AZA will synergize with VEN in a triple combination week-old female NSGS (NOD-scid IL2Rgnull3Tg(hSCF/hGM-
with efficacy superior to VEN/AZA or VEN/PEV alone. CSF/hIL3)) mice (The Jackson Laboratory) as described in the
Herein, we demonstrate that the triple combination of Online Supplementary Materials and Methods. Chimerism was
VEN/PEV/AZA induces robust activity in preclinical mod- assessed weekly in the peripheral blood, and, at time of tissue har-
els of AML that is superior to either agent alone or as com- vest, in the bone marrow and spleen. Animal experiments were
bination doublets. Leveraging AML cell lines and primary conducted in accordance with guidelines approved by the IACUC
AML patient samples ex vivo, we demonstrate that the at VUMC.
PEV/AZA combination induces NOXA to a greater extent
than PEV or AZA alone to further enhance VEN-mediated Cell line-derived xenografts
apoptosis. Apoptosis induced by the VEN/PEV/AZA com- Female fox chase SCID beige mice were employed for the flank
bination required PMAIP1, the gene encoding NOXA, xenograft model; female NOD SCID gamma mice were employed
since its deletion abrogated the activity of this triplet. in the systemic engraftment of OCI-AML2-Red-Fluc cell line
Importantly, the VEN/PEV/AZA combination enhanced (Charles River Laboratories) and performed as described in the
the kinetics of apoptosis compared to other treatment Online Supplementary Materials and Methods. Animal studies were
variations in AML cell lines and patient samples that man- conducted in accordance with the guidelines established by the
ifest in vivo to drive durable anti-leukemic activity in cell AbbVie Institutional Animal Care and Use Committee.

PEV/VEN/AZA therapy in preclinical AML
Flow cytometry the BCL2 family proteins and gene expression in AML
Red blood cells were lysed with EL buffer on ice (Qiagen), with cell lines. Treatment with PEV caused a robust upregula-
remaining cells washed and resuspended in 1X phosphate tion in NOXA protein levels in all five AML cell lines
buffered saline (PBS) with 1% bovine serum albumin (BSA) and (Figure 1C), and increased gene expression of its tran-
stained for 15 minutes with the conjugated antibodies listed in the script, PMAIP1, in the three of the AML cell lines
Online Supplementary Table S2. Cells were washed and submitted assessed (Figure 1D). The BH3-only protein PUMA was
for flow cytometric analysis using a 3-laser LSRII (Becton inconsistently induced across these cell lines at the pro-
Dickinson). tein or transcript level (Figure 1C and D). In contrast to
the BH3-only pro-apoptotic members, MCL1 was down-
Immunohistochemistry regulated in all cell lines treated with PEV (Figure 1C). In
Tissues were fixed in 4% paraformaldehyde for 48 hours and order to establish the importance of NOXA for either
stored in 70% ethanol, embedded in paraffin and sectioned at 5 PEV or VEN/PEV activity, we utilized three PMAIP1-defi-
µm after bone tissue was decalcified. Sections were dewaxed in cient AML cell lines.34 Deletion of PMAIP1 reduced PEV-
Xylene and rehydrated in successive ethanol baths. Standard induced death in OCI-AML5, Kasumi-1, and MV4-11
Mayer’s hematoxylin and eosin (H&E) staining was performed. cells (Figure 1E; Online Supplementary Figure S1B) and
Antigen retrieval using a standard pH 6 sodium citrate buffer abrogated the synergistic activity of VEN/PEV in all three
(BioGenex) was performed and sections were stained with mono- cell lines (Figure 1F and G). These findings illuminate the
clonal mouse anti-human CD45 (Dako, M0701, dilution 1:200) importance of NOXA for the synergistic activity
using M.O.M. Kit (Vector). between VEN and PEV.
Kinetics of caspase-3/-7 activation and cell death Venetoclax/pevonedistat/azacitidine induces

Activated caspase-3/-7 (aCasp-3/-7+) and cell death (DRAQ7+) BAX/BAK-dependent apoptosis in acute myeloid
was evaluated using an IncuCyte S3 (Sartorius) as described in leukemia cell lines
detail previously.34 Area under the curve (AUC) was calculated Previously, we demonstrated that NOXA is essential for
over the 48 hours period using Prism 8.0 (GraphPad). the combinatorial activity between VEN and AZA in AML
cell lines, providing an insight into the mechanism of
Gene expression VEN/AZA clinical activity in AML patients.34 We explored
100,000 AML cells (OCI-AML5, MV4-11, THP-1) were cultured the impact of adding PEV to the VEN/AZA combination
in 96-well plates three biological replicates were treated with to AML cell lines in vitro. A panel of seven AML cell lines
pevonedistat for 24 hours. QuantiGene Plex assay (Sigma- were treated with ten doses of VEN and three doses of
Aldrich ) was performed as instructed by the manufacturer. AZA. Upon the addition of PEV at 100 nM or 370 nM, the
overall cell viability decreased in the VEN/AZA combina-
Statistical analysis tion matrix, indicating an added benefit of PEV to the
An unpaired 2-tailed Student t-test was performed unless other- VEN/AZA cell killing in all seven cell lines (Figure 2A),
wise indicated. All statistical analyses were completed using Prism including in THP-1 cells where VEN/AZA was not syner-
8.0 software (GraphPad). The effects of VEN/PEV combinatorial gistic but VEN/PEV was (Figure 1A).34 Similarly, the addi-
activity were calculated using the zero interaction potency (ZIP) tion of AZA to VEN/PEV increased cell death in SET-2 and
synergy model, which compares observed and expected combina- U937 cells (Figure 2A). Thus, VEN/PEV/AZA triple combi-
tion effects.36–38 nation improves the activity observed with either the
VEN/PEV or VEN/AZA treatments, overcoming the inher-
ent resistance of some AML cells to these treatment dou-
Results blets.
In order to explore the kinetics of cell death with the
Deletion of PMAIP1 abrogates the synergistic activity VEN/AZA/PEV combination, caspase-3/-7 activation and
of pevonedistat and venetoclax in acute myeloid cell death (DRAQ7 uptake) were measured over time fol-
leukemia cell lines lowing the addition of individual agents, the double com-
We tested a panel of AML cell lines for the combinator- binations, or the triple combination. The triple
ial activity of VEN and PEV using a 10 by 3 concentration VEN/AZA/PEV treatment induced a more rapid activation
matrix, respectively (Online Supplementary Figure S1A). The of caspase-3/-7 and cell death in MV4-11 (Figure 2B) and
two agents were synergistic in 15 of 18 and highly syner- Kasumi-1 (Online Supplementary Figure 2A) compared to
gistic (d>5%) in nine of 18 AML cell lines (Figure 1A) as other variations of this treatment combination. Similarly,
assessed by the ZIP synergy model.38 In order to further the overall levels of caspase-3/-7 and cell death over the
validate this in vitro observation, a subcutaneous xenograft 48-hour period, as measured by the area under the kinetic
model of the MV4-11 cell line was established in the flank curve (AUCk), was significantly higher than the singlet or
of immunocompromised mice. The mice were divided doublet treatments (Figure 2C). We asked whether the cell
into four treatment groups and treated daily for two death induced by the triplet is dependent upon the intrin-
weeks with either PEV, VEN, or the combination of PEV sic apoptosis signaling pathway. OCI-AML5 cells deficient
and VEN. Both VEN and PEV each moderately inhibited in both BAX and BAK1 were generated using CRISPR-
MV4-11 tumor growth to similar degrees. However, the Cas9 clonal gene editing and the absence of BAX and BAK
two agents together displayed combinatorial activity protein expression confirmed (Figure 2D). The triplet com-
resulting in pronounced tumor regression and a tumor bination treatment in parental OCI-AML5 cells induced
growth delay (Figure 1B). significant caspase-3/-7 activation and cell death, that was
In order to explore the molecular drivers behind the completely abrogated by BAX/BAK1 deletion (Figure 2E;
VEN/PEV synergistic activity, we measured changes in Online Supplementary Figure S2B).

D. Cojocari et al.
A B
C D
Figure 1. Deletion of PMAIP1 abrogates the synergistic activity of pevonedistat and venetoclax in acute myeloid leukemia cell lines. (A) Synergistic activity between
venetoclax (VEN) and pevonedistat (PEV) as determined by the zero interaction potency (ZIP) model following 24 hours of treatment of a panel of acute myeloid
leukemia (AML) cell lines. (B) Tumor growth over time in the MV4-11 subcutaneous xenograft model treated (black bar) with vehicle control, VEN (50 mg/kg; 14-day
daily [QDx14]; orally [PO]), PEV (60 mg/kg; QDx14; intraperitoneal [IP]), or the two agents in combination (QD×14). Data are presented as the mean tumor volume
± standard error of the mean (SEM) from eight mice per treatment group. (C) Western blot analysis of NOXA, PUMA, BCL2, BCL-XL, and MCL1 proteins in a panel of
AML cell lines treated with PEV (OCI-AML5: 1 mM; Kasumi-1: 1.5 mM; MV4-11: 0.4 mM; OCI-AML2: 0.15 mM; U937: 2 mM) for 24 hours. (D) Gene expression analysis
of PMAIP1, BBC3, BCL2L1, MCL1, and BCL2 in OCI-AML5, MV4-11, and THP-1 cells following treatment with PEV at the indicated concentrations (OCI-AML5: 0.4 mM
and 1 mM; MV4-11: 0.2 mM and 0.4 mM) after 24 hour of treatment relative to the dimethyl sulfoxide (DMSO) control cells (t-test, *P<0.05, n=3). (E) Cell viability of
three parental or PMAIP1-/- AML cell lines after 24 hours of treatment with VEN and PEV in combination (n=3). (F) Synergy measured by ZIP model metric for the VEN
and PEV combination in three parental or PMAIP1-/- AML cell lines after 24 hours of treatment (n=3) (lower panel). (G) Visualization of the calculated 2D ZIP synergy
map for the parental and PMAIP1-/- MV4/11 from (F).

Azacitidine- and pevonedistat-induced NOXA tion factor ATF4 to enhance PMAIP1 expression.34,39 In
contributes to the combinatorial activity with order to understand the effects of combining AZA and PEV
venetoclax on the ISR’s ATF4-NOXA axis, we treated the parental and
NOXA is important for efficient VEN, PEV or AZA sin- PMAIP1 deficient OCI-AML5 cell lines for 24 hours with
gle-agent activity, as well as for VEN/AZA34 or VEN/PEV either agent alone or in combination. In parental cells, AZA
synergy (Figure 1F).35 However, PMAIP1 is induced by or PEV alone or in combination induced ATF4 or its tran-
AZA or PEV through distinct mechanisms. AZA treatment scriptional targets CHOP and NOXA and led to apoptosis,
induces the integrated stress response (ISR) pathway to as measured by levels of cleaved PARP (Figure 3A).
upregulate PMAIP1, whereas PEV stabilizes the transcrip- Similarly, AZA/PEV combination induced DDIT3 and
B C
D E
Figure 2. Adding azacitidine to venetoclax/pevonedistat treatment significantly decreases acute myeloid leukemia cell line viability in vitro that is dependent on
BAX/BAK-mediated apoptosis. (A) Cell viability matrix measure in a panel of acute myeloid leukemia (AML) cell lines following 24 hours of treatment with venetoclax
(VEN) (10 mM top dose 1:3 dilution, except OCI-AML2, MV4-11 top dose of 300 nM) and azacitidine (AZA) (0.3 and 1 mM) and pevonedistat (PEV) at the indicated
doses. (B) Activated caspase-3/-7 positive cells (aCasp-3/-7+) or dead cells (DRAQ7+) were counted over time following treatment with the VEN (1 nM), AZA (1 mM),
PEV (100 nM), or indicated combinations of these compounds in MV4-11 cells (n=5). (C) Area under the kinetic curves (AUCk) from (B) was calculated over a 48-hour
period and plotted as total positive cells (n=5). (D) Western blot analysis of BAX and BAK expression in the parental and BAX-/-/BAK1-/- OCI-AML5 cell lines. (E) OCI-
AML5 cells were treated with VEN (10 nM), AZA (3 mM), PEV (100 nM) or indicated combinations, and the AUC of the total aCasp-3/-7+ or DRAQ7+ cells calculated
over a 48-hour time period were plotted as total aCasp-3/-7+ (left) or DRAQ7+ (right) cells positive cells (n=5).

D. Cojocari et al.
A B
Figure 3. Azacitidine- and pevonedistat-induced NOXA contributes to the combinatorial activity with venetoclax. (A) Western blot analysis of total PARP, ATF4, CHOP,
and NOXA protein in the parental and the PMAIP1-/- OCI-AML5 cell line treated with azacitidine (AZA) (1 mM), pevonedistat (PEV) (0.37 mM) or both for 24 hours.
b-actin was used a protein loading control. (B) DDIT3 and PMAIP1 gene expression was measured in cell lines from (A) under the same conditions. (C) Western blot
analysis of ATF4, CHOP, eIF2a, phosphor eIF2a Ser51 and NOXA protein in the parental and the ATF4-/- OCI-AML5 cell line treated as indicated in (A). (D) Cell viability
of OCI-AML5 cell line following 24 hours of treatment with venetoclax (VEN) (10 mM top dose 1:3 dilution) and AZA (0.3 mM, 1 mM and 3 mM) and PEV (370 nM) (n=3).
(E) Cell viability as measured by area under the curve (AUC) of the VEN dose response curve (from C and Online Supplementary Figures S3A and B) of three parental
or PMAIP1-/- acute myeloid leukemia (AML) cell lines following 24 hours of treatment with VEN (10 mM top dose 1:3 dilution, MV4-11 top dose of 0.3 mM) and AZA
(0.3 mM, 1 mM and 3 mM) and PEV 100 nM and 370 nM (n=3). (F) Western blot analysis of total PARP, BAX, BAK, MCL1, NOXA protein in the parental and the
BAX-/-/BAK1-/- OCI-AML5 cell line treated as indicated in (A). DMSO: dimethyl sulfoxide.

PMAIP1 transcripts to a greater level than the single agent of ISR. Thus, ATF4 is required for the induction of NOXA
treatments in OCI-AML5 and Kasumi-1 cells (Figure 3B). In by AZA/PEV. In order to understand if NOXA is critical for
contrast, the PMAIP1-deficient OCI-AML5 cells displayed the PEV/VEN-mediated sensitization of AML cell lines to
reduced levels of cleaved PARP, while the ATF4 and CHOP VEN, we treated the parental and PMAIP1-deficient cell
induction was not affected by the gene deletion (Figure lines with the VEN/PEV/AZA triple combination and
3A). Upon ATF4 ablation in OCI-AML5, either agent measured cell viability after 24 hours. Relative to the
alone, or the combination, was unable to induce CHOP or parental cell lines, PMAIP1-deficiency reduced the potency
NOXA (Figure 3C). Furthermore, unlike AZA, PEV did not of VEN-mediated cell death alone or in combination with
induce significant phosphorylation of eIF2a at Ser 51, a AZA, PEV, or AZA/PEV in OCI-AML5 (Figure 3D and 3E),
marker of ISR activation, while still inducing ATF4 and Kasumi-1 (Online Supplementary Figure 3A) and MV4-11
NOXA. This suggests PEV stabilizes ATF4 in the absence (Online Supplementary Figure 3B) cell lines. NOXA can pro-
Figure 4. The venetoclax/pevonedistat/azacitidine triple-combination treatment induces durable responses in a systemic xenograft model of acute myeloid
leukemia. (A) Tumor growth from whole-body ROI bioluminescent signal (total photons/second) of systemically engrafted OCI-AML2-Red-Fluc tumor cells measured
over time and treated (black bar) with vehicle control, venetoclax (VEN) (50 mg/kg, 14-day daily [QDx14], orally [PO]), AZA (8 mg/kg, every 7 days for three treatments
[Q7D×3], intravenously [IV]), pevonedistat (PEV) (60 mg/kg, QD×14, intraperitoneal [IP]) or the combinations of the two or all three agents (n=6-8 mice per treatment
group). (B) Representative images of animals’ bioluminescent signal of the treatment cohorts from panel (A) showing significant delay in in vivo acute myeloid
leukemia (AML) growth with either single- or dual-treatment combinations, while no growth (BLI signal) was detected in the triple-combination treatment.

D. Cojocari et al.
A C
B D
Figure 5. The venetoclax/pevonedistat/azacitidine triple-combination treatment is efficacious in preclinical primary acute myeloid leukemia models. (A) Primary
acute myeloid leukemia (AML) samples from seven different patients with different mutational profiles treated ex vivo for 24 or 72 hours (venetoclax/azacitidine
[VEN/AZA] resistant patients) with pevonedistat (PEV) (0.3 mM), AZA (0.3 mM) and VEN (0.01 mM) alone, and combinations. (B) Western blot analysis of a primary
AML patient samples after 12 hours (AML008) or 24 hours (AML004, AML005, AML006 and AML009) of treatment with dimethyl sulfoxide (DMSO) control, AZA
alone, PEV alone, and PEV/AZA in combination. Protein levels of BCL2 family members MCL1, BCL2, NOXA, cleaved PARP, and b-actin. (C) Mice were injected with
2x106 patient primary AML 004 cells via tail vein on day 1, 24 hours after cesium irradiation and were treated (black bar) with vehicle, PEV (30 mg/kg; every other
day for 28 days [QOD×28]), AZA (1.5 mg/kg; once every day for 7 days [QD×7]), VEN (15 mg/kg; once every day for 28 days [QDx28]), VEN/AZA or triple combination
VEN/AZA/PEV. Human CD45 positive (hCD45+) cells were measured weekly by flow cytometry in peripheral blood (PB) from week 2 through week 12 post-transplant
(t-test, *P<0.05). (D) Percent of hCD45+ cells in bone marrow and spleen tissue were measured via flow cytometry on day of tissue harvest week 12 post-transplant.
(E) Immunohistochemistry of bone marrow (femur) and spleen (20X), stained with monoclonal antibody for hCD45 in experimental mice.
mote the degradation of MCL1 by the proteosome and The venetoclax/pevonedistat/azacitidine combination
executioner caspases can also cleave MCL1 during apopto- treatment is highly active in preclinical models of acute
sis.40 In order to understand the mechanism of MCL1 loss myeloid leukemia
upon treatment with either AZA or PEV, the OCI-AML5 The in vivo efficacy of the triple combination of PEV, VEN
cell line deficient in BAX and BAK1 were treated with these and AZA was tested in a systemic murine model that
agents (Figure 3F). MCL1 protein degradation induced by allowed monitoring the tumor burden of xenografted bio-
either AZA or PEV was not observed in the OCI-AML5 cell luminescent OCI-AML2-Red-Fluc cells. Animals were dis-
lines deficient in either PMAIP1 (Figure 3A), or BAX/BAK1 tributed into eight treatment cohorts and treated with
(Figure 3E). In the BAX-/- BAK1-/- cells, MCL1 was not either vehicle alone, VEN (50 mg/kg, QD×14, orally [PO]),
degraded despite NOXA upregulation (Figure 3F), thus 5-Aza (8 mg/kg, Q7D×3, intravenous [IV]), PEV (60 mg/kg,
MCL1 degradation by either AZA or PEV requires both QD×14, intraperitoneal [IP]) or the combinations of the
sensitization by NOXA and activation of apoptosis by two or all three agents. Animals were imaged at regular
BAX/BAK. Taken together, these data demonstrate that intervals until they reached endpoint (1×1010 photons/sec-
NOXA can be induced by both AZA and PEV and that it ond). The single-agent treatments were active in this
has a central role in driving their combinatorial activity model, and the doublets were more efficacious than the
with VEN. single-agents (Figure 4A). Most notably, the triplet combi-

nation of PEV, VEN and AZA demonstrated enhanced men of VEN/AZA in experimental models of AML. We
tumor growth inhibition compared to the singlet or dou- demonstrate that the VEN/PEV/AZA triple combination
blet treatment cohorts, driving durable responses with no requires the induction of the BH3-only protein NOXA to
evidence of tumor growth (zero of eight mice) by day 117, enhance the kinetics and depth of apoptosis in AML cell
93 days after treatment had ceased (Figure 4A and B). lines in vitro when compared to other treatment varia-
Importantly, none of the cohorts experienced a significant tions of these drug components. These observations are
decrease in body weight (Online Supplementary Figure 4A). reflected in vivo, where the VEN/PEV/AZA combination
In order to further explore the utility of the triplet induces durable anti-leukemic responses in sys-
VEN/AZA/PEV triple combination, we treated a panel of temic cell line-derived and patient-derived xenograft
primary AML patient samples with different mutational, models of AML.
karyotype profiles, and treatment failure (Online Although the addition of VEN to AZA in newly diag-
Supplementary Table S3) with PEV, AZA and VEN alone, nosed AML patients ineligible for intensive chemotherapy
and combinations of these agents (Figure 5A). Improved improved overall survival (14.7 months VEN/AZA vs. 9.6
combinatorial activity was noted in the triplet combination months AZA), a subset of AML patients present with
for all patient samples when compared to VEN/AZA. In shorter durations of response or never respond to treat-
order to test whether PEV can also improve efficacy in ment.23,41 A previous report suggested the potential of PEV
patient samples with significant resistance to VEN/AZA in to synergize with VEN in cell lines in vitro.35 In order to
vitro, we tested four patients with in vitro resistance to the build upon these observations and to explore the mecha-
doublet. In this subset of patients, the triplet combination nism of this interaction, we extensively explored the
also demonstrated superior efficacy compared to potential combinatorial activity extensively with, and
VEN/AZA alone (Figure 5A). Western blot analysis of five without AZA. VEN and AZA are effective in most AML
primary AML patient samples treated with PEV/AZA in cell lines in vitro and reflect clinical observations of dimin-
combination for 12 or 24 hours increased NOXA protein ished responses in cases of mutant TP53.34,41,42 Adding PEV
expression further than either drug alone, and coincided to the VEN/AZA treatment paradigm enhanced cell death
with a decrease in MCL1 protein without impacting BCL2 in AML cell lines and patient samples, consistent with our
expression (Figure 5B). In order to validate our ex vivo find- previous finding of NOXA induction in AML exposed to
ings in a patient-derived xenograft model, NSGS mice AZA,34 and consistent with synergistic NOXA induction
were transplanted with de novo cells from patient AML 004. with the combination of PEV and AZA. This coincided
Next generation sequencing of this patient’s AML revealed with diminished MCL1 protein which the primary target
a complex mutational profile including mutations in FLT3- of NOXA in the mitochondria. To that end, we saw
ITD, NPM1, IDH2, and DNMT3A. Chimerism was estab- enhanced killing of AML in cell lines and patient samples
lished, and 5 weeks post-transplant we began treating the in which VEN/PEV or then VEN/AZA combination activ-
mice with PEV, AZA, VEN, combination VEN/AZA (the ity was limited.
current standard of care) or the triplet combination at sub- In order to capture the response heterogeneity of AML
therapeutic doses of 30 mg/kg of PEV, 1.5 mg/kg of AZA, we tested the triple combination in a panel of primary
and 15 mg/kg of VEN for 28 days. Vehicle-treated mice AML samples from patients found ultimately to be refrac-
rapidly succumbed to leukemia by week 12 post-trans- tory to conventional chemotherapy, and/or VEN/DNMTi
plant (Figure 5C). At this point, the experiment was con- treatment (Online Supplementary Table S3). The triplet
cluded, and tissue was harvested for evaluation of human combination was found to effectively kill the malignant
chimerism in the bone marrow and spleen (Figure 5D). AML cells in ex vivo assays and in a disseminated AML
The triplet combination resulted in decreases of tumor patient-derived xenograft (PDX) model (AML004). Of
burden in the bone marrow (0.6±0.3%) beyond any single note, we observed ex vivo activity of all three agents in the
agent treatment (P<0.05; VEN: 79.0±7.8%; PEV: AML003 and AML005 patient cells even though in the
40.8±7.5%; AZA: 69.9±12.6%) or VEN/AZA alone clinic these patients eventually failed VEN/DNMTi treat-
(P<0.05; 8.5±5.8%) (Figure 5D). Staining with anti-hCD45 ment. Considering the heterogeneity that characterizes
antibody revealed lowest AML chimerism in the group AML and the clonal selection that occurs under existing
treated with the triplet combination (Figure 5E). Normal treatment strategies that can eventually contribute to dis-
tissues were unaffected in this experiment, and combina- ease relapse,43,44 we speculate that the breadth of activity
tion of the three agents neither led to any significant observed with VEN/PEV/AZA across AML cell lines and
effects on the weight of the mice (Online Supplementary patient samples harboring differing genetic mutations
Figure S4B), nor exhibited signs of stress. Ex vivo study of may enable more durable responses in AML patients.
the triplet combination did not lead to a significant reduc- An additional feature of the combinatorial activity of
tion in total colony formation in normal human CD34+ the VEN/PEV/AZA treatment was the increased rate of
cells (Online Supplementary Figure S5). caspase-3/-7 activation and the resulting AML cell death
over either agent as a monotherapy or as a combination
doublet. These in vitro observations translated in vivo
Discussion where the VEN/PEV/AZA treatment drove durable anti-
leukemic activity in a systemic model of OCI-AML2, with
Novel drug combinations of VEN with DNMTi or no evidence of tumor growth observed at 93 days post-
LDAC, have led to dramatic improvements in responses treatment cessation. Utilizing a PDX model of AML, we
in patients with AML.20,22 Despite these advances, some demonstrated that the VEN/PEV/AZA combination
patients do not respond or will relapse on this therapy reduced the leukemic burden within the spleen and bone
emphasizing a need to develop novel treatment strate- marrow to a greater extent than VEN, AZA or VEN/AZA
gies. We explored the impact of adding the NAE in combination. In efforts to minimize potential toxicity,
inhibitor, PEV, to the clinically relevant treatment regi- we lowered the doses of each agent used in this study,

D. Cojocari et al.
which if administered alone may be sub-therapeutic. While treatment with VEN/AZA in patients with AML
However, even at these low dosages, the VEN/PEV/AZA has been successful, additional therapies are needed to
treatment proved safe and effective, offering an opportu- prevent or rescue patients from relapse of their disease.
nity to potentially mitigate hematologic toxicity seen with Our studies provide mechanistic insight as to how addi-
azanucleosides or LDAC, and in recent combination trials tion of PEV to VEN/AZA is synergistic and suggest that
with VEN.23,45,46 this triple combination will be a promising treatment
PMAIP1 induction is critical to the activity of AZA and strategy. Clinical trials, including NCT03863257 and
PEV, and their respective synergy with VEN;34,35,39 howev- NCT04172844, are ongoing to assess the safety and effica-
er; the mechanism by which AZA and PEV induce cy of the triplet combination in patients with AML. The
PMAIP1 (NOXA) expression are distinct. PEV inhibition question of whether sequential dosing with PEV/AZA in
of NAE and the subsequent inactivation of CRL, leads to cases of VEN resistance, or in cases of de novo MCL1
an accumulation of the CRL substrates.47 Importantly, dependence, is tenable and effective in the clinic remains,
CRL substrates include the transcription factors MYC and and should be explored in future studies.
ATF4, which transcriptionally induce the expression of
PMAIP1.35,39 Recently, we showed that AZA induced cel- Disclosures
lular stress and ATF4/NOXA through the upstream acti- MRS receives research funding from Astex, Incyte, Takeda,
vation of the ISR pathway and eIF2a phosphorylation.34 TG Therapeutics; has equity in Karyopharm; serves on advisory
Here, we observed that ATF4, and its transcriptional tar- boards and DSMBs for AbbVie, BMS, Celgene, Karyopharm,
gets CHOP and NOXA were all induced by either AZA or Novartis, Ryvu, Sierra Oncology, Taiho, Takeda, TG
PEV, which when combined, enhanced NOXA expres- Therapeutics; and consults for Karyopharm and Ryvu. DC, JP,
sion further in AML cells and was associated with elevat- JH, YX, LJH, ERB and DCP are employees of AbbVie. DC, JP,
ed PARP cleavage and cell death. Deletion of PMAIP1 in YX, EB & DCP are stockholders of AbbVie Inc.
this combination decreased the magnitude of apoptosis,
measured by PARP cleavage, but not the ATF4/CHOP Contributions
induction. This implies that NOXA, and not ATF4 or its DC, BNS, DP and MS designed the study, performed exper-
other targets, is responsible for the ensuing apoptosis. iments and analyzed the data; JP, MA, JH, YZ, AG, LH, HR,
Furthermore, deleting PMAIP1 resulted in significant EB performed experiments and analyzed data; DP and MS
decrease in cell death induced by the VEN/AZA/PEV supervised the study. All authors contributed to the writing and
triple combination, indicating a critical role for NOXA in critical review of the manuscript and agreed on its submission.
the apoptosis-induction mechanism of this combination.
NOXA has its greatest affinity for MCL1 over BCL-XL Funding
and BCL2, and when upregulated, serves to reduce the BNS receives support from the Department of Health and
anti-apoptotic function of MCL1,48,49 subsequently prim- Human Services National Institutes of Health National Cancer
ing cells to venetoclax-mediated apoptosis. When AZA Institute under grant number 5K12CA090625. MRS is a
and PEV are combined, their additive effects on NOXA Leukemia and Lymphoma Society Clinical Scholar and is sup-
induction increases the kinetics and depth of venetoclax- ported by the E.P. Evans Foundation, Adventure Allie Fund, the
mediated caspase-3/-7 activation and cell death that pro- Biff Ruttenberg Foundation, the Beverly and George Rawlings
ceeds in a BAX/BAK-dependent manner. Directorship, and NIHP30 CA068485-19.
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ARTICLE Acute Myeloid Leukemia
Ferrata Storti Foundation Characteristics and outcome of patients with

core-binding factor acute myeloid leukemia
and FLT3-ITD: results from an international col-
laborative study
Sabine Kayser,1,2,3 Michael Kramer,4 David Martínez-Cuadrón,5,6 Justin Grenet,7
Klaus H. Metzeler,1,8 Zuzana Sustkova,9 Marlise R. Luskin,10 Andrew M.
Brunner,11 Michelle A. Elliott,12 Cristina Gil,13 Sandra Casal Marini,14 Zdeněk
Haematologica 2022 Ráčil,9,15 Petr Cetkovsky,16 Jan Novak,15 Alexander E. Perl,7 Uwe Platzbecker,1
Volume 107(4):836-843 Friedrich Stölzel,4 Anthony D. Ho,3 Christian Thiede,4 Richard M. Stone,10
Christoph Röllig,4 Pau Montesinos,5,6 Richard F. Schlenk2,3,17# and Mark J.
Levis18#
1
Medical Clinic and Policlinic I, Hematology and Cellular Therapy, University Hospital
Leipzig, Leipzig, Germany; 2NCT Trial Center, National Center of Tumor Diseases,
German Cancer Research Center (DKFZ), Heidelberg, Germany; 3Department of Internal
Medicine V, Heidelberg University Hospital, Heidelberg, Germany; 4Department of
Medicine I, University Hospital Carl-Gustav-Carus, Dresden, Germany; 5Hematology
Department, Hospital Universitari i Politècnic, La Fe, València, Spain; 6CIBERONC,
Instituto Carlos III, Madrid, Spain; 7Division of Hematology & Oncology, Abramson
Cancer Center, Perelman School of Medicine at the University of Pennsylvania,
Philadelphia, PA, USA; 8Laboratory for Leukemia Diagnostics, Department of Medicine
III, University Hospital, LMU Munich, Munich, Germany; 9Department of Internal
Medicine, Hematology and Oncology, Masaryk University and University Hospital Brno,
Brno, Czech Republic; 10Department of Medical Oncology, Dana-Farber Cancer Institute,
Boston, MA, USA; 11Massachusetts General Hospital, Boston, MA, USA; 12Division of
Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA;
13
Hospital General, Alicante, Spain; 14Department of Clinical Haematology, Centro
Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 15Institute of Hematology and
Blood Transfusion, Prague, Czech Republic; 16Department of Internal Medicine and
Presented in part at the 60th Annual Haematology, 3rd Faculty of Medicine, Charles University and Faculty Hospital Kralovske
Meeting of the American Society of Vinohrady, Prague, Czech Republic; 17Department of Medical Oncology, National Center
for Tumor Diseases (NCT), Heidelberg University Hospital, Heidelberg, Germany and
Hematology in Orlando (FL, USA) 18
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore,
December 8, 2019. MD, USA.
#
RFS and MJL contributed equally as co-senior authors.
Correspondence:
SABINE KAYSER ABSTRACT
s.kayser@dkfz-heidelberg.de
T
he aim of this study was to evaluate the prognostic impact of
Received: February 23, 2021. FLT3-ITD in core-binding factor acute myeloid leukemia (CBF-
Accepted: June 16, 2021. AML) in an international, multicenter survey of 97 patients of
Pre-published: August 5, 2021. whom 52% had t(8;21)(q22;q22) and 48% had
inv(16)(p13q22)/t(16;16)(p13;q22). The median age of the patients was
53 years (range, 19-81). Complete remission after anthracycline-based
https://doi.org/10.3324/haematol.2021.278645 induction (n=86) and non-intensive therapy (n=11) was achieved in
97% and 36% of the patients, respectively. The median follow-up was
©2022 Ferrata Storti Foundation 4.43 years (95% confidence interval [95% CI]: 3.35-7.39 years). The
Material published in Haematologica is covered by copyright.
median survival after intensive and non-intensive treatment was not
All rights are reserved to the Ferrata Storti Foundation. Use of reached and 0.96 years, respectively. Among intensively treated
published material is allowed under the following terms and patients, inv(16) with trisomy 22 (n=11) was associated with a favorable
conditions:
4-year relapse-free survival rate of 80% (95% CI: 59-100%) as com-
Copies of published material are allowed for personal or inter- pared to 38% (95% CI: 27-54%; P=0.02) in all other patients with CBF-
nal use. Sharing published material for non-commercial pur- AML/FLT3-ITD (n=75). Overall, 24 patients underwent allogeneic
hematopoietic cell transplantation (HCT), 12 in first complete remission
sect. 3. Reproducing and sharing published material for com- and 12 after relapse. Allogeneic HCT in first complete remission was
mercial purposes is not allowed without permission in writing not beneficial (P=0.60); however, allogeneic HCT seemed to improve
from the publisher.
median survival in relapsed patients compared to that of patients treat-
ed with chemotherapy (not reached vs. 0.6 years, respectively; P=0.002).
Excluding patients with inv(16) with trisomy 22, our data indicate that

Outcome of CBF-AML with FLT3-ITD
the outcome of CBF-AML patients with FLT3-ITD may be inferior to that of patients without FLT3-ITD
(based on previously published data), suggesting that prognostically CBF-AML patients with FLT3-ITD
should not be classified as favorable-risk. FLT3-inhibitors may improve the outcome of these patients.
Introduction ment-related mortality was significantly higher (hazard

ratio=6.76; P<0.001) than after chemotherapy.19 No bene-
Core binding factor acute myeloid leukemia (CBF- fit regarding RFS and OS was found for allogeneic HCT in
AML) is defined by the presence of either the entire study cohort. Two other studies compared the
t(8;21)(q22;q22)/ RUNX1/RUNX1T1 or inv(16) results of allogeneic versus autologous HCT in CBF-
(p13.1q22)/t(16;16) (p13.1;q22)/ MYH11-CBFB and is rec- AML.20,21 Gorin et al. reported a significantly higher
ognized by the World Health Organization classification relapse risk after autologous HCT than after allogeneic
as a separate entity within the category “AML with recur- HCT in patients with t(8;21) (28% vs. 15%, P=0.03) but
rent genetic abnormalities”.1 Both aberrations result in not in patients with inv(16) AML.20 Again, treatment-
formation of novel chimeric fusions involving genes of related mortality was significantly higher after allogeneic
the CBF complex, a master regulator of definitive HCT than afer autologous HCT in both t(8;21) patients
hematopoiesis.2 CBF-AML is associated with a favorable (24% vs. 6%, P=0.003) and in those with inv(16) (14% vs.
outcome, particularly if treated with repeated cycles of 2%, P=0.003), but the type of transplant did not affect
high-dose cytarabine as post-remission therapy.3-6 The 10- leukemia-free survival.20 A Japanese study also showed
year overall survival (OS) rate was reported to be 58% in comparable results for OS after allogeneic and autologous
FLT3 internal tandem duplication (FLT3-ITD)-negative HCT in CR1 for both t(8;21) and inv(16) AML.21
patients.7 Thus, CBF-AML is categorized into the favor- Nevertheless, the mutational status of FLT3-ITD was not
able-risk group according to the National Comprehensive taken into account in any of these reports.17-21
Cancer Network guidelines,8 as well as the European The prognostic impact of FLT3-ITD in CBF-AML is still
LeukemiaNet recommendations,9 regardless of the FLT3- a matter of debate. The objectives of our study were to
ITD mutational status. Nevertheless, 30-40% of patients characterize CBF-AML patients with FLT3-ITD within an
with CBF-AML experience relapse.3,7,10 international, multicenter cohort study and compare out-
FLT3-ITD mutations occur in roughly 5-10% of adults comes according to treatment strategies, with a specific
with CBF-AML.11 In a murine transplantation model the focus on the impact of allogeneic HCT, as compared to
co-transduction of FLT3-ITD with RUNX1-RUNX1T112 or conventional chemotherapy, on survival.
CBFB-MYH1113 promoted progression to AML, indicating
the cooperative nature of FLT3 aberrations. However, the
prognostic relevance of such aberrations in CBF-AML is Methods
still controversial. In a study of 176 patients with inv(16)
AML, those with FLT3 mutations (n=30) had an inferior Patients and treatment
OS compared to those with wild-type FLT3.14 Of note, in Information on 97 adult patients with CBF-AML diagnosed
the same analysis trisomy 22 was a favorable prognostic between 1996 and 2019 (prior to 2000, n=7; 2000-2010, n=39;
marker irrespective of concomitant FLT3 mutations,14 after 2010, n=51) was collected from eight study groups/institu-
confirming the findings of previous studies,3,10 in which tions in the USA and Europe. Participating centers were chosen
trisomy 22 was associated with a lower cumulative inci- upon network relationships of the first and last authors. Detailed
dence of relapse (CIR) than that in patients with only case report forms (including information on baseline characteris-
inv(16) (estimated long-term CIR rates of 42% and 66%, tics, chemotherapy, allogeneic HCT, response, and survival)
respectively; P = 0.02)3 and an excellent relapse-free sur- were collected from all participating centers. Inclusion criteria
vival (RFS) of 82%.10 The results of two other studies were adult CBF-AML patients with FLT3-ITD and all patients
were also in line with these findings.15,16 who fulfilled these criteria were included by the participating
Currently, it is unclear whether patients with CBF-AML groups/institutions. The diagnosis of AML was based on French-
and KIT or FLT3 mutations may benefit from allogeneic American-British Cooperative Group criteria,22 and, after 2003,
hematopoietic stem cell transplantation (HCT) in first on revised International Working Group criteria.23 Chromosome
complete remission (CR1). A meta-analysis of several banding was performed using standard techniques, and karyo-
prospective trials evaluating the impact of allogeneic types were described according to the International System for
HCT for AML in CR1 did not show a benefit of allogeneic Human Cytogenetic Nomenclature.24 FLT3 mutation screening
HCT on RFS or OS for favorable-risk AML (n=547) as for ITD and point mutations within the tyrosine kinase domain
compared to non-allogeneic HCT therapies including (TKD) was carried out at each institution as previously
post-remission chemotherapy, autologous HCT, or described.25,26 Data collection and analysis were approved by the
both.17 In line with these findings, Burnett et al. reported institutional review boards of the participating centers.
that CBF-AML patients who underwent allogeneic HCT
in CR1 had no survival benefit compared to patients not Treatment
receiving HCT.18 Another retrospective analysis of Eighty-six (89%) of the 97 patients received intensive induc-
younger (<60 years) AML patients with t(8;21) compared tion treatment either within clinical trials (n=30) or according to
the outcomes of 118 patients who received allogeneic local institutional standards (n=56). Treatment protocols includ-
HCT from a matched-related donor with the outcomes of ed the Study Alliance Leukemia AML60+ (n=2),27 AML96
132 patients treated with cytarabine-based chemothera- (n=18)28 and AML2003 (n=9)29 as well as the CALGB/Ratify trial
py.19 After allogeneic HCT, the risk of relapse was signifi- (n=1).30 Induction therapy of the 86 patients consisted of the
cantly lower (hazard ratio=0.47; P=0.014), but the treat- anthracycline/cytarabine based “7+3” regimen (n=62) or compa-

S. Kayser et al.
rable intensive treatment (n=24); additionally, five of the inten- somy 22 (n=11; median WBC 28.8x109/L; range, 3.9-
sively treated patients received midostaurin and four patients 186.7x109/L) than in those without trisomy 22 (n=36;
received gemtuzumab ozogamicin. median WBC 54.8x109/L; range, 2.7-298x109/L; P=0.18).
Eleven (11%) of the 97 patients were treated non-intensively.
Of these, five received azacitidine, either alone (n=2) or in com- Cytogenetic and molecular analyses
bination with venetoclax (n=2) or sorafenib (n=1); four patients The balanced translocation t(8;21)(q22;q22) was pre-
received fludarabine and low-dose cytarabine, one patient was sent in 50 (52%) of the 97 patients. It occurred as a sole
treated with tipifarnib and etoposide within a clinical trial and abnormality in 15 (30%) patients, while additional cyto-
one patient was treated with hydroxyurea only. genetic abnormalities were present in 35 (70%) patients,
Response was assessed according to International Working most frequently loss of a sex chromosome (n=26; loss of
Group recommendations.23 All clinical studies were approved by X or Y n=13, each), three or more abnormalities (n=10)
the institutional review boards of the participating centers. All and deletion of the long arm of chromosome 9 (del(9q),
patients provided written informed consent to participation in n=6). Of the del(9q) cases, all but one co-occurred within
one of the treatment trials or to therapy according to local stan- a karyotype with three or more abnormalities.
dards. An inv(16)(p13q22) (n=46) or t(16;16)(p13;q22) (n=1)
was detected in 47 (48%) patients. It was the sole abnor-
Statistical analyses mality in 25 (53%) patients, while concurrent cytogenetic
Survival endpoints including OS, RFS, CIR and cumulative abnormalities were present in the other 22 (47%) patients,
incidence of death in CR (CID) were defined according to the most frequently trisomy 22 (n=11), three or more abnor-
revised recommendations of the International Working Group.23 malities (n=7), trisomy 8 (n=8; all except one within a
Patients’ characteristics were compared with the Kruskal-Wallis karyotype with ≥3 abnormalities) as well as monosomy 7
rank sum test for continuous variables and Fisher exact test for or deletion of the long arm of chromosome 7 (del(7q); n=4;
categorical variables. The median follow-up time was computed all within a karyotype with ≥3 abnormalities).
using the reverse Kaplan-Meier estimate.31 The Kaplan-Meier
method was used to estimate the distribution of RFS and OS.32
Confidence interval (CI) estimates for survival curves were
Table 1. Baseline characteristics of patients with acute myeloid
based on the cumulative hazard function using the Greenwood leukemia and core-binding factor leukemia.
formula for variance estimation. Log-rank tests were employed
to compare survival curves between groups. A Cox proportional All patients Inv(16) t(8;21) P-value
hazards regression model was used to identify prognostic vari-
(n=97) (n=47) (n=50)
ables for RFS.33 CIR and CID and their standard errors were com- Median age, years 53 50 53.5 0.95
puted according to the method described by Gray34 and included (range) (19-81) (19-81) (22-77)
only patients attaining CR. The effect of allogeneic HCT on OS Female, n. (%) 45 (46) 28 (56) 17(36) 0.07
as a time-dependent intervening event was tested using the
Median WBC, 109/L 20.5 43.4 14.3 <0.001
Mantel-Byar method.35 The method of Simon and Makuch was (range) (1.8-298) (2.7-298) (1.8-153.4)
applied to estimate survival distributions with respect to time- Missing 2 - 2
dependent interventions.36 The individuals at risk were initially
Median Hb, g/dL 8.6 8.7 8.4 0.1
all represented in the chemotherapy group. If patients received
(range) (4.6-14.3) (5.6-14.3) (4.6-12.0)
an allogeneic HCT, they were censored at this time point in the
Missing 5 2 3
chemotherapy group and further followed up within the allo-
geneic HCT group. Median platelets, 109/L 33 33 33 0.92
(range) (7-372) (7-261) (7-373)
All statistical analyses were performed with the statistical
Missing 5 2 3
software environment R, version 3.3.1, using the R packages
prodlim, version 1.5.7, and survival, version 2.39-5.37 Median BM blasts, % 60 61 58 0.49
(range) (0-98) (0-98) (17-96)
Missing 8 2 6
Results Cytogenetics, n. (%)
CBF as sole abn 41 (42) 26 (55) 15 (30) 0.01
Study cohort CBF + other abn 56 (58) 21 (45) 35 (70)
Demographic and clinical data were collected from 97 Trisomy 22 12 (12) 11 (23) 1 (2) 0.002
patients (Study Alliance Leukemia, n=46; Spanish Trisomy 8 7 (7) 5 (11) 2 (4) 0.26
Programa Español de Tratamientos en Hematología Disease type, n. (%)
[PETHEMA], n=20; Johns Hopkins University, Baltimore, De novo AML 87 (90) 42 (89) 45 (90)
n=8; Perelman School of Medicine at the University of Secondary AML 2 (2) 1 (2) 1 (2) 0.99
Pennsylvania, n=6; University of Munich, n=6; Czech Therapy-related AML 8 (8) 4 (9) 4 (8)
Leukemia Centers, n=6; Dana-Faber Cancer Institute and Median FLT3-ITD
Massachusetts General Hospital, Boston, n=3; and Mayo allelic ratio 0.35 0.32 0.35
Clinic Rochester, n=2) diagnosed with CBF-AML (range) 0.003-50 0.003-50 0.005-34 0.99
between 1996 and 2019. The median age of the patients Missing 20 9 11
was 53 years (range, 19-81 years) and 45 patients (46%) FLT3-TKD
were female. The patients’ baseline characteristics are N. (%) 10 (21) 8 (29) 2 (10) 0.16
summarized in Table 1. Median white blood cell (WBC): Missing 49 19 30
count was higher in patients with inv(16)/t(16;16) than in abn: aberration; allo: allogeneic; AML: acute myeloid leukemia; BM: bone marrow; CBF:
core-binding factor; FLT3: fms-related tyrosine kinase 3; Hb: hemoglobin; TKD: tyrosine
patients with t(8;21). In addition, median WBC count in kinase domain; WBC: white blood cell count. Results may not add-up to 100 due to
patients with inv(16)/t(16;16) was lower in those with tri- rounding.

The FLT3-ITD allelic ratio was available in 77 (79%) and there was one treatment-related death. In those
patients and the median allelic ratio was 0.35 (range, relapsing after chemotherapy, allogeneic HCT was per-
0.003-50). Median WBC count was higher in patients formed in 12 patients: eight in second CR (CR2) and four
with a high allelic ratio than in those with a low allelic with active disease.
ratio (31.7x109/L vs. 16x109/L, P=0.02). FLT3 mutational status was available in 17 (50%) of 34
The median FLT3-ITD size and number of ITD clones relapsed patients who received intensive treatment. Of
were available for 29 (30%) patients. The median FLT3- these, eight (47%) were still FLT3-ITD-positive.
ITD size was 39 (range, 3-120) base-pairs and most of the Interestingly, one of the ITD-positive patients developed
patients harbored one clone (1 clone, n=24; 2 clones, n=4, a new FLT3-TKD mutation.
3 clones, n=1). Besides the FLT3-ITD, ten (21%) of 48
patients with available data also harbored a FLT3-TKD Characteristics of patients undergoing allogeneic
(Table 1). hematopoietic cell transplantation
Overall, an allogeneic HCT was performed in 24 (25%)
Response to induction therapy of the 97 patients, either in CR1 (n=12; inv(16), n=4;
Data on response to induction therapy were available t(8;21), n=8) or CR2 (n=8; inv(16), n=5; t(8;21), n=3), or
for all 97 patients. Of the intensively treated patients with active disease (n=4; inv(16), n=2; t(8;21), n=2).
(n=86), CR after induction therapy was achieved in 84 Thirteen patients received myeloablative conditioning,
(98%), including one patient who achieved CR after sal- including total body irradiation in eight patients; addi-
vage therapy with 1 g cytarabine every 12 h on 4 days as tionally eight patients received reduced-intensity condi-
well as mitoxantrone 12 mg/day on 3 days. Early death tioning (missing, n=3). The stem cell source was a
occurred in two (2%) patients; none of the intensively matched related donor in 11 cases, a matched unrelated
treated patients had refractory disease. All patients, who donor in ten cases, a haplo-identical donor in two, and
received “7+3” treatment, either with midostaurin (n=5) unknown in one of the 24 patients.
or gemtuzumab ozogamicin (n=4), achieved CR.
Eleven patients were treated less intensively because of Cumulative incidences of relapse and death in
their older age (median: 72 years; range, 40-81 years) or complete remission, and survival
comorbidities. Of these 11 less intensively treated The median follow-up of the entire cohort was 4.43
patients, four (36%) achieved CR, one had a partial remis- years (95% CI: 3.35-7.39 years). The median and 4-year
sion, four were refractory and two patients died early. OS of the entire cohort were 4.48 years (95% CI: 2.48-not
The four patients who achieved CR had been treated reached) and 51% (95% CI: 41-64%), respectively.
with a hypomethylating agent (n=1), venetoclax + azaci- In intensively treated patients RFS and OS were not dif-
tidine (n=1) and fludarabine + low-dose cytarabine (n=2). ferent between patients with inv(16) and those with
t(8;21) (P=0.70 and P=0.80, respectively) (Figure 1).
Further therapy including intensive consolidation and Furthermore, CIR (P=0.26) (Figure 2A) and CID (P=0.96)
allogeneic hematopoietic cell transplantation (Figure 2B) were comparable in patients proceeding to
Seventy-two (86%) of 84 intensively treated patients in allogeneic HCT in CR1 or not. However, in relapsed
CR1 received intensive chemotherapy consolidation con- patients survival was dismal without allogeneic HCT
sisting of high-dose cytarabine with or without addition- (n=22) irrespective of CBF-AML type, with a median sur-
al chemotherapy (mitoxantrone and/or amsacrine, n=25). vival of 0.6 years after relapse (95% CI: 0.31-1.11 years),
Precise information on applied consolidation cycles was and none of the patients survived beyond 2 years. In con-
available for 54 patients. Of those, ten patients received trast, in relapsed patients proceeding to allogeneic HCT
four cycles of consolidation, 14 patients received three either in CR2 or with active disease the median survival
cycles, seven received two cycles and 23 patients received was not reached and survival at 4 years was 53% (95%
one cycle. For analysis, we compared patients who CI: 30-94% Figure 3). In a Mantel-Byar analysis including
received one or two consolidation cycles with those who allogeneic HCT performed after relapse as a time-depen-
received more than two cycles of consolidation. There dent event, survival after relapse was significantly
was no difference in CIR between patients who received improved by allogeneic HCT (P=0.002).
one or two cycles and those who received more than two Since supportive care might have had an influence on
cycles (P=0.97). One of the transplanted patients received outcome, we performed a Cox regression analysis. This
maintenance with gilteritinib for 2 years after allogeneic analysis revealed no impact of date of diagnosis either as
HCT within a randomized trial. In addition, one patient a continuous variable (P=0.92) or as a dichotomized vari-
with intensive chemotherapy consolidation received able (on the year 2010; P=0.23).
maintenance with midostaurin. The median survival of non-intensively treated patients
Twelve (14%) patients proceeded to allogeneic HCT in was 0.96 years (95% CI: 0.24-not reached) and none of
CR1 with five of the transplanted patients receiving some these patients survived beyond 3 years.
consolidation chemotherapy prior to their transplant. Exploratory subset analysis revealed trisomy 22 in
There was no difference in baseline characteristics, such patients with inv(16) as a significant prognostic factor for
as median WBC count, median age and median FLT3-ITD RFS (n=11; P=0.02) (Figure 4A); the outcome of these
allelic ratio, between patients proceeding to allogeneic patients was favorable with a 4-year RFS rate of 80% (95%
HCT in CR1 and those given consolidation chemothera- CI: 59-100%), whereas all other CBF patients had a high
py, (data not shown). relapse rate resulting in a 4-year RFS rate of 38% (95% CI:
Among the patients consolidated with chemotherapy, 27-54%, P=0.02) (Figure 4A). In addition, OS tended to be
relapses occurred in 31 and there were six treatment- higher in patients with inv(16) and trisomy 22 (P=0.10)
related deaths after consolidation. In patients consolidat- than in those with all other CBF-AML (Figure 4B).
ed with allogeneic HCT in CR1 three patients relapsed Other relevant prognostic factors, such as type of CBF-

S. Kayser et al.
AML, older age (≥60 years), WBC count, platelet count, ment strategies, with a specific focus on the impact of allo-
trisomy 8, complex karyotype, and high FLT3-ITD allelic geneic HCT, as compared to conventional chemotherapy,
ratio (≥0.5) were not identified as significant variables on survival.
either for RFS or OS (Table 2). In addition, loss of the Y Secondary chromosome aberrations can be detected in
chromosome in patients with t(8;21) had no impact on more than 60% of AML cases with t(8;21) and in 35% to
outcome (RFS, P=0.7; OS, P=0.3). Trisomy 22 was the 40% of those with inv(16). In line with published data,10,38
only variable with a significant effect on the RFS endpoint the most frequent secondary chromosome aberration in our
(hazard ratio=0.22; P=0.04) (Table 2). cohort of t(8;21) AML patients was loss of a sex chromo-
some, whereas the most frequent secondary chromosome
aberration in inv(16) AML was trisomy 22. In addition, we
Discussion found a higher WBC count in patients with inv(16) than in
those with t(8;21).
The focus of our study was to characterize adult CBF- In contrast to previous reports there was no impact of
AML patients with FLT3-ITD in an international, multicen- WBC count, older age (≥60 years) or loss of the sex chromo-
ter cohort study and compare outcomes according to treat- some10,19,39 on outcome.
A B
Figure 1. Kaplan-Meier plots of survival in intensively treated patients according to type of core-binding factor acute myeloid leukemia. (A) Relapse-free survival.
(B) Overall survival.
A B
Figure 2. Plots of cumulative incidence of relapse and cumulative incidence of death according to treatment strategy in first complete remission. (A) Cumulative
incidence of relapse. (B) Cumulative incidence of death. Only patients attaining complete remission are included. Treatment strategy is divided into consolidation
chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HCT) in first complete remission (CR1).

In our cohort, remission rate after intensive treatment ITD-positive patients as compared to 58% after 10 years in
was very high, as was reported in CBF-AML without FLT3- those with wild-type FLT3.7 In addition, the outcome of
ITD,3,10 suggesting that CBF-AML is highly chemosensitive intensively treated patients was not affected by CBF sub-
regardless of a concurrent FLT3-ITD. We confirmed the type, inv(16) and t(8;21), or CR1 consolidation approach
excellent prognosis of patients with inv(16) and trisomy (chemotherapy or transplantation). These results might
22,3,10,14 despite the additional presence of a FLT3-ITD. To argue for the benefit of repeated cycles of intensive
date, it is unclear why patients with an inv(16) and trisomy chemotherapy as post-remission treatment, i.e., high-dose
22 so rarely relapse after intensive induction and consolida- cytarabine in this subgroup of patients, although we would
tion therapy. Obviously, leukemic clones harboring both like to emphasize that this needs to be validated in a larger
abnormalities are very chemosensitive. Our study adds to cohort.
previous knowledge that despite the proliferative signal Biologically, a FLT3-ITD in CBF-AML seems to impair
induced by a FLT3-ITD40 and the chemoresistance induced the favorable prognosis, comparable to its negative impact
by high FLT3-ITD allelic ratios41 patients with inv(16) and in acute promyelocytic leukemia, at least in those patients
trisomy 22 remain extremely chemosensitive. The underly- not treated with all-trans retinoic acid and arsenic
ing pathogenetic mechanism by which trisomy 22 exerts its trioxide.42,43 Despite the limitation that data on measurable
prognostic impact remains elusive. residual disease were not available in our cohort, outcome
Regarding the outcome of intensively treated CBF was inferior compared to published data in FLT3-ITD-neg-
patients exhibiting a FLT3-ITD without trisomy 22, results ative patients.7 Thus, the FLT3 mutational status should be
were dismal with a RFS rate of 38% after 4 years. The taken into account when classifying CBF-AML; patients
relapse rate in these patients was high and confirmed find- with FLT3-ITD should not be classified within the favor-
ings from previous studies.3,10,14 In comparison, an OS rate of
58% after 10 years was reported in FLT3-ITD-negative
patients.7 In our cohort OS was 51% after 4 years in FLT3- A
Figure 3. Simon Makuch plot of overall survival measured from the date of
relapse in relapsed patients illustrating the impact of allogeneic hematopoietic
stem cell transplantation as a time-dependent event. Allo-HCT: allogeneic
hematopoietic stem cell transplantation.
Table 2. Univariable Cox models on relapse-free and overall survival.

RFS OS
HR P-value HR P-value
Type of CBF-AML 0.89 0.72 0.91 0.80
Older age (≥60 years) 0.61 0.14 0.63 0.20
Log (WBC)
10 0.42 0.22 1.43 0.34
Platelets 1.00 0.54 1.00 0.50
Complex karyotype 0.68 0.32 0.96 0.91
High FLT3-ITD allelic ratio (≥0.5) 1.19 0.63 1.83 0.13
Trisomy 8 0.94 0.92 1.53 0.48
Trisomy 22 0.22 0.04 0.35 0.15 Figure 4. Kaplan-Meier plots of survival of patients with inversion 16 and tri-
somy 22 as compared to all other core-binding factor acute myeloid leukemia
AML: acute myeloid leukemia; CBF: core-binding factor; FLT3: fms-related tyrosine patients. (A) Relapse-free survival. (B) Overall survival. CBF-AML: core-binding
kinase 3; HR: hazard ratio; ITD: internal tandem duplication; OS: overall survival; RFS: factor acute myeloid leukemia.
relapse-free survival, WBC: white blood cell count..

S. Kayser et al.
able-risk category.8,9 Rather, these patients might be candi- should not be classified within the favorable-risk catego-
dates for targeted treatment with tyrosine kinase ry. Our data suggest that allogeneic HCT should be the
inhibitors as well as intensive chemotherapy.30,44 In addi- preferred approach in relapsed patients. CBF-AML with
tion, there is evidence that gemtuzumab ozogamicin in FLT3-ITD represents a further therapeutic target for tyro-
combination with chemotherapy particularly benefits sine kinase inhibitors as well as gemtuzumab ozogamicin
patients with FLT3-ITD mutations45 as well as patients and should be included in combined FLT3-
with CBF-AML.46 However, in our cohort only a few inhibitor/CD33-antibody trials (ClinicalTrials.gov
patients were treated with either midostaurin or gem- Identifier: NCT04385290).
tuzumab ozogamicin, so the effect of these drugs on out-
come could not be evaluated. The impact of midostaurin Disclosures
in combination with gemtuzumab ozogamicin on out- No conflicts of interest to disclose.
come is currently being evaluated within a phase I/II trial
(ClinicalTrials.gov Identifier: NCT04385290). Contributions
The survival of relapsed patients was dismal without SK and RFS were responsible for the concept of this paper, con-
allogeneic HCT, irrespective of CBF-AML type, with a tributed to the literature search data collection, analyzed and inter-
median survival of 0.6 years after relapse and none of the preted data, and wrote the manuscript. MJL was responsible for
patients survived beyond 2 years. In contrast, in patients the concept of this paper, contributed to the literature search data
proceeding to allogeneic HCT after relapse, either in CR2 or collection, contributed patients, analyzed and interpreted data, and
with active disease, the median survival had not been critically revised the manuscript. CTh performed research and crit-
reached and survival at 4 years was 53%, arguing that allo- ically revised the manuscript. MK, DM-C, JG, KHM, ZS, MRL,
geneic HCT should be the preferred approach in relapsed AMB, MAE, CG, SCM, ZR, PC, JN, AEP, FS, ADH, UP,
patients.3,10,21 However, we would like to emphasize that RMS, CR, and PM contributed patients and critically revised the
retrospectively collected data have serious limitations since manuscript. All authors reviewed and approved the final
the factors for allocating patients to allogeneic HCT, such as manuscript.
co-morbidities, individual assessment of the treating physi-
cian, choice of conditioning, and availability of a donor, Funding
remain unknown and these factors need to be taken into SK was supported by the Olympia-Morata fellowship program
account when evaluating the value of allogeneic HCT in from the Medical Faculty of the Heidelberg University. MJL is sup-
our series. ported by a grant from the NCI (NCI Leukemia SPORE P50
In conclusion, despite a high remission rate patients CA100632). ZS, ZR, PC and JN were supported by the Ministry
with FLT3-ITD had an inferior outcome compared to of the Czech Republic, grant n. 15-25809A. The authors also
those without FLT3-ITD, based on previously published acknowledge support from Leipzig University for Open Access
data on CBF-AML. Thus, CBF-AML with FLT3-ITD Publishing.
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ARTICLE Bone Marrow Transplantation
Ferrata Storti Foundation Refined HLA-DPB1 mismatch with molecular

algorithms predicts outcomes in hematopoietic
stem cell transplantation
Jun Zou,1* Piyanuch Kongtim,2* Betül Oran,3 Vasilis Kosmoliaptsis,4 Yudith
Carmazzi,1 Junsheng Ma,5 Liang Li,5 Gabriela Rondon,3 Samer Srour,3 Hannah C.
Copley,4 David Partlow,1 Stefan O. Ciurea,2 Uri Greenbaum,3 Qing Ma,3 Elizabeth
J. Shpall,3 Richard E. Champlin3# and Kai Cao1#
Haematologica 2022 1
Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center,
Volume 107(4):844-856 Houston, TX, USA; 2Division of Hematology/Oncology, Department of Medicine, Chao
Family Comprehensive Cancer Center, University of California, Irvine, CA, USA;
3
Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas
MD Anderson Cancer Center, Houston, TX, USA; 4Department of Surgery, University of
Cambridge, and NIHR Cambridge Biomedical Research Centre, Cambridge, UK and
5
Department of Biostatistics, The University of Texas MD Anderson Cancer Center,
Houston, TX, USA
*JZ and PK contributed equally as co-first authors.
REC and KC contributed equally as co-senior authors.
#
ABSTRACT
H
LA-DPB1 mismatches between donor and recipient are common-
ly seen in allogeneic hematopoietic stem cell transplantation
from an unrelated donor. HLA-DPB1 mismatch, conventionally
determined by the similarity of the T-cell epitope (TCE), is associated
with an increased risk of acute graft-versus-host disease (GVHD) and a
decreased risk of disease relapse. We investigated the clinical impact of
HLA-DPB1 molecular mismatch quantified by mismatched eplets (ME)
and the Predicted Indirectly Recognizable HLA Epitopes Score (PS) in a
cohort of 1,514 patients receiving hematopoietic stem cell transplants
from unrelated donors matched at HLA-A, -B, -C, -DRB1/3/4/5, and -
DQB1 loci. HLA-DPB1 alloimmunity in the graft-versus-host direction,
determined by high graft-versus-host ME/PS, was associated with a
Correspondence: reduced risk of relapse (hazard ratio [HR]=0.83, P=0.05 for ME) and
increased risk of grade 2-4 acute GVHD (HR=1.44, P<0.001 for ME),
JUN ZOU
jzou@mdanderson.org whereas high host-versus-graft ME/PS was only associated with an
increased risk of grade 2-4 acute GVHD (HR=1.26, P=0.004 for ME).
Notably, in the permissive mismatch subgroup classified by TCE group-
Received: April 14, 2021. ing, high host-versus-graft ME/PS was associated with an increased risk
Accepted: July 9, 2021. of relapse (HR=1.36, P=0.026 for ME) and grade 2-4 acute GVHD
Pre-published: August 26, 2021. (HR=1.43, P=0.003 for PS-II). Decision curve analysis showed that graft-
versus-host ME outperformed other models and provided the best clinical
net benefit for the modification of acute GVHD prophylaxis regimens in
https://doi.org/10.3324/haematol.2021.278993 patients with a high risk of developing clinically significant acute GVHD.
In conclusion, molecular assessment of HLA-DPB1 mismatch enables
©2022 Ferrata Storti Foundation separate prediction of host-versus-graft or graft-versus-host alloresponse
quantitatively and allows further refinement of HLA-DPB1 permissive-
All rights are reserved to the Ferrata Storti Foundation. Use of ness as defined by conventional TCE grouping.
conditions:
Copies of published material are allowed for personal or inter- Introduction
Currently, allogeneic hematopoietic stem cell transplantation (HSCT) is the only
sect. 3. Reproducing and sharing published material for com- curative therapy for many hematologic malignancies. Although modern immuno-
mercial purposes is not allowed without permission in writing suppressive therapy and transplant interventions have significantly improved
from the publisher. non-relapse mortality (NRM) over years,1 as a major complication after HSCT,
acute graft-versus-host disease (GVHD) occurs in 20 to 80% of recipients with
15% mortality.2 It is well established that patients who undergo allogeneic HSCT

Refined HLA-DPB1 mismatch with molecular algorithms
from an HLA-mismatched unrelated donor are more like- through the indirect recognition pathway, Predicted
ly to have a higher incidence of acute GVHD and subop- Indirectly Recognizable HLA Epitopes (PIRCHE), with
timal clinical outcomes.3-5 Among patients who have PIRCHE score (PS)-I representing CD8+ T-cell alloreactiv-
received HLA-A, -B, -C, -DRB1, and -DQB1 matched ity and PS-II representing CD4+ T-cell alloreactivity, is
(10/10) grafts from unrelated donors, the disparity widely and successfully used for this purpose.25
between the donor and recipient at the HLA-DPB1 locus In the present study, we sought to comprehensively
is associated with an increased risk of GVHD but is coun- validate the molecular mismatch algorithms in predicting
terbalanced by a reduced risk of relapse.6,7 the risks associated with HLA-DPB1 mismatches in a rel-
Given the weak linkage disequilibrium between the DP atively large cohort of patients with malignant disease
locus and DR/DQ loci, mismatching at the HLA-DPB1 who underwent HSCT from unrelated donors.
locus is observed in about 75-90% of transplants from Furthermore, we hypothesized that in silico quantification
unrelated donors regardless of matching at other HLA could refine the current definition of TCE grouping, espe-
loci.7-10 Pioneering studies have classified HLA-DPB1 mis- cially in the permissive or nonpermissive mismatch sub-
matches as permissive or nonpermissive using the func- groups, given that significantly different T-cell cross-reac-
tional toxicity assay and by analyzing the similarity of T- tivities are seen in various HLA-DPB1 alleles within the
cell epitopes (TCE).11,12 The initial experimental hypothe- same subgroup.11
sis has been confirmed clinically and translated into a
donor selection algorithm; permissive HLA-DPB1 mis-
matches are associated with elicited alloreactivity result- Methods
ing in a beneficial graft-versus-leukemia (GVL) effect with
clinically tolerable GVHD.13,14 This approach has signifi- Patients and transplant characteristics
cantly expanded the likelihood of finding suitable unrelat- Our cohort included consecutively treated patients with
ed donors and reduced the risks of mortality by avoiding hematologic malignancies who were 18 years of age or older
donors with nonpermissive mismatches.7,15,16 Although and underwent allogeneic HSCT at The University of Texas MD
the TCE model assigns permissiveness based on T-cell Anderson Cancer Center (UTMDACC) between June 2005 and
alloreactivity within the same or from different immuno- December 2018. All patients in our analysis received HSCT from
genicity groups,11 another partially overlapping model an HLA-A, -B, -C, DRB1, -DQB1, -DRB3/4/5 matched unrelated
predicts HLA-DPB1 immunogenicity with similar success donor to minimize the confounding alloreactivity caused by
by analyzing expression levels of the specific HLA-DPB1 HLA mismatch from other loci. Clinical and laboratory data
allele.17,18 were collected from electronic medical records.
Modern HLA molecular matching methods may open All patients provided written informed consent for HSCT in
new avenues for alloimmune risk assessment and help to accordance with the Declaration of Helsinki. A retrospective
quantitatively refine the traditional TCE grouping. data review protocol and a waiver of informed consent were
Additionally, the different direction of HLA-DPB1 non- approved by the UTMDACC Institutional Review Board.
permissive mismatches defined by the TCE model, i.e.,
either in the host-versus-graft (HVG) or graft-versus-host HLA typing and ME and PS analyses
(GVH) direction, appears to have a similar impact on the Patients included in the study had donor and recipient HLA
risk of GVHD and mortality in HSCT from unrelated typing performed at the HLA-A, -B, -C, DRB1, -DRB3/4/5, -
donors.7,8,16 Although the underlying mechanism of non- DQB1, and -DPB1 loci using sequence-based typing methods at
permissive mismatch in the HVG direction remains high resolution.26 ME load at the HLA-DPB1 locus was measured
unclear, recent compelling evidence showed that periph- using the HLAMatchmaker module incorporated in HLA Fusion
eral host T cells present in the skin and gut are primed by software v4.3, which identifies theoretically predicted eplets
donor-derived antigen-presenting cells and contribute to based on crystallized HLA molecule models27 and identifies ME
the development of GVHD.19-21 Computational prediction by comparing donor and recipient eplets. The analyses were per-
methods could separately assess immunogenicity from a formed separately in both the GVH and HVG directions.22 Eplet
donor’s or recipient’s perspective in a quantitative man- repertoires are listed in the HLA Epitope Registry
ner, which might shed light on the alloreactive mecha- (http://www.epitopes.net/downloads.html). The PS for mismatched
nisms that mediate GVHD risk and the GVL effect in HLA-DPB1 in the GVH direction was calculated using the HSCT
HSCT from HLA-DPB1 mismatched donors. module from the PIRCHE online matching service
HLAMatchmaker, one of the best-studied molecular (http://www.pirche.com/pirche/#/). The PS for mismatched HLA-
matching strategies, compares eplets, which are the key DPB1 in the HVG direction was calculated by inverting the
structural component of epitopes, between the donor and patient and donor in the input fields using the same HSCT mod-
recipient. The amount of mismatched eplets (ME) ule.
between donor and recipient has been shown to correlate
with the level of immune response and is associated with HLA-DPB1 permissiveness defined by the TCE model
clinical outcomes in patients who have undergone hap- HLA-DPB1 mismatches between the donor and the recipient
loidentical HSCT.22 As HLAMatchmaker focuses mainly were classified into permissive and nonpermissive mismatches
on surface-accessible positions, TCE that are derived according to TCE algorithms (version 2.0) on the IPD-IMGT/HLA
from polymorphisms on the non-exposed region of HLA website (https://www.ebi.ac.uk/ipd/imgt/hla/dpb.html).28 As previous-
molecules could be overlooked.23,24 Alloreactivity in trans- ly described,26 the direction of HLA-DPB1 mismatch, either in the
plantation is critically dependent on T-cell responses via GVH or HVG direction, was assigned. Transplants were therefore
the indirect recognition pathway in which polymorphic classified into four groups: (i) HLA-DPB1 matched, (ii) permissive
HLA-derived peptides are presented to T cells. Although mismatched, (iii) nonpermissive mismatched in the HVG direc-
various approaches have been described to predict TCE tion, and (iv) nonpermissive mismatched in the GVH direction.

J. Zou et al.
Statistical analysis permissive mismatch group. Likewise, the median ME,

The primary outcome was acute GVHD and secondary out- PS-I, and PS-II values in the HVG direction were consid-
comes were overall survival, progression-free survival, relapse, erably higher in the HVG nonpermissive mismatch group
NRM, and neutrophil engraftment. than in the GVH nonpermissive mismatch and permissive
Univariate and multivariable Cox proportional hazards regres- mismatch groups.
sion was used to determine the impact of baseline characteris- No or weakly positive correlations were seen between
tics, PS, ME, and HLA-DPB1 matching on survival outcomes, GVH and HVG ME, PS-I, and PS-II values, indicating that
while univariate and multivariable sub-distributional hazards ME/PS from the donor perspective were different from
regression was used to analyze cumulative incidence outcomes, ME/PS from the recipient perspective, whereas positive
including relapse, NRM, acute GVHD, and engraftment. All correlations were observed between PS-I and PS-II values
regression models were tested for proportional hazards assump- and between ME and PS values in the same direction
tion and interaction terms. Each PS, ME, and HLA-DPB1 match (GVH or HVG) (Online Supplementary Figure S1). The
group with a P value <0.1 in the univariate analysis was ana- number of patients in the low and high PS and ME groups
lyzed in separate multivariable regression models adjusted for and TCE model are summarized in Online Supplementary
significant baseline characteristics. PS and ME were analyzed as Tables S1 and S2.
both continuous variables and categorical variables (low versus
high), and they were analyzed only as categorical variables in Impact of HLA-DPB1 matching status defined by TCE,
multivariable analyses. To determine the optimal cutoff for low ME, and PS on post-transplant outcomes
versus high PS and ME groups, the concordance probabilities of In the entire cohort, molecular mismatches in the GVH direction
PS and ME for acute GVHD prediction were tested at the 25th, were associated with a reduced risk of relapse and increased risk of
50th, and 75th percentile cutoffs. The cutoffs at the 50th percentile GVHD and NRM, whereas mismatch in the HVG direction was asso-
were selected for the analysis to maximize the concordance ciated only with increased risk of GVHD without relapse protection.
probability. Results from multivariable analyses showed that HLA-
The discrimination power of the TCE, ME, and PS models on DPB1 mismatches by TCE grouping, ME, PS-I, and PS-II
acute GVHD was compared using the Harrell C-concordance in both the GVH and HVG directions were strongly asso-
index. A decision-curve analysis29,30 was performed to assess the ciated with an increased risk of clinically significant acute
net clinical benefit of all models in deciding on GVHD regimen GVHD after adjustment for significant baseline character-
modification. istics (Figure 1A). Using conventional TCE grouping,
Outcome definitions and details of the statistical analysis are compared with the HLA-DPB1 matched group, those
described in the Online Supplementary Material. with permissive mismatch, GVH nonpermissive mis-
match, and HVG nonpermissive mismatch had an
increased risk of grade 2-4 acute GVHD (permissive: haz-
Results ard ratio [HR]=1.42, 95% confidence interval [95% CI]:
1.15-1.76, P=0.001; GVH nonpermissive: HR=1.99, 95%
Patients’ characteristics and HLA-DPB1 matching CI: 1.55-2.55, P<0.001; HVG nonpermissive: HR=1.80,
status defined by TCE and in silico methods 95% CI: 1.38-2.35, P<0.001).
The analysis included 1,514 patients with a median age Using the median cutoff of ME, the risk of grade 2-4
of 56 years (range, 18-79). The characteristics of the acute GVHD was 1.44 (95% CI: 1.23-1.68, P<0.001) and
patients and their transplants are listed in Table 1. The 1.26 (95% CI: 1.08-1.48, P=0.004) times higher in those
majority of patients received a peripheral blood graft with high ME in the GVH and HVG direction, respective-
(62%) and GVHD prophylaxis with tacrolimus and ly, than in those with low ME in the same direction.
mycophenolate (83%). Seventy-four percent of patients Similarly, having a high PS in the GVH direction was
received anti-thymocyte globulin as a part of GVHD pro- associated with an increased risk of grade 2-4 acute
phylaxis. The variables that were significantly different GVHD (PS-I: HR=1.39, 95% CI: 1.19-1.63, P<0.001; PS-II:
between subgroups were bone marrow stem cell source HR=1.40, 95% CI: 1.19-1.64, P<0.001). Having a high PS
(with 29% in the GVH nonpermissive group versus 37.6% in the HVG direction was also associated with an
in the whole group) and the year of HSCT. The number increased risk of grade 2-4 acute GVHD (PS-I: HR=1.32,
of transplants with nonpermissive mismatch was signifi- 95% CI: 1.12-1.54, P=0.001; PS-II: HR=1.24, 95% CI:
cantly reduced in recent years (2014-2018) compared to 1.05-1.45, P=0.009).
the previous years (27.6% versus 36.5%, respectively), The associations of ME, PS-I, and PS-II in the GVH
likely due to the awareness of the adverse effect of non- direction with grade 2-4 acute GVHD risk were indepen-
permissive mismatch. dent of the associations of ME, PS-I, and PS-II in the HVG
HLA-DPB1 permissive mismatch was present in 43.0% direction with grade 2-4 acute GVHD. However, higher
of patients, and nonpermissive HLA-DPB1 mismatches in risks of grade 2-4 acute GVHD were seen in patients who
the GVH and HVG directions were noted in 17.7% and had high ME, PS-I, or PS-II in both the GVH and HVG
15.1% of patients, respectively. The median follow-up directions than in those with low ME, PS-I, or PS-II in
duration in 695 surviving patients was 57.1 months both directions (Figure 1A, Online Supplementary Figure
(range, 3.4-148.4). S2A).
ME, PS-I, and PS-II were quantified in both HVG and For NRM, HLA-DPB1 nonpermissive mismatch in
GVH directions (Table 1). High concordances between either the GVH direction (HR=1.67, 95% CI: 1.24-2.27,
the functional TCE grouping and in silico methods were P=0.001) or HVG direction (HR=1.46, 95% CI: 1.05-2.03,
noted. The median ME, PS-I, and PS-II values in the GVH P=0.025) was associated with a significantly increased
direction in the GVH nonpermissive mismatch group risk of NRM compared with that in the matched group,
were significantly higher than the corresponding values whereas no association was seen between NRM and per-
in the HVG nonpermissive mismatch group and in the missive mismatch status. The strong association of high

Table 1. Clinical characteristics of patients who underwent hematopoietic stem cell transplantation from unrelated donors.
HLA-DPB1 match by TCE grouping
Characteristic Entire cohort, Match, Permissive GVH nonpermissive HVG nonpermissive P
n=1514 n=366 mismatch, mismatch, mismatch,
n=651 n=269 n=228
Median age in years (range) 56 (18-79) 55 (18-76 ) 56 (18-76) 56 (20-77) 57 (20-79) 0.972
Age >50 years, n (%) 991 (65.5) 237 (64.8) 437 (67.1) 172 (63.9) 145 (63.6) 0.673
Donor age in years (range) 30 (18-71) 30 (18-63) 30 (18-58) 30 (18-59) 29 (19-71) 0.387
Donor age >40 years, n (%) 288 (19.0) 59 (16.1) 122 (18.8) 61 (22.7) 46 (20.2) 0.207
Female, n (%) 614 (40.6) 141 (38.5) 259 (39.8) 120 (44.6) 94 (41.2) 0.447
Donor-recipient sex combination, n (%) 0.566
Female to female 178 (11.8) 42 (11.5) 74 (11.4) 35 (13.0) 27 (11.8)
Female to male 211 (13.9) 48 (13.1) 97 (14.9) 42 (15.6) 24 (10.5)
Male to female 436 (28.8) 99 (27.0) 185 (28.4) 85 (31.6) 67 (29.4)
Male to male 689 (45.5) 177 (48.4) 295 (45.3) 107 (39.8) 110 (48.3)
ABO matching, n (%) 0.259
Match 724 (47.8) 177 (48.4) 313 (48.1) 129 (48.0) 105 (46.1)
Minor mismatch 351 (23.2) 77 (21.0) 159 (24.4) 56 (20.8) 59 (25.9)
Major mismatch 333 (22.0) 81 (22.1) 147 (22.6) 62 (23.0) 43 (18.9)
Bidirectional mismatch 106 (7.0) 31 (8.5) 32 (4.9) 22 (8.2) 21 (9.2)
Donor-recipient CMV serostatus (n=1510), n (%) 0.725
NR-NR 192 (12.7) 52 (14.2) 83 (12.8) 33 (12.3) 24 (10.5)
NR-R 734 (48.6) 182 (49.9) 303 (46.8) 137 (50.9) 112 (49.1)
R-NR 99 (6.6) 23 (6.3) 47 (7.3) 18 (6.7) 11 (4.8)
R-R 485 (32.1) 108 (29.6) 215 (33.2) 81 (30.1) 81 (35.5)
Diagnosis, n (%) 0.376
AML/MDS 673 (44.5) 170 (46.5) 293 (45.0) 107 (39.8) 103 (45.2)
Other hematologic malignancies 841 (55.5) 196 (53.6) 358 (55.0) 162 (60.2) 125 (54.8)
DRI, n (%) 0.710
Low 228 (15.1) 63 (17.2) 89 (13.7) 42 (15.6) 34 (14.9)
Intermediate 600 (39.6) 139 (38.0) 269 (41.3) 97 (36.1) 95 (41.7)
High 518 (34.2) 130 (35.5) 219 (33.6) 96 (35.7) 73 (32.0)
Very high 168 (11.1) 34 (9.3) 74 (11.4) 34 (12.6) 26 (11.4)
HCT-CI, median (range) 3 (0-11) 3 (0-11) 2 years (0-11) 3 years (0-10) 2 years (0-11) 0.261
HCT-CI ≥3, n (%) 766 (50.6) 187 (51.1) 325 (49.9) 150 (55.8) 104 (45.6) 0.152
Prior AlloHSCT, n (%) 36 (2.4) 8 (2.2) 14 (2.2) 8 (3.0) 6 (2.6) 0.878
Prior AutoHSCT, n (%) 120 (7.9) 30 (8.2) 52 (8.0) 21 (7.8) 17 (7.5) 0.990
HSCT protocol, n (%) 0.274
Clinical trial protocol 962 (63.5) 234 (63.9) 407 (62.5) 164 (61.0) 157 (68.9)
Standard of care 552 (36.5) 132 (36.1) 244 (37.5) 105 (39.0) 71 (31.1)
Conditioning regimen intensity, n (%) 0.223
MA 1024 (67.6) 245 (66.9) 454 (69.7) 183 (68.0) 142 (62.3)
RIC/NMA 490 (32.4) 121 (33.1) 197 (30.3) 86 (32.0) 86 (37.7)
Stem cell source, n (%) 0.001
PB 945 (62.4) 202 (55.2) 411 (63.1) 191 (71) 141 (61.8)
BM 569 (37.6) 164 (44.8) 240 (36.9) 78 (29) 87 (38.2)
GVHD regimen (n=1513), n (%) 0.540
Tacrolimus/methotrexate 1268 (83.8) 295 (80.6) 547 (84.2) 229 (85.1) 197 (86.4)
PTCY 185 (12.2) 55 15.0 79 (12.2) 29 (10.8) 22 (9.7)
Others 60 (4.0) 16 (4.4) 24 (3.7) 11 (4.0) 9 (4.0)
ATG, n (%) 1121 (74.0) 266 (72.7) 477 (73.3) 205 (76.2) 173 (75.9) 0.657
Year of HSCT, n (%) <0.001
2005-2009 359 (23.7) 68 (18.6) 163 (25.0) 72 (26.8) 56 (24.6)
2010-2013 531 (35.1) 105 (28.7) 229 (35.2) 103 (38.3) 94 (41.2)
2014-2018 624 (41.2) 193 (52.7) 259 (39.8) 94 (34.9) 78 (34.2)
Quantified ME, PS-I, and PS-II, median (range)
GVH DP ME 4 (0-22) 0 (0-0) 5 (0-22) 9 (0-19) 5 (0-21) <0.001
GVH PS-I 0 (0-14) 0 (0-0) 1 (0-13) 3 (0-9) 1 (0-14) <0.001
GVH PS-II 2 (0-28) 0 (0-0) 3 (0-22) 8 (0-28) 2 (0-27) <0.001
HVG DP ME 4 (0-20) 0 (0-0) 5 (0-19) 5 (0-20) 9 (1-19) <0.001
HVG PS-I 0 (0-17) 0 (0-0) 1 (0-14) 0 (0-17) 3 (0-10) <0.001
HVG PS-II 1 (0-34) 0 (0-0) 3 (0-34) 1 (0-22) 8(0-25) <0.001
Notes and abbreviations on following page.

J. Zou et al.
Note: Percentages may not add up to 100 because of rounding. P values of categorical variables were from the Fisher exact or c2 test. P values of continuous variables were from
analysis of variance or the Kruskal-Wallis test. There were four missing data points for donor-recipient cytomegalovirus serostatus and one missing data point for the graft-ver-
sus-host disease regimen. HSCT: hematopoietic stem cell transplantation; AlloHSCT: allogeneic hematopoietic stem cell transplantation; AutoHSCT: autologous hematopoietic
stem cell transplantation; TCE: T-cell epitope; GVH: graft-versus-host; HVG: host-versus-graft; CMV: cytomegalovirus; NR: nonreactive; R: reactive; AML: acute myeloid leukemia; MDS:
myelodysplastic syndrome; DRI: Disease Risk Index; HCT-CI: Hematopoietic Cell Transplant-Comorbidity Index; MA: myeloablative; RIC: reduced-intensity conditioning; NMA: non-
myeloablative; PB: peripheral blood; BM: bone marrow; GVHD: graft-versus-host disease; PTCY: post-transplant cyclophosphamide; ATG: antithymocyte globulin; DP ME: HLA-DPB1
mismatched eplets; PS-I, Predicted Indirectly Recognizable HLA Epitopes score I; PS-II, Predicted Indirectly Recognizable HLA Epitopes score II.
GVH PS-I and GVH PS-II with grade 2-4 acute GVHD risk HLA-DPB1 nonpermissive mismatch in the GVH direc-
resulted in an increased risk of NRM (GVH PS-I: tion was associated with not only an increased risk of
HR=1.31, 95% CI: 1.07-1.60, P=0.008; GVH PS-II: acute GVHD but also a reduced risk of relapse (HR=0.64,
HR=1.34, 95% CI: 1.10-1.63, P=0.004), whereas HVG PS- 95% CI: 0.47-0.86, P=0.003), whereas permissive mis-
I (HR=1.22, 95% CI: 1.01-1.49, P=0.041), but not HVG match and HVG nonpermissive mismatch were not sig-
PS-II, was associated with an increased risk of NRM, and nificantly associated with risk of relapse.
neither GVH nor HVG ME was significantly associated Similar results were seen in patients with high GVH
with NRM. In the analysis of combined groups, NRM risk ME, PS-I, and PS-II, which were associated with reduced
was highest in those with high GVH and high HVG PS-I risk of relapse (ME: HR=0.83, 95% CI: 0.70-0.99, P=0.05;
(HR=1.48, 95% CI: 1.15-1.91, P=0.002) and in those with PS-I: HR=0.82, 95% CI: 0.68-0.98, P=0.032; PS-II:
high GVH and high HVG PS-II (HR=1.50, 95% CI: 1.16- HR=0.79, 95% CI: 0.66-0.95, P=0.011), whereas HVG
1.94, P=0.002) (Figure 1B). ME, PS-I, and PS-II were not associated with a reduced
Figure 1. Figure continued on following page.

Figure 1. Forest plots showing results from multivariable analyses of the impact of molecular mismatch scores (ME, PS-I, PS-II) and traditional T-cell epitope group-
ing on outcomes, stratified by the mismatch in the graft-versus-host and host-versus-graft direction. (A) Acute graft-versus-host disease grade 2-4. (B) Non-relapse
mortality. (C) Relapse. (D) Overall survival. Dots and bars in the forest plots represent adjusted hazard ratios and 95% confidence intervals. PS and ME were cate-
gorized into low and high groups using the median as a cutoff point. ME: mismatched eplets, PS: Predicted Indirectly Recognizable HLA Epitope score; GVH: graft-
versus-host: HVG: host-versus-graft; GVHD: graft-versus-host disease.
risk of relapse (Figure 1C). Relapse risk was significantly alloimmunity predicted by ME or PS, in either the HVG
lower in patients with high GVH ME combined with low or the GVH direction, was associated with a trend of
HVG ME than in patients with low ME in both directions increased risk of grade 2-4 acute GVHD (Figure 2A). In
(Figure 1C, Online Supplementary Figure S2B). particular, HVG PS-II was associated with a significantly
Neither HLA-DPB1 mismatch permissiveness nor increased risk of grade 2-4 acute GVHD (HR=1.43, 95%
molecular mismatches were found to be associated with CI: 1.13-1.82, P=0.003). This finding was further con-
overall survival (Figure 1D, Online Supplementary Table firmed by our analysis of combined groups, in which a
S3), progression-free survival (Online Supplementary Table significantly increased risk of grade 2-4 acute GVHD was
S4), or engraftment in the present study cohort. observed in the group with high ME (Figure 2B) or PS-II
in both directions. However, high GVH ME or PS without
In the permissive mismatch group, GVH alloimmunity determined concurrent HVG alloimmunity was not associated with
by ME and PS was associated with an increased risk of GVHD, an increased risk of acute GVHD.
whereas HVG alloimmunity determined by ME and PS was associat- Similar to what we observed in the entire cohort, no
ed with an increased risk of relapse and GVHD. anti-leukemia benefit was associated with HVG allore-
Consistent with the previous report,26 permissive mis- sponse assessed by ME or PS. Moreover, high ME in the
match represented the largest subgroup in our cohort of HVG direction was associated with an increased risk of
patients who underwent HSCT from unrelated donors. relapse in the permissive mismatch group (HR=1.36, 95%
Results from the multivariable analyses showed that the CI: 1.02-1.76, P=0.026) (Figure 2C), and this was more

J. Zou et al.
pronounced in the group with high HVG ME coupled grade 2-4 acute GVHD (HR=2.82, 95% CI: 1.41-5.62,
with low alloimmunity in the GVH direction (Figure 2D). P=0.003) (Figure 3B).
Molecular mismatches assessed by ME or PS were not No significant association between the molecular mis-
associated with the risk of NRM, overall survival, or pro- match factors and relapse (Online Supplementary Figure
gression-free survival in this permissive mismatch sub- S3), NRM, engraftment, overall survival, or progression-
group. free survival was identified.
In the GVH nonpermissive mismatch group, ME in the GVH direc- In the HVG nonpermissive mismatch group, ME and PS-I in the GVH
tion was associated with a higher incidence of grade 2-4 acute direction were associated with worse NRM without an increased risk
GVHD, and HVG ME could synergistically contribute to this risk. of GVHD
Alloimmunity quantified by ME appeared to be more None of the mismatch factors was associated with the
clinically relevant than alloimmunity quantified by PS in risk of relapse or acute GVHD in the HVG nonpermissive
the GVH nonpermissive mismatch group. Results from mismatch group with high HVG alloimmunity settings
the multivariable analyses showed that high ME in the (Online Supplementary Figure S4A, B). Although no associ-
GVH direction was associated with an increased risk of ation with the risk of acute GVHD was identified, alloim-
grade 2-4 acute GVHD (HR=1.64, 95% CI: 1.16-2.31, munity in the GVH direction determined by ME and PS-I
P=0.005) (Figure 3A). Although HVG ME itself was not was associated with an increased risk of NRM (ME:
associated with the risk of acute GVHD, those with high HR=1.90, 95% CI: 1.18-3.07, P=0.008, Figure 4A; PS-I:
ME in both directions had a significantly increased risk of HR=1.60, 95% CI:1.04-2.60, P=0.024, Figure 4B), indicat-
Figure 2. Figure continued on following page.

Figure 2. Forest plots showing results from the multivariable analyses of the impact of molecular mismatch scores (ME, PS-I, and PS-II) on outcomes in the per-
missive mismatch group, stratified by the mismatch in the graft-versus-host and host-versus-graft direction. (A) Acute graft-versus-host disease (GVHD) grade 2-4.
(B) Adjusted cumulative incidence of acute GVHD grade 2-4. (C) Relapse. (D) Adjusted cumulative incidence of relapse. Dots and bars in the forest plots represent
adjusted hazard ratios and 95% confidence intervals. PS and ME were categorized into low and high groups using the median as a cutoff point. ME: mismatched
eplets, PS: Predicted Indirectly Recognizable HLA Epitope score; GVH: graft-versus-host: HVG: host-versus-graft; GVHD: graft-versus-host disease; HR: hazard ratio.
ing that the increased risk of NRM observed here may not 0.560, 0.556, 0.545, 0.541, 0.542, and 0.566, respectively.
be mostly attributed to GVHD. Additionally, a lower inci- Moreover, decision curve analysis29 was conducted to
dence of neutrophil engraftment was observed in the compare the clinical application of different matching
group with high ME in the HVG direction, likely models. We found that ME in the GVH direction outper-
attributable to the alloimmunity towards the graft formed other models, including the conventional TCE
(HR=0.73, 95% CI: 0.56-0.96, P=0.028 for low GVH ME + model, and provided the best net clinical benefit for the
high HVG ME). modification of the acute GVHD prophylaxis regimen in
patients with a high risk of developing clinically signifi-
Predictive performance of the TCE, ME, and PS models cant acute GVHD (Figure 5).
Results from the concordance test showed that the ME
in the GVH direction provided better discriminative ability
for the prediction of clinically significant acute GVHD with Discussion
a concordance index of 0.595 compared with other mod-
els. The values of the concordance index of GVH PS I, Relapse and GVHD remain two major causes of mor-
GVH PS II, HVG ME, HVG PS I, HVG PS II, and TCE were bidity and mortality in patients with hematologic malig-

J. Zou et al.
nancies undergoing HSCT. It has been accepted that ed that HLAMatchmaker and PIRCHE can be used to
donor T-cell–mediated alloimmune responses are the key assess histocompatibility in HSCT at the molecular level.
mediators of beneficial GVL and adverse GVHD effects. Using the decision curve analysis method that incorpo-
A better understanding of T-cell alloreactivity in patients rates clinical considerations, it was found that ME in the
receiving HSCT would help to minimize the risk of GVH direction has advantages over other predictive mod-
GVHD while still preserving GVL activity. With recent els including the conventional TCE model, in aiding the
progress in bioinformatics and molecular HLA typing, in decision whether or not to modify the acute GVHD pro-
silico prediction of immunogenicity has evolved rapidly, phylaxis regimen. In patients with a high risk of develop-
and several algorithms with a different focus have been ing clinically significant acute GVHD predicted by high
shown to be predictive of outcomes in patients who have ME in both GVH and HVG directions, the addition of
undergone HSCT.22,31 therapy based on T-cell depletion to the prophylactic reg-
In the present comprehensive study in a cohort of imen may reduce the incidences and intensity of GVHD.
patients with hematologic malignancies, we demonstrat- Moreover, ME and PS can quantitatively refine the con-
Figure 3. Forest plots showing results from the multivariable analyses of the impact of molecular mismatch scores (ME, PS-I, PS-II) on outcomes in patients with
HLA-DPB1 nonpermissive mismatch in the graft-versus-host (GVH) direction, stratified by ME GVH and host-versus-graft combinations. (A) Acute graft-versus-host
disease (GVHD) grade 2-4. (B) Adjusted cumulative incidence of acute GVHD grade 2-4. Dots and bars in the forest plots represent adjusted hazard ratios and 95%
confidence intervals. PS and ME were categorized into low and high groups using the median as a cutoff point. ME: mismatched eplets, PS: Predicted Indirectly
Recognizable HLA Epitope score; GVH: graft-versus-host: HVG: host-versus-graft; GVHD: graft-versus-host disease; HR: hazard ratio.

ventional TCE grouping, so the finding here will aid pri- host T cells secrete higher levels of inflammatory
oritization of the donors even within the same TCE cytokines and contribute to GVHD in addition to graft T-
group. cell immunity.19,21 For the first time, we demonstrate that
Using the HLA-DPB1 TCE model, Fleischhauer et al. the direction of alloreactivity may be better reflected by
concluded that mismatches in different directions (HVG ME or PS in different directions. The elicited GVH allore-
versus GVH) did not differ in terms of acute GVHD and activity defined by PS and ME seems to contribute to
mortality risk.32 However, bidirectional mismatches GVL along with GVHD, whereas HVG alloreactivity is
seemed to work synergistically and were associated with likely to augment GVHD without the anti-leukemia
an increased risk of GVHD. How to reconcile HVG effect. In the HLA-DPB1 permissive mismatch group, the
alloimmunity remains unclear, because host T cells in cir- largest subgroup of patients within our cohort, the elicit-
culation are believed to be depleted by conditioning regi- ed HVG alloreactivity appears to counteract the anti-
mens during HSCT. Recent studies indicate that peripher- leukemia effect exerted by GVH alloimmunity, discourag-
al host T cells resident in the skin and gut are stimulated ing the use of donors with a high load of HVG ME/PS in
by the mismatched HLA and, as a result, the activated patients with HLA-DPB1 permissive mismatch. These
Figure 4. Adjusted cumulative incidence of non-relapse mortality in patients with HLA-DPB1 nonpermissive mismatch in the host-versus-graft direction. (A)
Stratified by the number of mismatched eplets (ME) in the graft-versus-host (GVH) direction. (B) Stratified by Predicted Indirectly Recognizable HLA Epitopes score-I
(PS-I) in the GVH direction. HR: hazard ratio.

J. Zou et al.
Figure 5. The clinical net benefit of the TCE,

ME, and PS models in deciding to modify
graft-versus-host disease (GVHD) prophylaxis
regimen for patients with a high risk of devel-
oping clinically significant acute GVHD in
comparison with a “treat/modify all” and
“treat/modify none” strategy. Y-axis repre-
sents the net clinical benefit (positive values)
or risk (negative values) of using model-guid-
ed GVHD regimen modification in comparison
with no GVHD regimen modification (net clini-
cal benefit =0). The X-axis represents thresh-
old probabilities of acute GVHD grade 2-4 at
100 days after transplantation. TCE: T-cell
epitopes; ME: mismatched eplets, PS:
Predicted Indirectly Recognizable HLA
Epitope score; GVH: graft-versus-host: HVG:
host-versus-graft; aGVHD: acute graft-versus-
host disease.
findings not only assist donor selection and risk stratifica- that a significant number of polymorphic amino acids,
tion in HSCT from unrelated donors but also provide especially in the b-sheet and a-3 domain, were not co-
valuable insights into the mechanism and process of localized.42 Therefore, an optimized algorithm that con-
alloimmunity in this setting. siders both direct and indirect alloresponses would be
In agreement with recent studies on DP mismatches more predictive of risks or benefits in the context of
using the TCE model33 or DP expression model,34 associ- HSCT with HLA-mismatched donors.
ations of the nonpermissive mismatch and overall sur- Unlike the TCE and the expression model that has
vival or transplant-related mortality were not found in been extensively studied and shown to be clinically rele-
our cohort. This is perhaps attributable to a high degree vant for HSCT in several high power studies,7,8,17,18 the
of HLA matching degree in the cohort, recent advances molecular mismatching algorithms have been primarily
in GVHD prophylaxis and reduced incidence of severe studied in the solid organ transplant setting in the assess-
GVHD. The majority of our patients received in-vivo T- ment of antibody-mediated rejection. The predictive
cell depletion which may lessen the alloresponse derived value of ME or PIRCHE was only reported in a few small
from DP mismatch and reduce the severity and incidence studies in HSCT settings.38,43-45 and further validation is
of acute GVHD.35 Additionally, several recent studies warranted before routine clinical application. The het-
documented an improved outcome with post-transplant erogeneity of the cohort and retrospective nature of the
cyclophosphamide in patients receiving not only hap- current study may have biased our results.
loidentical transplants but also in transplants from In conclusion, molecular HLA disparity and subse-
matched unrelated donors,36 it may be particularly effec- quent alloresponse assessed by in silico methods are use-
tive for individuals with high ME/PS due to the profound ful in the prediction of clinical outcomes. In addition to
effect of this treatment on GVHD outcomes compared conventional TCE grouping, additional information pro-
with conventional GVHD prevention regimens.37 vided by ME and PS can be used to refine the permissive-
However, due to the low number of patients who ness of HLA-DPB1 mismatches. In the present study,
received post-transplant cyclophosphamide in the cur- high alloimmunity in both the HVG and GVH directions,
rent study, future large prospective studies are warranted revealed by high PS or ME, is associated with an
to confirm our hypothesis. increased risk of GVHD. Nevertheless, only GVH ME or
The predictive value of the HLAMatchmaker and PS was associated with a reduced risk of relapse. An inte-
PIRCHE algorithms has been demonstrated in HSCT grated study in which patients’ immune cells are charac-
from HLA-mismatched unrelated donors or haploidenti- terized and comprehensively analyzed will provide
cal donors.24,31,38 Although HLAMatchmaker mainly deeper and better insights into the process of GVH
focuses on epitopes directly recognized by B cells, allore- response and the contribution from host T cells.
active T-cell clones that are specific to certain eplets iden-
tified by HLAMatchmaker have also been found,39-41 sug- Disclosures
gesting that HLAMatchmaker reveals many polymorphic No conflicts of interest to disclose.
residues overlapping in both B-cell epitopes and T-cell
epitopes. Consistent with a previous study,42 we Contributions
observed a considerable correlation between ME load JZ, PK, REC, and KC designed the study and contributed to
and PS. However, the disparity determined by ME load collecting and interpreting the data and writing the manuscript;
appears to be more clinically relevant in our study. JZ and PK wrote the initial draft of the manuscript; PK, JM, and
Analysis of the topographic location of immunogenic LL contributed to the statistical analysis and interpretation of
amino acids identified with both methods demonstrated statistical results and reviewed and approved the manuscript;

BO, VK, YC, SS, HCC, DP, SOC, and QM contributed to the University of Texas MD Anderson Cancer Center, for editing
data collection and analysis and reviewed and approved the this article.
manuscript; GR contributed to data collection and reviewed and
approved the manuscript; BO, SS, UG, EJS, and REC con- Funding
tributed to the treatment of patients and reviewed, edited, and VK acknowledges funding from an NIHR Fellowship (PDF-
approved the final version of the manuscript. 2016-09-065). This research was partially supported by the
Cancer Center Support Grant of MD Anderson (NIH:
Acknowledgments P30CA016672 to L.L.).
The authors would like to thank Kevin Harrell and Dr. Jar-
How Lee from Thermo Fisher Scientific for their help in eplet Data-sharing statement
analysis for this manuscript. We thank Erica Goodoff, Senior For data sharing, contact the corresponding author:
Scientific Editor in the Research Medical Library at The jzou@mdanderson.org
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Cell Therapy & Immunotherapy ARTICLE
Comparison of immune reconstitution Ferrata Storti Foundation

between anti-T-lymphocyte globulin and
posttransplant cyclophosphamide as acute
graft-versus-host disease prophylaxis in
allogeneic myeloablative peripheral blood
stem cell transplantation
Radwan Massoud, Nico Gagelmann, Ulrike Fritzsche-Friedland, Gaby Zeck,
Silke Heidenreich, Christine Wolschke, Francis Ayuk, Maximilian Christopeit Haematologica 2022
and Nicolaus Kröger Volume 107(4):857-867
Department of Stem Cell Transplantation, University Medical Center Hamburg-
Eppendorf, Hamburg, Germany
ABSTRACT
A
nti-T-cell lymphocyte globulin (ATLG) and posttransplant
cyclophosphamide (PTCy) are now widely used strategies to pre-
vent graft-versus-host disease (GVHD) after allogeneic stem cell
transplantation. Data comparing immune reconstitution (IR) between
ATLG and PTCy is scarce. This retrospective study conducted at the
University Medical Center Hamburg-Eppendorf (UKE) compares PTCy
(n=123) and ATLG (n=476) after myeloablative allogeneic peripheral blood
stem cell transplant. Detailed phenotypes of T, B natural killer (NK), natu-
ral killer T (NKT) cells were analyzed by multicolor flow at day 30, 100
and 180 posttransplant. Incidence of infections, viral reactivations, GVHD
and relapse were collected. Neutrophil engraftment was significantly
delayed in the PTCy group (median day 12 vs. day 10, P<0.001) with a
high incidence of infection before day+100 in the PTCy arm but a higher
Epstein-Barr virus reactivation in the ATLG arm and comparable
cytomegalovirus reactivation. Overall incidence of acute GVHD was sim-
ilar but moderate/severe chronic GVHD was seen more often after PTCy
(44% vs. 38%, P=0.005). ATLG resulted in a faster reconstitution of CD8+ Correspondence:
T, NK, NKT and gdT cells while CD4 T cells and B cells reconstituted faster NICOLAUS KRÖGER
after PTCy. Similar reconstitution was observed for T-regulatory cells and nkroeger@uke.uni-hamburg.de
B cells. Non-relapse mortality relapse incidence, disease-free survival, and
overall survival did not differ significantly between both arms. Even
Received: September 10, 2020.
though differences in IR were related to a decreased incidence of infection
and moderate/severe cGVHD in the ATLG group they had no impact on Accepted: March 31, 2021.
any of the other long-term outcomes. However, it remains undetermined Pre-published: April 8, 2021.
which regimen is better as GVHD prophylaxis.
Introduction
Allogeneic stem cell transplantation (allo-SCT) is a potentially curative treat- Material published in Haematologica is covered by copyright.
ment strategy for hematological diseases.1 This is attributed mainly to the graft- All rights are reserved to the Ferrata Storti Foundation. Use of
versus-tumor effect derived from transferring the donor’s immune system to the conditions:
recipient.2 However, the benefit of allo-SCT may be offset by increased transplant- https://creativecommons.org/licenses/by-nc/4.0/legalcode.
related mortality (TRM) especially due to graft- versus-host disease (GVHD).3 In an Copies of published material are allowed for personal or inter-
attempt to decrease the incidence of GVHD physicians employ a multitude of nal use. Sharing published material for non-commercial pur-
strategies, including in vivo T-cell depletion (TCD) with pretransplant anti-T-lym- https://creativecommons.org/licenses/by-nc/4.0/legalcode,
phocyte globulin (ATLG)4,5 and/or posttransplant cyclophosphamide (PTCy).6-8 sect. 3. Reproducing and sharing published material for com-
GVHD prophylaxis with PTCy decreases the incidence of graft rejection and mercial purposes is not allowed without permission in writing
GVHD by impairing the function of alloreactive T cells.9 However, data is scarce from the publisher.
on the effect of PTCy effect on immune reconstitution (IR) post-allo-SCT, espe-
cially when compared to the standard use of ATLG as a TCD tool.4,5,10,11 In this

R. Massoud et al.
study we aim to compare the IR kinetics and transplant by standard descriptive statistical methods. c2 test was used to
outcomes between ATLG and PTCy as TCD strategies in compare categorical variables, whereas continuous variables
patients undergoing allo-SCT with myeloablative condi- were compared using Student’s t-test. We defined disease-free
tioning (MAC) and peripheral blood stem cells (PBSC). survival (DFS) as survival without relapse or progression of
hematological disease; we censored patients without disease or
progression at the time of the last follow-up. We defined OS and
Methods NRM as death from any cause, and without evidence of relapse
respectively. We used the Kaplan-Meier method to calculate the
This retrospective study conducted at the University Medical probabilities of moderate/severe cGVHD relapse-free survival,
Center Hamburg-Eppendorf (UKE) with a primary outcome to DFS and OS; and the cumulative incidence functions were used
compare IR between PTCy versus ATLG in vivo TCD in adult to estimate RI and NRM in a competing risk setting. All analysis
patients who received MAC PBSC allo-SCT. Secondary out- was performed using SPSS version 26.0 and ACCorD.
comes included incidence of viral reactivations, engraftment,
infections, acute GVHD (aGHVD), chronic GVHD (cGVHD),
non-relapse mortality (NRM), progression-free survival (PFS), Results
overall survival (OS). All patients signed written informed con-
sents for treatment, and the study was approved by the Patients and transplant characteristics
Institutional Review Board of UKE. In order to have comparable groups we selected only
MAC regimens were defined according to working group def- patients receiving myeloablative conditioning for their
inition.12 ATLG (Grafalon®, Neovii, Switzerland) was given at a first allo-SCT with only PBSC as a stem cell source. From
dose of 30 mg/kg for related donor or 60 mg/kg for unrelated 2005 to 2019, 599 patients were included in the study.
donor with a trend in recent years to give the lower dose for Four hundred and seventy-six patients received ATLG,
both groups. All ATLG doses were fractionated between days - with 34% and 66% receiving 30 mg/kg and 60 mg/kg
4 to -1. PTCy was administered as 50 mg/kg/day. Posttransplant ATLG respectively. One hundred and twenty-three
immunosuppression was given on days +3 and +4 combined patients received PTCy. The median age at transplant was
with calcineurin inhibitor (tacrolimus for unrelated donor or 53 years (range, 18-75 years) in both groups. Seventy nine
haplo-identical donor, and cyclosporine for related donor) and percent and 72% were transplanted from a full match
mycophenolate mofetil for mismatched transplants. Similar sup- donor (HLA10/10) in the ATLG and PTCy group, respec-
portive care was used for all patients per institutional guidelines tively. All patients, donor and transplant characteristics
including antimicrobial prophylaxis consisting of fluoro- are listed in Table 1.
quinelone for bacterial infections, trimethorpin-sulfamethoxa-
zole or pentamidine for Pnemocystis jiroveci, micafungin for fungal Transplant outcomes
infections and acyclovir for viral infections. Patients were All transplant outcomes are summarized in Table 2.
screened weekly for cytomegalovirus (CMV) and Epstein-Barr
virus (EBV) by blood polymerase chain reaction (PCR). Engraftment
Neutrophil engraftment was defined as the first 3 consecutive Platelet and neutrophil engraftment were significantly
days with a measure of an absolute neutrophil count >0.5x109/L. delayed in the PTCy group when compared to the ATLG
Platelet engraftment was defined as the first consecutive days group; with a median of 12 days (range, 8-36 days) to
with a platelet count >20x109/L without transfusion support. neutrophil in the ATLG versus 16 days (range, 12-27 days)
Acute GVHD was graded according to standard criteria.13 in PTCy group (P<0.001); and a median of 15 days (range,
Chronic GVHD was graded according to National Institute of 8-99 days) to platelet engraftment in the ATLG versus 21
Health (NIH) criteria routinely at every visit after transplanta- days (range, 9-99 days) in the PTCy group (P=0.024).
tion.14
Cytomegalovirus infections and Epstein-Barr virus
Infections were defined as any microbial testing with a reactivation
positive result and requiring therapy. We observed no significant differences in incidence of
As per institution guidelines, blood samples were collected for CMV reactivation before day 100 (ATLG 46%, PTCy
each patient on days 30, 100 and 180 post-allo-SCT. Samples 50%). The overall incidence of infection before day 100
were used directly after red blood cell lysis following 10 minutes was significantly higher in the PTCy group (91%) when
of incubation with erythrocyte lysing reagent without fixative. compared to the ATLG group (75%), P<0.001. The inci-
Immunophenotypes were assessed using four color cytometry dence of EBV reactivation before days 100 in the ATLG
using mouse anti-human antibodies for the following cells: T group was higher than the PTCy group (33% vs. 16%,
lymphocytes (CD3+), activated T lymphocytes P<0.001).
(CD3+HLADR+), T-helper cells (CD3+/CD4+), cytotoxic T cells
(CD3+/CD8+), B lymphocytes (CD19+), B-lymphocyte subpop- Graft-versus-host disease
ulations (CD19+CD5+CD1d+)(CD19+CD27+), naïve B cells The cumulative incidences of aGVHD grade 2-4 and 3-
(CD19+CD27-CD10+), natural killer (NK) cells (CD56+CD3-), 4 were similar between the two groups with 36% and
natural killer T (NKT) cells (CD56+CD3+), naïve T-helper cells 15% in the ATLG group and 40% and 12 % in the PTCy
(CD4+CD45RA+), memory T-helper cells (CD4+CD45R0+), group.
naïve cytotoxic T cells (CD8+CD45RA+), memory cytotoxic T The cumulative incidence of all grade cGVHD were
cells (CD8+CD45R0+), gdT cells (gdTCR+,CD3+), regulatory T similar between the two groups, 15% and 27% in the
cells (CD4+CD25+CD127low-neg). ATLG and PTCy groups respectively, however we
observed a higher cumulative incidence of moderate and
Statistical analyses severe cGVHD in the PTCy group, 38% ATLG versus
All data was retrospectively collected, and was summarized 44% PTCy (P=0.005).

Immune reconstitution with ATLG vs. PTCy
Table1. Patients and transplant characteristics. continued from previous coloum
Patients ATLG PTCy P Immune suppression <0.001

N (%) N(%) CNI+MMF 413 (87) 77 (62)
CNI+MTX 57 (12) 0 (0)
Total patients 476 (100) 123 (100) CNI 1 (0.2) 23 (19)
ATLG dose other 5 (1) 23 (19)
30 mg/kg 162 (34) ATLG: anti T-cell lymphocyte globulin; PTCy: and post-transplant cyclophosphamide;
60 mg/kg 314 (66) NS: statistically not significant; ALL: acute lymphoblastic leukemia; AML: acute
myeloid leukemia; CML: chronid myeloid leukemia; MDS: myelodysplastic syndrome;
Mean age (Standard Deviation) 50 (14) 50 (13) NS MDS-MPN: myelodysplastic syndrome - myeloproliferative neoplasm; HL: Hodgkin
lymphoma; NHL: non Hodgkin lymphoma; MM: multiple myeloma; PMF: primary
Disease <0.001 myelofibrosis; Other AL: other acute leukemia; ECOG: Eastern Cooperative Oncology
ALL 27 (10) 35 (29) Group performance status; KI: Karnofsy index: CMV: cytomegalovirus; D-: donor with
AML 206 (43) 23 (19) negative CMV serology; D+: donor with positive CMV serology; R-: recipient with neg-
CML 16 (3) 1 (1) ative CMV serology; R+: recipient with positive CMV serology; MRD: matched related
door; MMRD: mismatched related donor; MUD: matched unrelated donor; MMUD:
MDS 43 (9) 2 (2) mismatched unrelated donor; SD: standard deviation; TBI: total body irradiation; CNI:
MDS-MPN 6 (1) 4 (3) calcineurin inhibitor; MMF: mycophenolate mofetil; age in years.
HL 4 (1) 2 (2)
NHL 75 (16) 13 (11) Table 2. Transplant outcomes.
MM 64 (13) 38 (31) Transplant outcomes
PMF 12 (3) 2 (2) ATLG PTCy P
Other AL 3 (1) 3 (2) N (%) N(%)
ECOG NS
Patients 476 (100) 123 (100)
0 115 (30) 23 (20)
1 241 (63) 81 (72) Leukocytes engraftment 12 (8-36) 16 (12-27) <0.001
2 23 (6) 9 (8) median days (range)
3 3 (1) 0 (0) Platelet engraftment median 15 (8-99) 21 (9-99) 0.024
Mean KI at SCT (Standard Deviation)86 (12) 83 (11) 0.011 days (range)
Mean donor age (Standard Deviation)36 (12) 37 (14) NS CMV reactivation [15 in ATLG, 54 214 (46) 60 (50) NS
in PTCy missing]
Donor/ recipient CMV serology NS
D-/R- 151 (32) 41 (34) EBV reactivation [34 in ATG 131 (33) 19 (16) <0.001
D+/R+ 194 (41) 58 (47) missing, 4 in PTCy missing]
D-/R+ 62 (13) 9 (7) Overall incidence of infection 344 (75) 109 (91) <0.001
D+/R- 68 (14) 15 (12) by day 100
Donor-Recipient sex NS NRM at 3 years 16% 30% 0.006
No mismatch 322 (67) 75 (61) Relapse incidence 34% 29% NS
Male-Male 241 (51) 59 (48)
Female-Female 81 (17) 16 (13) DFS at 3 years 50% 42% NS
Mismatch 154 (32) 48 (39) OS at 3 years 65% 58% NS
Male - Female 101 (21) 32 (26) Moderate/severe cGVHD 40% 27% NS
Female - Male 53 (11) 16 (13) relapse-free survival
ABO incompatibility NS NS: statistically non-significant; NRM: non-relapse mortality; OS: overall survival; DFS:
Isogroup 182 (39) 57 (47) disease-free survival; cGVHD: chronic graft-versus-host disease; EBV: Epstein-Barr
virus; CMV: cytomegalovirus; PTCy: posttransplant cyclophosphamide; ATLG: anti T-
Minor 127 (27) 23 (19) cell lymphocyte globulin; SD: standard deviation.
Major 111 (24) 33 (27)
Bidirectional 48 (10) 8 (7)
Median year of transplant (range) 2013 2015 <0.001 Non-relapse mortality
(2005-2019) (2005-2019) PTCy was associated with a higher NRM when com-
Type of transplant pared to ATLG on univariate analysis, in addition a posi-
Related 77 (16) 45 (37) <0.001 tive patient CMV serology, patient age >52 years, donor
Unrelated 399 (84) 78 (63) age >34 years and female donor were also associated
Full match (HLA10/10) 377 (79) 88 (72) NS with higher NRM. On multivariate analysis, NRM was
Mismatch (HLA <10/10) 99 (21) 35 (29) NS not affected by ATLG and PTCy; only donor sex, patient
MRD 74 (16) 31 (25) <0.001 age and CMV serology had a significant impact on NRM
MMRD 3 (1) 14 (11) (Table3).
MUD 303 (64) 57 (46)
MMUD 96 (20) 21 (17)
Relapse, disease-free survival and overall survival
Mean CD34 x10 /kg (SD) infused 11.55 (64)
6
7.18 (2) NS After a median follow-up of 16 months (range, 1-169
Conditioning details <0.001 months) we observed no significant differences in terms
Busulfan based 256 (54) 29 (24) of DFS (at 3 years ATLG 51%, PTCy 42%, P=0.3), relapse
TBI based 130 (27) 55 (45) incidence (ATLG 34% vs. PTCy 29%, P=0.261), OS (65%
Other 90 (19) 39 (32) vs. 58%, P=0.663) or moderate/severe cGVHD relapse-
TBI 146 (31) 55 (45) 0.003 free survival at 3 years was (ATLG 40% vs. PTCy 27%,
TBI dose P=0.068) between the two groups.
≤10 Gy 44 (9) 21 (17) 0.001
>10 Gy 102 (21) 34 (28) NS Immune reconstitution
We observed a faster reconstitution of CD3 T lympho-

R. Massoud et al.
cytes (CD3+) (P<0.05) and activated T cells Table 3. Multivariate non-relapse mortality
(CD3+/HLADR+) after ATLG than PTCy (P<0.05) (Figure Multivariate NRM HR (95% CI)
1A and B). In addition, as shown in Figure 2 and the P-value
Online Supplementary Table S1 the reconstitution of cyto-
toxic T cells (CD3+/CD8+) and also of naïve cytotoxic T ATLG vs. PTCy 1.6 (0.98-2.48)
0.061
cells (CD3+/CD8+/CD45RA+) (P=0.017) was significant-
ly faster in the ATLG group (on day 90, P=0.002), while Patient CMV serology 1.73 (1.09-2.75)
the reconstitution of memory cytotoxic T cells negative vs. positive 0.02
(CD3+/CD8+/CD45R0+) was comparable (Figure 2). In Patient age 1.69 (1.05-2.73)
contrast to cytotoxic CD8+ positive cells, CD3 helper <52 vs. ≥ 52 0.03
cells (CD3+/CD4+) had a trend for faster reconstitution in Donor Age 1.32 (0.81-2.13)
the PTCy group (Figure 3), which was sustained for naïve <34 vs. ≥ 34 0.26
(CD4+/CD45RA+) (P=0.002) as well as for memory Donor sex 1.61 (1.01-2.59)
helper cells (CD4+/CD45R0+) (P<0.001). The reconstitu- Male vs. Female 0.048
tion of B cells (CD19+) was similar in the ATLG and CD34 x106/kg 0.748 (0.46-1.2)
PTCy group (Figure 4A) and a trend for faster reconstitu- <7.2 vs. ≥ 7.2 0.24
tion of naïve B-cells (CD19+/CD27-/CD10+) was
observed in the PTCy group (Figure 4B). NK-cell reconsti- ECOG 1.4 (0.87-2.14)
3 vs. 0-2 0.17
tution was faster on day 30 in the ATLG group (P<0.001),
however the values on day 100 and 180 were similar after NRM: non-relapse mortality; HR: hazard ratio; CI: confidence interval; ATLG:
ATLG and PTCy (Figure 5A). Furthermore, NKT cells and anti T-cell lymphocyte globulin; PTCy: posttransplant cyclophosphamide;
gdT cells reconstitution was faster in the ATLG group ECOG: Eastern Cooperative Oncology group.
(P<0.05) at all time points (Figure 5B and C), while there
were no significant differences in regulatory T-cell
immune reconstitution between the two groups. Retiere et al. observed a rapid NK recovery within day
All our data is summarized in the Online Supplementary 30 after allo-HSCT in the ATLG and PTCy group, howev-
Table S1. All our findings were confirmed in a donor sub- er while they reported an increase in NK-cell counts in
group analysis (Online Supplementary Table S2). the ATLG group, they did not observe any effect of the
donor type on these values and concluded the recovery
rate was a direct effect of the difference in GVHD pro-
Discussion phylaxis between the two groups.15 Our results fall in line
with Retiere’s, we observed rapid reconstitution of NK
Although ATLG and PTCy are widely used for GVHD cells at day 30, and we observed a significantly higher
prevention in allo-SCT, data is scarce on their impact on percentage and absolute count of NK cells at day 30 in the
immune reconstitution and only one small prospective ATLG group. This was validated by our donor subgroup
study using RIC PBSC has compared immune reconstitu- analysis which allows us to conclude that this was a
tion PTCy to ATLG so far.15 In this retrospective study, direct effect of the difference in the TCD strategy. This
we compared the influence of ATLG to PTCY on immune supports the hypothesis that ATLG spares NK cells while
reconstitution and transplant outcomes after myeloabla- PTCy targets them.15 Rubio et al. reported that early
tive allogeneic PBSC transplant. In our study, we recovery of NKT cells post T-cell-repleted allo-SCT, and a
observed some strong differences in terms of cell counts, high NKT-cell dose in the graft are associated with pro-
immune reconstitution, infections moderate/severe tection from aGVHD.36,37 In addition Tae et al. report an
cGVHD and EBV reactivation between the two groups. increase in the incidence of aGVHD and of relapse in
Since NK and gdT cells have a protective role against patients with lower NKT-cell counts post-allo-SCT.38
many bacterial and viral infections including CMV16-32 the Retiere et al. did not observe any significant differences in
longer period of aplasia and the decreased numbers of NK the NKT-cell population between ATLG and PTCy.15
and NKT cells in the PTCy group can explain the higher However, in our study, we observed a higher number and
incidence of infections before day 100 in this group. percentage of NKT cells in the ATLG group when com-
One of the most recent studies of gdT-cell recovery and pared to PTCy and this was confirmed by our subgroup
their association with transplant outcomes was conduct- donor analysis. Nonetheless, we did not observe any sig-
ed on 102 pediatric patients with acute leukemia.33 They nificant differences in the incidence of relapse or aGVHD,
reported significantly improved PFS and OS in patients while we observed a significantly lower incidence of
with elevated gdT cells, these findings have also been moderate and severe cGVHD in the ATLG group when
reported in adults.34,35 In addition they reported a signifi- compared to the PTCy group.
cantly lower incidence of infections with a total absence Servais et al. studied the impact of ATLG on IR post
of bacterial infections in the high gdT-cell group.33 Our MAC PBSC allo-SCT.39 They looked precisely at memory
findings fall in line with the literature. We observed an and naïve T cells and they observed that ATLG selective-
early recovery of the gdT-cell population in both groups ly depletes naïve CD4+ T and naïve CD8+ T cells where-
independent of the donor subtype. In addition, gdT cell as it does not significantly impact memory Tcells. 39 Our
were consistently higher in the ATLG group in all evalua- results fall in line with Servais et al., as we observed a
tions when compared to the PTCy group, which may progressive increase in the naïve to memory ratio both in
explain the decreased overall incidence of infections in the CD4+ T cells and CD8+ T cells, indicating that the
this group. Even though our study was not designed for effect of ATLG effect is more pronounced on naïve T
long term outcomes, we observed no significant differ- cells than on memory T cells. However, in our study the
ence in DFS or OS between the two groups. effect of ATLG on naïve and memory CD4+ T cells was

A
Figure 1. Comparison between
ATLG and PTCy regarding
immune reconstitution of (A) acti-
vated T cells CD3+/HLADR+) and
(B) all T cells (CD3+) P*=P-value
at day 30; P**=P-value at day
100; P***=P-value at day 180; %:
percentage of cells; Absolut: abso-
lute number of cells. TLG: anti T-
cell lymphocyte globulin; PTCy:
and post-transplant cyclophos-
phamide.
more pronounced than PTCy. In our study the reconsti- static peripheral expansion (HPE) of donor T cells trans-
tution of CD8+ T cells was faster than that of CD4+ T- ferred with the graft and from the novel production of
cells with CD8+ T cells recovering by day 100, while the naïve T cells in the thymus.40,41 In patients receiving MAC
CD4+ T-cell reconstitution was not observed at the last most of the T cells originate from HPE, and given that
evaluation of the immune profile on day 180. In addi- ATLG persist for several weeks in circulation,42,43 it can be
tion, the CD4+/CD8+ ratio did not return to normal in hypothesized that ATLG selectively depletes donor
either of the two groups, which indicates incomplete naïve T cells while it spares other T-cell populations.
recovery of the CD4+ T-cell compartment. These find- This differential cytotoxic activity of ATLG has been
ings were confirmed by subgroup analysis according to demonstrated in vitro.44 In addition, HPE occurs more
the donor. asymmetrically between T cells, with CD8+ T cells hav-
It is well established that reconstitution of the T-cell ing higher proliferating potential by HPE when com-
compartment after allo-HSCT arises from both homeo- pared to CD4+ T cells.39 This may explain the decreased

R. Massoud et al.
A
Figure 2. Comparison between ATLG and
PTCY regarding immune reconstitution of
CD8+ cells. (A) Total CD8+ T cells; (B) naïve
CD8+ T cells; (C) memory CD8+ T cells. P*=P-
value at day 30; P**=P-value at day 100;
P***=P-value at day 180; %: percentage of
cells; Absolut: absolute number of cells.
ATLG: anti T-cell lymphocyte globulin; PTCy:
and post-transplant cyclophosphamide.

A
Figure 3. Immune reconstitution for CD4+ T-
cell ATLG versus PTCy. (A) Total CD4+ T cells;
(B) naïve CD4+ T cells; (C) memory CD4+ T
cells. P*=P-value at day 30; P**=P-value at
day 100; P***=P-value at day 180; %: per-
centage of cells; Absolut: absolute number of
cells. ATLG: anti T-cell lymphocyte globulin;
PTCy: and post-transplant cyclophosphamide.

R. Massoud et al.
A
Figure 4. Comparison between ATLG and
PTCy regarding immune reconstitution of B
cells(A) Total B cells; (B) naïve B cells. P*=P-
value at day 30; P**=P-value at day 100;
P***=P-value at day 180; %: percentage of
cells; Absolut: absolute number of cells.
ATLG: anti T-cell lymphocyte globulin; PTCy:
and post-transplant cyclophosphamide.
CD4+ T-cell population and increased CD8+ T-cell num- The higher percentage of T lymphocytes and activated
bers in the ATLG group. Another explanation for the lymphocytes in the ATLG group may be explained by the
decreased number of CD8+ T cells in the PTCy group is higher CD8+ T-cell and NTK-cell reconstitution observed
the ability of PTCy to selectively target proliferating NK in this group.
and CD8+ T cells more than CD4+ T cells.15 These find- It has been proven in animal models that Tregs suppress
ings were all validated in the donor subgroup analyses GVHD without decreasing GVL,45 and that they accelerate
which makes it safe to assume that the observed discrep- post-transplant T-cell immune reconstitution in murine
ancies between the ATLG and PTCy groups can be models.46 In our study Tregs persisted after transplant and
attributed to the difference in the TCD between the we observed no significant differences in Treg reconstitu-
groups. From our findings, we can hypothesize that tion post-allo-HSCT between the two groups. This may be
PTCy has less impact on all the CD4+ T cells, while it explained by the cyclophosphamide resistance of Tregs
has increased activity against CD8+ T cells, which was conferred by their increase in the expression of aldehyde
expressed by higher proliferation of CD4+ T cells in the dehydrogenase for cyclophosphamide detoxification
PTCy and CD8+ T cells in the ATLG group. In addition, which allow them to persist posttransplant in the PTCy
we can hypothesize that ATLG has a more pronounced setting,10,15 and by the selective sparing of Tregs by ATLG.39
effect on memory CD8+ and CD4+ T cells than PTCy. After allo-HSCT the numbers of total B cells normalize

A Figure 5. Comparison between ATLG and

PTCy regarding immune reconstitution of
innate immune system (A) B natural killer
(NK) cells; (B) natural killer T (NKT) cells; (C)
gdT cells. P*=P-value at day 30; P**= P-value
at day 100; P***=P-value at day 180; %: per-
centage of cells; Absolut: absolute number of
cells. ATLG: anti T-cell lymphocyte globulin;
PTCy: and post-transplant cyclophosphamide.

R. Massoud et al.
within 3 months to 1 year.47-49 However, the reconstitution in a subgroup analysis according to donor type. However,
in the first year compromise mainly transitional and naïve our findings should be confirmed in more homogenous
subsets and memory B cells occurs much later. Our results prospective studies.
show normalization of the total B-lymphocyte count at
day 60, but no complete recovery of naïve and memory B- Conclusion
cells at last follow-up. In addition, we observed a higher Acknowledging the bias associated with our study
percentage and count of naïve B cells in the PTCy group at especially its retrospective nature, while taking into con-
day 100. sideration the large sample size, it is safe to conclude that
Even though we observed a higher incidence of NRM in a better CD8+ T-cell, NK-cell, NKT-cell and gdT-cell
the PTCY group on univariate analysis, this did not persist reconstitution is observed in the ATLG group while
on multivariate analysis (hazard ratio 1.6; 95% Confidence improved CD4+ recovery is a hallmark of the PTCy
Interval: 0.98-2.48; P=0.061). This difference can be group. Even though these findings have been translated
explained by a higher proportion of high risk patients into a decreased incidence of infection and
(higher proportion of mismatch transplants and ALL) in the moderate/severe cGVHD in the ATLG group they had no
PTCY group when compared to ATLG. impact on any of the other long-term outcomes. So, it
Given the retrospective nature of our study and the het- remains undetermined which TCD strategy is better to
erogeneity of our patient population especially the differ- consider and results from well-designed randomized
ences in donor types, ATLG dosing, immune suppression studies are needed.
regiments and conditioning regimens, it should be noted
that our study is prone to bias especially with univariate Disclosures
analysis. In an attempt to reduce bias and render the pop- No conflicts of interest to disclose.
ulation more homogenous we selected consecutive
patients undergoing allo-SCT only with MAC regimens Contributions
and PBSC as a stem cell source. In addition, we conducted RM and NK designed the study, analyzed data, interpreted
subgroup analysis in which we found anecdotal differ- results, and wrote the manuscript; NG, UFF, GZ, SH, CW, FA,
ences in clinical outcomes and IR between the 30 mg/kg and MC collected and analyzed data. All authors approved the
and 60 mg/kg ATLG dose, and we confirmed our findings final version of the manuscript.
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ARTICLE Chronic Lymphocytic Leukemia
Ferrata Storti Foundation The complex karyotype landscape in chronic

lymphocytic leukemia allows the refinement of
the risk of Richter syndrome transformation
Andrea Visentin,1,2 Laura Bonaldi,3 Gian Matteo Rigolin,4
Francesca Romana Mauro,5 Annalisa Martines,3 Federica Frezzato,1,2
Stefano Pravato,1,2 Leila Romano Gargarella,1,2 Maria Antonella Bardi,4
Maurizio Cavallari4, Eleonora Volta,4 Francesco Cavazzini,4 Mauro Nanni,5
Monica Facco,1,2 Francesco Piazza,1,2 Anna Guarini,5 Robin Foà,5
Haematologica 2022 Gianpietro Semenzato,1,2 Antonio Cuneo4 and Livio Trentin1,2
Volume 107(4):868-876 1
Hematology and Clinical Immunology Unit, Department of Medicine, University of Padua,
Padua; 2Veneto Institute of Molecular Medicine, Padua; 3Immunology and Molecular
Oncology Unit, Veneto Institute of Oncology IOV-IRCSS, Padua; 4Hematology section,
Department of Medical Sciences, Azienda Ospedaliera-Universitaria, Arcispedale S.
Anna, University of Ferrara, Ferrara; 5Hematology division, Department of Precision and
Translational Medicine, "Sapienza" University, Rome, Italy
ABSTRACT
C
omplex karyotype (CK) at chronic lymphocytic leukemia (CLL)
diagnosis is a negative biomarker of adverse outcome. Since the
impact of CK and its subtypes, namely type-2 CK (CK with major
structural abnormalities) or high-CK (CK with ≥5 chromosome abnor-
malities), on the risk of developing Richter syndrome (RS) is unknown,
we carried out a multicenter real-life retrospective study to test its prog-
nostic impact. Among 540 CLL patients, 107 harbored a CK at CLL diag-
nosis, 78 were classified as CK2 and 52 as high-CK. Twenty-eight
patients developed RS during a median follow-up of 6.7 years. At the
time of CLL diagnosis, CK2 and high-CK were more common and pre-
dicted the highest risk of RS transformation, together with advanced
Binet stage, unmutated (U)-IGHV, 11q-, and TP53 abnormalities. We
Correspondence: integrated these variables into a hierarchical model: high-CK and/or CK2
LIVIO TRENTIN patients showed a 10-year time to RS (TTRS) of 31%; U-IGHV/11q-
livio.trentin@unipd.it /TP53 abnormalities/Binet stage B-C patients had a 10-year TTRS of
12%; mutated (M)-IGHV without CK and TP53 disruption a 10-year
ANDREA VISENTIN TTRS of 3% (P<0.0001). We herein demonstrate that CK landscape at
andrea.visentin@unipd.it
CLL diagnosis allows the risk of RS transformation to be refined and we
recapitulated clinico-biological variables into a prognostic model.
Received: January 5, 2021.
Accepted: May 21, 2021.
Pre-published: June 3, 2021. Introduction
Chronic lymphocytic leukemia (CLL), the most common leukemia in western

https://doi.org/10.3324/haematol.2021.278304 countries, is a remarkably heterogeneous disease, with some patients never requir-
ing treatments and others with a highly aggressive and/or rapidly progressive clin-
ical course.1,2 Richter syndrome (RS) is the transformation of CLL into an aggres-
©2022 Ferrata Storti Foundation sive lymphoma, most commonly resembling diffuse large B-cell lymphoma
Material published in Haematologica is covered by copyright. (DLBCL) or Hodgkin lymphoma (HL) variants.3,4 It is characterized by fast growing
All rights are reserved to the Ferrata Storti Foundation. Use of lymphadenopathies, 18-fluorodeoxyglucose (FDG) positron emission tomography
conditions:
computerized tomography (PET-CT)-avid masses, B symptoms, worsening per-
https://creativecommons.org/licenses/by-nc/4.0/legalcode. formance status and increased lactate dehydrogenase levels.5 It is a challenging
Copies of published material are allowed for personal or inter- task to distinguish RS from progressive CLL and it is even more difficult to study
nal use. Sharing published material for non-commercial pur- prognostic markers since the frequency of RS transformation affects 2%-10% of
CLL patients.5
sect. 3. Reproducing and sharing published material for com- Several studies have proved that chromosome banding analysis is able to refine
mercial purposes is not allowed without permission in writing the prognostic stratification of CLL compared to fluorescence in situ hybridization
from the publisher. (FISH) analysis. In fact, 22%-36% of CLL cases with ‘normal’ FISH carry a chro-
mosomal aberration following stimulated karyotypic analyses. In particular, com-
plex karyotype (CK), defined by the presence of at least three chromosome lesions

Complex karyotype subtypes and RS
in the same clone, is detectable in 14%-34% of CLL then harvested following standard procedures. Karyotype was
cases.6-9 The presence of a CK is both a negative prognos- described after the analysis of at least 20 G-banded metaphases
tic and predictive biomarker associated with an adverse using IKAROS software (MetasYstems, Altlhusseim, Germany),
outcome6,10 and worse response to chemoimmunothera- according to international guidelines (ISCN 2016). The definition
py,7, 11 as well as to novel agents,12,13 regardless of the CLL- of a complex karyotype (CK) was defined by the presence of three
IPI score or IGHV mutational status.8 However, the CK or more chromosome abnormalities in the same clone.6,8,23,24
itself is a heterogeneous quantitative and qualitative cyto- According to the literature, CK2 is represented by CK cases with
genetic category that includes numerical (i.e., monosomies major structural rearrangements that are unbalanced transloca-
and trisomies) and structural abnormalities (i.e., balanced tions, chromosome additions, insertion, duplications, ring, dicen-
and unbalanced translocations, marker chromosomes, tric and marker chromosomes, whereas complex karyotypes with
isochromosomes, deletions, insertions and additions). balanced translocations, deletions, monosomies or trisomies is
Recently, collaborative studies have demonstrated that defined as type-1 (CK1).16 High-CK cases were those presenting at
among CK cases assessed at CLL diagnosis, those harbor- least five chromosome abnormalities.14 Chromosome abnormali-
ing five or more chromosome abnormalities (high-CK)14 or ties found in only one metaphase were not considered as clonal,
those with major structural abnormalities, also called and were excluded. Karyotypes were reported by local cytogeneti-
type-2 CK (CK2),15,16 identify highly aggressive disease cists (AM, MAB and MN) and reviewed by LB and AV.
subsets with a poor outcome; the latter is also character- Detail descriptions of IGHV mutational status,25-29 an assess-
ized by a peculiar mRNA expression profile.15,17 Indeed, ment of stereotyped B-cell receptor (BCR),30,31 cytogenetics by flu-
most of the patients included in these retrospective studies orescence in situ hybridization (FISH),26,32 TP53 gene mutation,22
were managed with chemoimmunotherapy.14-16 However, and NOTCH1 c.7544_7545delCT analysis33 are available in the
the presence of CK has rarely been associated to the devel- Online Supplementary Methods.
opment of RS18 and, to date, it is unknown whether CK
subtypes, namely high-CK or CK2, could help to identify Statistical analysis
patients at a higher risk of developing an RS at CLL diag- Categorical variables were compared by the c2 test (for Binet
nosis. stages and FISH) or the Fisher exact test (gender, treatment, TP53
In this multicenter retrospective study, we documented, and IGHV), when appropriate. Continuous variables (median age)
for the first time, that the presence of a CK at CLL diagno- were compared using the Mann-Whitney test. TTRS was calculat-
sis is associated with an increased risk of developing an ed starting from the date of CLL diagnosis to RS transformation
RS. In particular, patients with CK2 and high-CK had the (event) or last known follow-up (censored).19,34 Overall Survival
highest likelihood of RS transformation. Finally, we reca- (OS) was calculated starting from the date of CLL or RS diagnosis,
pitulated clinico-biological variables associated with RS when specified, to death for any cause, or to last known follow-
into a prognostic model defining three statistically distinct up. Survival analyses were performed by the Kaplan-Meier
classes of risk of developing RS, the lowest risk for IGHV method and the Log-rank test was used to compare survival
gene mutated (M-IGHV) being patients without any CK curves between groups. The Cox regression model was employed
subtypes and an absence of TP53 abnormalities, and the to estimate hazard ratios (HR). The Cox proportional hazard
highest risk for patients harboring highCK and/or CK2 assumption was assessed based on the scaled Schoenfeld residu-
subtypes. als. The stability of our model was internally validated by the
bootstrap 0.632 method with B=540. The Harrell concordance
index (c-index; 1.0 indicates a perfect discrimination, while a value
Methods of 0.5 indicates equivalence to chance) was used to compare our
prognostic model.35 The prediction error was calculated as 1 - c-
Study design index, corrected for optimism and estimated using the 0.632 boot-
Inclusion criteria for this study were diagnosis of CLL according strap method.36 Akaike information criterium (AIC) was calculat-
to the 2008 iwCLL guidelines,19 histologically confirmed diagnosis ed using the AIC function with R (an open-source statistical pack-
of RS (diffuse large B-cell lymphoma or high-grade B-cell lym- age downloadable from http://www.r-project.org).37 A P value >0.05
phoma), age >18 years and chromosome banding analysis per- was considered as not significant.
formed within one year of diagnosis in patients without features
of disease progression. Data included in the comparative analysis
were gender, age, Binet stage,19 CLL treatment prior to RS, 11q22- Results
23 deletion by FISH,20 IGHV gene mutational analysis21 and TP53
abnormalities including gene deletions (deletion 17p13) or muta- Patients’ characteristics
tions,22 and b2-microglobulin level >3.5 mg/L. The primary end- We gathered data from 540 treatment-naive CLL
point was the impact of overall CK, CK2 and highCK on the time patients with chromosome banding analysis assessed
to Richter syndrome (TTRS) transformation. The correlation of RS within 12 months from diagnosis at three Italian centers
with clinical and biologic variables and their impact on TTRS were (Table 1). The median age at diagnosis of the whole case
secondary endpoints. This study was approved by the local series was 63±12 years, 61% were male, 75% showed
research ethics committee and informed consent was obtained Binet A stage, the median b2-microglobulin was 2.93
from all patients. mg/L, 57% of patients were U-IGHV, 11% harbored TP53
abnormalities (8% 17p13 deletion and 3% only TP53
Chromosome banding analysis mutation) and 20% a CK (Figure S1A). NOTCH1 muta-
Cytogenetic analysis was performed on peripheral blood after a tion was assessed in 47 patients at CLL diagnosis and it
72h exposure to 500 mM CpG ODN DSP30 (Roche, Risch, CH) was found in two subjects who further developed RS.
mitogen + 20U/mL IL2 (Roche). Cultures were exposed overnight Two hundred and fifty-two patients subsequently
to 0.1 mg/mL colcemid (Gibco® Karyomax Colcemid, received at least one line of therapy - 31% FCR (fludara-
ThermoFisher, Waltham, MA, USA) to obtain metaphases and bine, cyclophosphamide, rituximab), 17% BR (bendamus-

A. Visentin et al.
Table 1. Clinical and biological features of patients.

Population RS no RS P
(n=540) (n= 28) (n=512)
Age (years)
median±sd 63±12 63±9.8 63±12 0.8793
Age at RS (years)
median±sd 68±12 68±12 - n.a.
Gender
Female 211 (39%) 11 (39%) 200 (39%) >0.9999
Male 329 (61%) 17 (61%) 312 (61%)
Binet stage
A 407 (75%) 15 (54%) 392 (77%) 0.0113
B-C 133 (25%) 13 (46%) 120 (23%)
b2-microglobulin (mg/L)*
median±sd 2.9±1.5 3.2±0.98 2.9±1.6 0.1216
IGHV status
M-IGHV 232 (43%) 6 (21%) 225 (44%) 0.019
U-IGHV 309 (57%) 22 (79%) 287 (56%)
FISH
13q or Normal 404 (75%) 19 (68%) 385 (75%) 0.0861
+12 84 (15%) 3 (11%) 81 (16%) 0.0415+
11q- 52 (10%) 6 (21%) 46 (9%)
TP53 abn
Normal 482 (89%) 19 (68%) 463 (90%) 0.0043
Disrupted 58 (11%) 9 (32%) 49 (10%)
KARYOTYPE
no CK 433 (80%) 14 (50%) 419 (82%) 0.0002
CK 107 (20%) 14 (50%) 93 (18%)
QUALITATIVE
no CK 433 (80%) 14 (50%) 419 (82%) <0.0001
CK1 29 (5%) 1 (4%) 28 (5%)
CK2 78 (14%) 13 (46%) 65 (13%)
QUANTITATIVE
0 165 (30%) 3 (11%) 162 (32%) <0.0001
1-2 269 (50%) 11 (39%) 258 (50%)
3-4 54 (10%) 3 (11%) 52 (10%)
≥5 52 (10%) 11 (39%) 41 (8%)
RS SCORE
low-risk 212 (39%) 3 (11%) 209 (41%) <0.0001
Int.-risk 247 (46%) 12 (43%) 235 (46%)
high-risk 81 (15%) 13 (46%) 68 (13%)
RS: Richter syndrome; sd: standard deviation; M-IGHV: mutated IGHV gene; U-IGHV: unmutated IGHV gene; 11q-: del11q22-23 by interphase FISH analysis; TP53 abn: TP53 abnor-
malities include deletions and/or mutations; CK: complex karyotype; CK1: type-1 CK; CK2: type-2 CK; highCK: ≥5 chromosome abnormalities; high-risk: CK2 and/or highCK; int.-
risk: U-IGHV/11q-/TP53abn/Binet B-C; low-risk: M-IGHV without CK and TP53 wild type; n.a.: not applicable; * data available from 520 (96%) patients, 26 (93%) who developed
an RS and 494 (96%) who did not transform; + Analysis between subgroups with 11q- and others.
tine, rituximab), 10% ibrutinib, 5% chlorambucil plus an malities and 52 (10%) were classified as high-CK (i.e., ≥5
anti-CD20 monoclonal antibody, 2% venetoclax, 35% chromosome lesions) (Online Supplementary Figure S1A and
other treatments such as FC or chlorambucil single agent S2A, Table 1). In particular, a high-CK was more common
as first line therapy - and 90 died during the follow-up. in CK2 than in CK1 patients, being present in 63% of
According to the qualitative CK subtype, 29 of 107 patients harboring a CK2 subtype but in only 10% of CK1
(27%) patients displayed a CK1 and 78 (73%) a CK2 patients (P<0.0001, Online Supplementary Figure S2A).
(Online Supplementary Figure S1A, Table 1) whereas, As a preliminary step for our further analysis, we con-
according to the number of chromosome lesions, 165 firmed the established prognostic role of overall CK, CK
(30%) patients had a normal karyotype (i.e., 46,(XX) or with major unbalanced abnormalities (i.e., CK2) and
46,(XY) for females and males, respectively), 268 (50%) highCK in our dataset (Figure S2B-D). The 10-year OS
had one or two lesions, and 54 (10%) three or four abnor- was 54% and 79% for CK and no-CK patients, respective-

Table 2. Hazard ratios (HR) for the time to Richter syndrome.

Univariate analysis Multivariate analysis
HR 95% C.I. P HR 95% C.I. P
TTRS
≥65 years 1.4 0.6-2.9 0.4289 - - -
Age+ 1.02 0.9-1.1 0.2120 - - -
Male 1.0 0.5-2.1 0.9379 - - -
b2MG high* 1.8 0.6-5.6 0.2925 - - -
Binet B-C 3.9 1.6-9.6 0.0024 2.9 1.4-6.3 0.0039
U-IGHV 4.0 1.9-8.6 0.0004 4.5 1.8-11.3 0.0011
+12 0.8 0.3-2.4 0.6675 - - -
11q- 4.6 1.3-16.7 0.0215 2.8 1.1-6.9 0.0285
TP53 abn 9.5 2.9-31.4 0.0002 3.9 1.8-8.7 0.0008
CK 7.4 3.0-18.3 <0.0001 4.7 2.2-9.9 <0.0001
CK2 8.8 4.9-19.8 <0.0001 5.6 2.7-11.8 <0.0001
High-CK 9.9 6.5-22.9 <0.0001 6.9 3.3-14.9 <0.0001
RS MODEL
Low-risk 1.0 - - 1.00 - -
Int.-risk 4.0 1.4-11.4 0.0101 3.4 1.5-7.5 0.0023
High-risk 13.6 7.1-20.9 <0.0001 9.2 4.7-17.3 <0.0001
TTRS: time to Richter syndrome; b2MG high: beta2-microglobulin >3.5mg/L; U-IGHV: unmutated IGHV gene; TP53 abn: TP53 abnormalities include deletions and/or mutations;
CK: complex karyotype; CK1: type-1 CK; CK2: type-2 CK; highCK: ≥5 chromosome abnormalities; High-risk: CK2 and/or highCK; Int.-risk: U-IGHV/11q-/TP53abn/Binet B-C; Low-
risk: M-IGHV without CK and TP53 wild type; n.a.: not applicable; + : age considered as continuous variable; * : data available from 520 (96%) patients, 26 (93%) who developed
an RS and 494 (96%) who did not transform.
ly (P<0.0001, Online Supplementary Figure S2B); 48% vs. time. As shown in Figure 1A, 2.6%, 12% and 13% of
72% vs. 79% for CK2, CK1 and no-CK (P<0.0001, Online patients developed an RS within five, ten and 15 years
Supplementary Figure S2C), respectively; 44% vs. 64% vs. after CLL diagnosis, respectively. We observed that
70% vs. 90% for patients with ≥5 (i.e., high-CK), 4-3, 2-1 patients with a CK, overall (Figure 1B) and its subtypes
and without chromosome abnormalities (P<0.0001, (Figure 1C-D), had a very high risk of developing an RS.
Online Supplementary Figure S2D), respectively. The estimated ten-year TTRS to be 25% vs. 8%
(P<0.0001), 38% vs. 8% (P<0.0001) and 41% vs. 8%
Clinico-biological features of patients who developed (P<0.0001) for patients with CK vs. no-CK, CK2 vs. other
a RS transformation patients (i.e., CK1 or noCK), highCK vs. other patients
Twenty-eight (5.2%) patients developed a histologically (i.e., 3-4 or 1-2 or, 0 chromosome abnormalities) respec-
confirmed RS over a median follow-up of 6.7 years (Figure tively (Figure 1C-D and S3G-H). Multivariate analysis
S1B). The median age at RS diagnosis was 68 years (range revealed that CK overall was associated with a more than
38-84), 61% were male, 75% had received a CLL treat- four-fold higher risk of developing an RS (HR 4.7, 95% CI
ment in the past, 79% were U-IGHV, 32% presented TP53 2.2-9.9, P<0.0001). This risk was even higher for CK sub-
abnormalities, 50% harbored a CK at CLL diagnosis, types, being more than five-fold (HR 5.6, 95% CI 2.7-11.8,
which included 46% and 39% of CK2 and high-CK sub- P<0.0001) and seven-fold (HR 6.9, 95% CI 3.3-14.9,
types, respectively. Eight cases showed deletion of 9p21.3, P<0.0001) higher for patients harboring CK2 and high-CK
i.e., the locus of CDKN2A gene, all with a CK. In particu- subtypes, respectively (Table 2). Other variables associat-
lar, 8 of 8 were classified as CK2 and 6 of 8 as high-CK ed with TTRS at univariate and multivariate analysis were
subtype. Only one patient, who developed RS, received Binet stage B-C, U-IGHV, 11q-, TP53 abnormalities (Table
ibrutinib frontline. We also observed that more patients 2, Online Supplementary Figure S3A-E).
who developed an RS displayed an advanced Binet stage Among CK2 and/or high-CK patients (n=81), 32 (39%)
at CLL diagnosis (P=0.0113) and were enriched in U-IGHV patients carried TP53 abnormalities and 52 (63%) an
(P=0.0191), TP53 abnormalities (P=0.0043), CK overall U-IGHV status. We found that TP53 abnormalities and
(P=0.0002), CK2 (P<0.0001) and high-CK (P<0.0001) cases IGHV status mildly impact on the risk of developing RS
as compared to patients who did not develop an RS (Table among CK2 and/or high-CK subgroup, but the difference
1, Figure S1C). Age at CLL diagnosis (median age 63.5 and was not statistically significant (Figure S4A-B, P=0.1150
63.3 years), gender distribution (both 61%), trisomy of and P=0.1405, respectively). These data suggest that CK
chromosome 12 (11% and 16%), b2-microglobulin (medi- subtypes per se represent a stronger prognosticator of RS
an levels 3.2mg/L and 2.9mg/L) and stereotyped BCR transformation than conventional biologic markers such
(10.7% vs. 9.8%) had a superimposable distribution as TP53 disruption and U-IGHV conformation.
among patients with and without an RS transformation The median OS from CLL diagnosis for the whole pop-
(Table 1). ulation was not reached and the estimated ten-year OS
was 73% (Figure S5A). Patients who developed an RS had
Prognosticators of Richter Syndrome a shorter OS (Figure 2A). The median OS was seven years
The cumulative incidence of RS slowly increases over vs. not reached and the estimated ten-year OS was 16%

A. Visentin et al.
A B
C D
Figure 1. Kaplan Meyer curves of time to Richter syndrome. The upper-left (A) panel shows the time to Richter syndrome (RS) transformation for the whole popula-
tion. Patients with a CK overall (B), CK2 (C) or high-CK (D) have a significantly increased a risk of developing an RS compared to the other patients (Log-rank test,
P<0.0001).
vs. 79% for patients who developed an RS vs. those who to the identification of three statistically different groups.
did not transform (Figure 2E, P<0.0001), respectively. These were ranked from the shortest to the longest TTRS,
Variables that were associated with a higher risk of death as follows: 81 (15%) patients were classified as high-CK
in multivariate analysis are summarized in Table S1. and/or CK2, and had a five-year and ten-year TTRS of
The median time from CLL diagnosis to RS transforma- 13% and 31%; 247 (46%) patients displayed a U-IGHV
tion was 5.3 years. ranging from 0.10 years to 10.8 years. status or 11q- or TP53 disruption or Binet stage B-C, and
In only one patient was RS was diagnosed within six showed the five-year and ten-year TTRS of 0.9% and
months of CLL diagnosis. The median OS from RS trans- 12%; 212 (39%) patients were M-IGHV without CK and
formation was 5.3 months and the two-year OS was only TP53 abnormalities, and had a five-year and ten-year
20% (Figure 2B). The OS from RS was not affected by the TTRS of 0.7% and 3% (Figure 3, P<0.0001). Multivariate
presence of a CK at CLL diagnosis nor its subtypes (Figure analysis confirmed that the former subgroup (i.e., high-CK
S5B-C). The two-year OS from RS was 28% vs. 10% for and/or CK2) was associated with the highest risk of RS
CK2 cases and other patients (i.e., CK1 and no-CK) transformation (HR 9.2, 95% CI 3.8-46, P<0.0001) com-
(P=0.3317) and 24% vs 16% for high-CK cases and other pared to the low-risk group, one which is characterized by
patients, respectively (i.e., <5 chromosome abnormalities) the presence of M-IGHV without CK and TP53 abnormal-
(P=0.9864) (Figure S5C-D). No traditional prognostic ities. Patients with U-IGHV or 11q- or TP53 abnormalities
markers could foresee the risk of death after RS diagnosis or Binet stage B-C had an intermediate risk, with a three-
in our population (Table S2). fold higher risk of RS compared to low-risk patients (HR
3.4, 95% CI 1.5-7.5, P=0.0023) (Table 2). Our model was
A Richter syndrome prognostic model also internally validated using the bootstrap 0.632 method
By integrating CK subtypes, TP53 abnormalities, 11q showing a prediction error of 0.26. Finally, the c-index for
deletion, IGHV mutational status and Binet stages based our proposed model was 0.81 for TTRS and the Akaike
on HR values, we developed a hierarchical model leading information criterium was 286. These results indicate that

A B
Figure 2. Kaplan Meyer curves of overall survival. The left panel (E) shows the overall survival analysis for patients with RS transformation and those who did not
develop an RS (no RS). Patients who developed an RS had a shorter overall survival, calculated from CLL diagnosis (Log-rank test, P<0.0001). The right (F) panel
shows the overall survival after RS transformation, confirming these patients’ very poor prognosis.
our model had a good prediction accuracy for the risk of mutation and a stereotype BCR subset #8.44,45 To date, the
developing an RS; higher than of the CLL-IPI38 (c-index impact of CK at CLL diagnosis on the risk of developing
0.69, prediction error 0.28, Akaike information criterium RS has been investigated in only a few studies.46,47
301) and the Barcelona-Brno39 (c-index 0.74, prediction The German CLL study group has recently reviewed the
error 0.25, Akaike information criterium 292) scores accu- clinical features of RS patients as part of their clinical
racies applied to our population (Figure S4C-D). Based on trials.34 In this study, 3.5% of CLL developed an RS trans-
the lower Akaike score, our RS prognostic model better formation after a median observation time of 4.4 years.
predicts the risk of developing an RS than the available The median age at RS was 65 years and the median OS
comparators. after RS was 9.4 months, which was significantly longer
for HL compared to the DLBCL variant (median OS 83
months vs. 8.7 months, respectively). Adverse risk factors
Discussion at trial enrollment, such as 17p13 deletion by FISH, high
b2-microglobulin and CLL-IPI scores were more common
In this multicenter retrospective study, we demonstrat- in patients who developed an RS34 while NOTCH1
ed that patients harboring a CK at CLL diagnosis, in par- mutations and stereotype #8 were not recurrent in RS
ticular those with CK2 and/or high-CK, are characterized cases.34 Conversely, among the 204 RS from the Mayo
by the highest risk of developing an RS transformation. clinic, the median OS after RS diagnosis was 12 months.48
Subsequently, by integrating data of CK subtypes with In a multivariate Cox regression analysis, prior CLL treat-
other clinical and biologic variables associated with the ment and older age, but not TP53 disruption, were associ-
risk of RS, we were able to define an RS prognostic model. ated with a shorter OS.48 The results of our real-life study
To minimize selection and attrition biases, as well as are in line with the GCLLSG and Mayo clinic reports, even
imprecise reporting of data inherent to observational stud- though our patients were slightly older; this could explain
ies, we asked the clinicians to report all patients who per- the shorter OS in our RS cohort (median survival after RS
formed stimulated cytogenetic analysis within the first is 5.3 months). Comparable survival rates, between six
year of diagnosis. We analyzed the reported data and per- and 12 months, have been observed in other retrospective
formed computerized and manual consistency checks on analyses.34,44,48 In addition, advanced Binet stage, U-IGHV
each case report form. and 11q- were also significantly associated with an RS risk
RS is a rare and an aggressive complication of CLL in our patients.
patients, affecting between 2% and 10% of CLLs.34 Most Chromosome banding analysis in CLL is capable of
RS patients are elderly, have a poor performance status identifying chromosomal abnormalities that are missed by
and suffer from several comorbidities which limit the use FISH analysis, sometimes fulfilling CK criteria.6,24,49,50
of intensive chemoimmunotherapy.40 Since the majority Genomic microarrays have also emerged as a valuable tool
of patients are primary refractory to first-line treatment for genome-wide studies in CLL. However, in a recent
and only a few are able to undergo allogenic stem cell study, no significant differences emerged in patients’ clas-
transplantation procedures, the reported estimated sur- sification, time to first treatment, OS and prediction accu-
vival after a diagnosis of RS is usually less than one year, racy between chromosome banding analysis and genomic
even with the introduction of targeted-therapy41,42 and microarrays.51 The prognostic and predictive role of CK,
immune checkpoint inhibitors.43 For these reasons, the defined by the presence of at least three chromosomal
standard of care of patients with RS remains a primary lesions, is evident at diagnosis,6,8 as well as at disease pro-
unmet need. Known biologic risk factors for the develop- gression7 and in relapsed/refractory patients treated with
ment of RS are TP53 and CDKN2A aberrations, NOTCH1 ibrutinib13,52 or venetoclax.12 Of note, CK was not a prog-

A. Visentin et al.
Figure 3. The Richter syndrome scoring system. Kaplan-Meier curve of time to Richter syndrome transformation according to the Richter syndrome scoring system.
Patients were classified at high-risk if they were high-CK and/or CK2 at CLL diagnosis (blue curve); at intermediate-risk if they displayed unmutated IGHV status (U-
IGHV), 11q22-23 deletion (11q), TP53 abnormalities (including deletions or mutations, TP53 abn) or Binet stage B-C (grey curve); at low-risk if they were IGHV mutat-
ed (M-IGHV) patients without CK and wild-type TP53 gene (TP53 not deleted non mutated) (orange curve).
nostic marker of survival on multivariate analysis for adverse impact on the R-EPOCH regimen. A recent study
patients treated frontline with ibrutinib±rituximab53 while from Ohio State University found that six of nine patients
treatment with idelalisib plus rituximab seems to have a with a near-tetraploidy (four copies of most chromo-
comparable efficacy in R/R patients with and without somes) karyotype developed an RS.46 At multivariate
CK.54-56 CK has been found in 14%-35% of CLL depend- analysis, near-tetraploidy and CK predicted ibrutinib dis-
ing on the study in question,6,10 and identifies a heteroge- continuation due to transformation.46 Although the exact
neous cytogenetic category in terms of quantitative and mechanism that favors the development of an RS in
qualitative characteristics. Data from the literature has patients with CK is unknown, the strong association
documented that the presence of at least five chromoso- between CK and TP53 abnormalities, short telomere
mal aberrations is associated with a very aggressive clini- length and, consequently, increased chromosome instabil-
cal course independent of the IGHV status and TP53 ity could play a relevant role.18,58
lesions.14 Our collaborative group has previously demon- Thanks to stimulated chromosome banding analysis,
strated that almost 70% of CK cases harbor major struc- we were able to identify a CK in 20% of 540 CLL patients
tural aberrations such as unbalanced translocations and and could demonstrate that patients harboring a CK2 or a
ring or marker chromosomes.15 This subset, called CK2, high-CK had a six and seven-fold increased risk of devel-
was associated with a higher incidence of TP53 aberra- oping RS. We therefore suggest that the integration of CK
tions, chemo-refractoriness, early relapse after chemoim- subtypes, together with IGHV mutational status, TP53
munotherapy, and a shorter OS at multivariate analysis.15 abnormalities, 11q22-23 deletion and Binet stage, may
In addition, the prognostic and predictive accuracy of CK allow the prognostic risk of RS transformation (Figure 3)
subtypes is enhanced when it is combined with IGHV to be refined. Indeed, we could show that M-IGHV
mutational status.16 Interestingly, a recent analysis of the patients without any CK subtypes and a wild-type TP53
international CLL14 clinical trial suggests that the fixed- gene are characterized by a very low risk of developing
duration combination of obinutuzumab plus venetoclax RS, at only 0.7% five years from CLL diagnosis. On the
seems to overcome the negative predictive impact of CK, other hand, patients with CK subtypes, both CK2 and/or
both in terms of undetectable minimal residual disease high-CK, are characterized by the highest risk of develop-
rates and progression-free survival.57 ing RS, with 31% of them experiencing a disease transfor-
The presence of CK has been sporadically linked to the mation within ten years of diagnosis. In addition, our
development of RS.18 In a retrospective study on CLL model seems to better predict the risk of RS transforma-
patients treated with FCR, one of four cases with RS had tion than the available scoring systems. Our results, like
a CK.9 Anderson et al.12 found a CK in 48% of the 25 most data found in the literature, derived from a cohort of
patients who progressed on venetoclax, including eight of patients treated mainly with chemoimmunotherapy, a
17 patients with RS. Rogers et al.,47 reported a CK in 67% choice made partly due to their longer follow-up.
patients who developed an RS and found that a CK had an Although the cumulative incidence of RS among patients

treated with chemo/chemoimmunotherapy seems to be and MN performed cytogenetic tests; FF, MF and AG performed
higher than patients treated with BTK or BCL2 inhibitors, cytofluorimetric and IGHV analysis; FRM, GMR, PF, GS, RF,
this difference was not statistically significant (P=0.3337, AC and LT visited patients, provided intellectual inputs and
Online Supplementary Figure S5E). In addition, after valida- reviewed the article.
tion by an independent cohort of patients treated frontline
with targeted drugs and in a prospective study, our prog- Funding
nostic model might be used in the follow-up management This work was supported by funds from Associazione Italiana
of patients with CLL. In particular, patients with a CK2 per la Ricerca sul Cancro (A.I.R.C.) projects to LT (IG-25024),
and/or high-CK should be carefully monitored for the Gilead fellowship program 2018 to LT, Special Program
development of an RS during their follow-up. ‘Metastatic disease: the key unmet need in oncology’, AIRC
5x1000 (No. 21198) to RF, Fondo di Ateneo per la Ricerca 2016,
Disclosures 2017 of the University of Ferrara to GMR and FC, Fondo di
AV received honoraria from Janssen, Abbvie, Italfarmaco. LT Incentivazione alla Ricerca 2017 of the University of Ferrara to
received research funding by Gilead, Roche, Janssen and GMR, Ministero dell’Istruzione, dell’Università e della Ricerca
Takeda, advisory board for Roche, Shire and Abbvie, PRIN 2015 to AC (2015ZMRFEA). AV received a research fel-
Astrazeneca. GMR received research funding by Gilead. FRM lowship from the University of Padua supported by ONLUS
advisory board for Janssen, Shire and Abbvie. AC advisory Ricerca per Credere nella Vita (RCV) odv, Padua, Italy.
board and speaker bureau for Roche, Abbvie, Gilead and This study was approved by the local research ethics committee
Janssen. GS board member of Abbvie, Roche, Janssen and of Padua hospital and informed consent was obtained from all
Celgene. RF advisory board or speaker bureau for Roche, Abbvie, patients.
Celgene, Incyte, Amgen, Janssen, Gilead and Novartis.
Data sharing statement
Contributions The datasets generated and analyzed during the current study
AV designed the study, performed statistical analysis, visited are not publicly available due to the data protection and lack of
patients and wrote the article; SP, LRG, MC, EV and FC and consent from the patients. Access to data is strictly limited to the
provided intellectual inputs and visited patients; LB, AM, MAB researchers who have obtained permission for data processing.
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evaluation of the comparative effectiveness Clinical characteristics and outcomes of Leukemia. 2019;33(9):2183-2194.

Chronic Lymphocytic Leukemia ARTICLE
IGHV-associated methylation signatures more Ferrata Storti Foundation

accurately predict clinical outcomes of chron-
ic lymphocytic leukemia patients than IGHV
mutation load
Dianna Hussmann,1 Anna Starnawska,1,2,3,4 Louise Kristensen,5 Iben
Daugaard,1,6 Astrid Thomsen,1 Tina E. Kjeldsen,1 Christine Søholm Hansen,2,7
Jonas Bybjerg-Grauholm,2,7 Karina Dalsgaard Johansen,8 Maja Ludvigsen,8,9
Thomas Kristensen,5 Thomas Stauffer Larsen,10 Michael Boe Møller,5 Charlotte
Guldborg Nyvold,11 Lise Lotte Hansen1 and Tomasz K. Wojdacz1,12,13
Haematologica 2022
Volume 107(4):877-886
1
Department of Biomedicine, Aarhus University, Aarhus, Denmark; 2The Lundbeck
Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, Denmark;
3
Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, Denmark; 4Center
for Genomics and Personalized Medicine, CGPM, Aarhus University, Aarhus, Denmark;
5
Department of Pathology, Odense University Hospital, Odense, Denmark; 6Department
of Pathology, Aarhus University Hospital, Aarhus, Denmark; 7Department for Congenital
Disorders, Statens Serum Institut, Copenhagen, Denmark; 8Department of Hematology,
Aarhus University Hospital, Aarhus, Denmark; 9Department of Clinical Medicine, Aarhus
University, Aarhus, Denmark; 10Department of Haematology, Odense University Hospital,
Odense, Denmark; 11Haematology-Pathology Research Laboratory, Research Unit for
Haematology and Research Unit for Pathology, University of Southern Denmark and
Odense University Hospital, Odense, Denmark; 12Aarhus Institute of Advanced Studies,
Aarhus University, Aarhus, Denmark and 13Independent Clinical Epigenetics Laboratory,
Pomeranian Medical University, Szczecin, Poland
ABSTRACT
C
urrently, no molecular biomarker indices are used in standard care
to make treatment decisions at diagnosis of chronic lymphocytic
leukemia (CLL). We used Infinium MethylationEPIC array data
from diagnostic blood samples of 114 CLL patients and developed a pro-
cedure to stratify patients based on methylation signatures associated
with mutation load of the IGHV gene. This procedure allowed us to pre-
dict the time to treatment with a hazard ratio (HR) of 8.34 (95% confi-
dence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: Correspondence:
2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases
TOMASZ K. WOJDACZ
for which the two classification procedures gave discrepant results tomasz.wojdacz@pum.edu.pl
showed that these cases were incorrectly classified using IGHV status.
Moreover, methylation-based classification stratified patients with dif-
ferent overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not pos- Received: January 31, 2021.
sible using IGHV status. Furthermore, we assessed the performance of Accepted: May 21, 2021.
the developed classification procedure using published Pre-published: June 3, 2021.
HumanMethylation450 array data for 159 patients for whom informa-
tion on time to treatment, overall survival and relapse was available.
Despite 450K array methylation data not containing all the biomarkers https://doi.org/10.3324/haematol.2021.278477
used in our classification procedure, methylation signatures again strati-
fied patients with significantly better accuracy than did IGHV mutation ©2022 Ferrata Storti Foundation
load regarding all available clinical outcomes. Thus, stratification using Material published in Haematologica is covered by copyright.
IGHV-associated methylation signatures may provide better prognostic All rights are reserved to the Ferrata Storti Foundation. Use of
power than IGHV mutation status. published material is allowed under the following terms and
conditions:
Introduction nal use. Sharing published material for non-commercial pur-
Most patients diagnosed with chronic lymphocytic leukemia (CLL) have asymp- sect. 3. Reproducing and sharing published material for com-
tomatic, early-stage disease at the time of diagnosis but the subsequent disease mercial purposes is not allowed without permission in writing
course is highly variable, with some patients experiencing early progression and from the publisher.
others living for many years with indolent disease.1 Immediate treatment after
diagnosis does not seem to improve patients’ survival.2-5 Consequently, to reduce

D. Hussmann et al.
unnecessary harmful complications following therapy, nostic significance. In this study, we developed a proce-
the majority of CLL patients are managed with a “watch dure for classifying patients based on methylation
and wait” strategy,6 and treatment is only initiated at dis- changes associated with IGHV mutation load, comparing
ease progression. This is assessed according to clinical the prognostic power of this classification procedure to
symptoms defined by the Rai and Binet staging systems.7- predict clinical outcomes with that of patients’ stratifica-
9
However, with the advent of new therapies it is well- tion based on IGHV mutation load alone.
recognized that some patients can potentially benefit
from earlier intervention.9
Molecular biomarker-based indices, as opposed to clin- Methods
ical staging, are likely to reflect the complex biology of
CLL and, therefore, predict patients’ outcomes more accu- Clinical material
rately.10 However, the development of biomarker-based Our cohort of patients has already been described;23,24 the
indices in CLL is still ongoing. Recent large multicenter patients’ clinicobiological characteristics are summarized in
studies, investigating the prognostic power of various Table 1 (see Online Supplementary File, Patient Cohort Section). The
known molecular and clinical biomarkers, have proposed Ethics Committee of the Region of Southern Denmark approved
two new biomarker indices: the International Prognostic the study (approval number: S-20100128).
Index for Chronic Lymphocytic Leukemia (CLL-IPI) and
the International Prognostic Score for Early-stage CLL Genome-wide DNA methylation analysis
(IPS-E).11,12 The CLL-IPI index is based on TP53 aberrations, To assess genome-wide DNA methylation, we analyzed 400
IGHV mutation status, b2-microglobulin concentration, ng of DNA with the Illumina Infinium MethylationEPIC
clinical Rai/Binet stage, and age, with TP53 aberrations Beadchip (EPIC) array. Raw data were processed in R using the
predicting overall survival (OS) most accurately in multi- RnBeads package25 with default filtering settings including the
variable modeling.11 However, lesions affecting the TP53 removal of probes, which were: (i) outside CpG context; (ii)
locus are rather rare and other studies have shown that overlapping single-nucleotide polymorphisms; (iii) targeting sex
IGHV-mutated patients with TP53 locus aberrations expe- chromosomes; (iv) missing b-values; (v) showing a standard
rience a rather indolent disease course.13-15 deviation of b-values <0.005; and (vi) cross-reactive probes.26 b-
The IPS-E index was developed for early-stage patients values were normalized using the BMIQ method27 followed by
with asymptomatic disease and time to first treatment noob background correction.28 We assessed the sample purity
(TTFT) as a primary outcome.12 This index includes IGHV using the methylomic data,19 and included only patients’ sam-
status, absolute lymphocyte count and palpable lymph ples with at least 85% B cells (n=114) to limit the impact of cell-
nodes. TP53 status did not show independent prognostic type composition.
power in this index, which indicates that this biomarker
may provide no clinical relevance for predicting TTFT for Bioinformatic and statistical analyses
early-stage patients. Bioinformatic and statistical analyses were performed in R
In both of the above indices, stratification of patients version 3.6.1, Stata/SE 15.0 (StataCorp, TX, USA), and Qlucore
into mutated (M-CLL) or unmutated (U-CLL), according Omics Explorer 3.4 (Qlucore, Lund, Sweden). We used linear
to IGHV mutation load, plays a central role.16 regression to test the association between methylation levels at
It is well-established that the CLL methylome reflects, individual CpG loci (b-values) and mutation load of IGHV (as
to a large extent, the natural history of the B cell.17-20 percentage identity to germline sequence to avoid specific cut-
Recent studies have also shown that the CLL methylome off29) for a total of 671,684 CpG, using P<10-8 as recommended
can guide the stratification of patients experiencing differ- for methylomic studies.30 Only CpG with qualitative methyla-
ent clinical outcomes both at diagnosis18,19,21 and in clinical tion changes defined as an interquartile range of minimum 0.80
trials.20 Specifically, Kulis et al. and, subsequently, Queirós were included in subsequent analysis (Online Supplementary File,
et al. have shown that methylation signatures can stratify Section 1).
CLL patients into three groups experiencing different clin- The primary clinical endpoint used to develop the classification
ical outcomes: the n-CLL (naïve B-cell-like CLL), i-CLL procedure was time to treatment (TTT). CpG with methylation
(intermediate CLL), and m-CLL (memory B-cell-like CLL) levels associated with TTT were selected using Cox regression
subgroups.18,21 The identified methylation signatures were with the significance threshold of P<10-7; this was chosen to iden-
closely related to IGHV mutation status, with the n-CLL tify the most associated CpG and to control for false-positive
and m-CLL subgroups consisting mainly of U-CLL results. CpG independently associated with TTT were identified
patients and M-CLL patients, respectively. The new i- in a multivariable Cox regression model using a backward elimi-
CLL subgroup included borderline M-CLL and U-CLL nation procedure with P<0.05. Classification of IGHV mutation
patients, as they were found to display both an interme- load (IGHV status) into mutated (M-CLL) and unmutated (U-CLL)
diate load of mutations in the IGHV gene and intermedi- was based on 98% identity cutoff to the germline sequence.16 The
ate clinical outcomes.18,21 Further studies of i-CLL patients strength of association between two classification methods was
have shown that certain molecular features are enriched quantified by the odds ratio (OR) using Woolf approximation to
in this group of patients, such as poor-prognostic subset calculate 95% confidence intervals (CI).
#2 characteristics.21,22 The subset #2 i-CLL cases seem to Secondary clinical endpoints were OS and relapse.31 The prog-
constitute an aggressive subgroup of i-CLL with clinical nostic accuracy of a classification method in predicting the clin-
prognosis resembling the prognosis of n-CLL patients.22 ical outcomes was evaluated using hazard ratios (HR) from uni-
Thus, the diagnostic utility of this classification needs to variate and multivariable Cox regression models, and by Kaplan-
be studied further. Meier plots combined with log-rank tests and estimation of
The above findings clearly indicate that methylation median time to event. The Cox regression model assumptions
signatures of CLL cells are largely associated with the were tested using Schoenfeld residuals, and P values <0.05 were
mutation load of the IGHV gene and that they have prog- considered as statistically significant results.

IGHV-associated methylation in CLL prognostics
Validation of EPIC microarray data with This analysis also showed that methylation status of the
methylation-sensitive high resolution melting individual CpG sites predicted the clinical outcomes of
The microarray data were validated using methylation-sensi- patients with very similar accuracy (Online Supplementary
tive high-resolution melting.32 The details of the assay design can File, Figures S2 and S3), and that none of the CpG sites was
be found in Online Supplementary Methods, Section 2. uniformly informative to predict short TTT (Online
Supplementary Figure S1). Then, to combine the information
Stratification of patients using IGHV-associated from all nine CpG sites, we counted the number of CpG
methylation signatures from 450K data that predicted a short TTT for each patient and compared
We used data from an independent CLL cohort (n=159)33 previ- the HR between groups of patients with a different num-
ously published by Kulis et al.18 and Queirós et al.21 to test whether ber of the CpG sites predicting short TTT. We performed
HumanMethylation450 BeadChip (450K) data are sufficient to this analysis for a series of different b-value cutoffs for indi-
stratify patients using our procedure. vidual CpG sites to allow us to establish a b-value cutoff at
which the final stratification of patients was most accurate
(Online Supplementary Figures S4 and S5).
Results
Table 1. Clinicobiological characteristics of the patients with chronic
Identification of methylation signatures that lymphocytic leukemia.
independently predict short time to treatment
To investigate whether IGHV-associated methylation Variable N (%)
signatures can more accurately classify patients with Age 114
aggressive disease at diagnosis than IGHV mutation sta- Median [range], years 71 [49-92]
tus, we first used linear regression and identified 4,518 Age ≤65 years 37 (32 %)
sites (CpG) in the EPIC array dataset at which the methy- Age >65 years 77 (68 %)
lation levels (b-values) were associated with the IGHV Sex 114
mutation load (Figure 1A). Due to both technical and bio- Male 72 (63 %)
logical limitations of quantitative methylation measure- Female 42 (37 %)
ments in clinical material (for a detailed description, see Binet stage 114
Online Supplementary File, Section 1), we focused our anal- A 77 (68 %)
ysis on 147 sites of the 4518 CpG at which we also B+C 37 (32 %)
observed qualitative methylation changes (defined as an ZAP70 expression* 114
interquartile range of b-values >0.8) (Figure 1B). As TTT Low 67 (59 %)
was the primary clinical indicator of aggressive disease in High 47 (41 %)
our study, we then used Cox regression to identify 44
CD38 expression† 114
CpG among these 147 sites at which the level of methy- Low 84 (74 %)
lation (b-values) were associated with an increased haz- High 30 (26 %)
ard of short TTT (Figure 1C). Moreover, as biomarkers
that independently predict clinical outcomes are most Trisomy 12 112
Absent 101 (90 %)
useful in clinical practice, we applied multivariable Cox
Present 11 (10 %)
regression analysis, performed as a backward elimination
model, to select CpG sites at which the methylation lev- Del(11q) 114
els independently predicted TTT (Figure 1D). This analy- Absent 101 (89 %)
sis resulted in a final set of nine CpG sites with six CpG Present 13 (11 %)
located in gene bodies of REPS1 (cg21740960), RRM2B Del(13q) 113
(cg00395579), SMYD3 (cg07395110), IL1B (cg07250315), Absent 55 (49 %)
UBE2R2 (cg02198280), and ATP9B (cg21394039); two Present 58 (51 %)
CpG did not annotate to any known gene (cg03282117 NOTCH1 mutation 114
and cg00185137) and one CpG was located in the S-shelf Absent 110 (96 %)
of a CpG island in the LMBR1 promoter (cg12032915). Present 4 (4 %)
TP53 aberration‡ 114
Development of a methylation-based classification Absent 103 (90 %)
procedure Present 11 (10 %)
Next, we assessed whether the methylation status of IGHV status** 114
one of the nine selected CpG sites is sufficient to stratify M-CLL 72 (63 %)
the patients accurately into two groups with different TTT, U-CLL 42 (37 %)
or whether combining the information from all CpG sites Time to treatment 114
stratifies patients more accurately. A detailed description of Median [range], months 51.3 [0.1-126]
these analyses is provided in the Online Supplementary File, Number of treated patients 64 (56 %)
Section 3. Briefly, we used TTT as the primary outcome and Overall survival 114
estimated the power of the methylation changes at each Median [range], months 98.2 [0.4-144]
CpG site to predict TTT using the HR from the Cox regres- Median follow-up time [range], months 98.9 (0.4-144)
sion analysis. These analyses showed that hypomethyla- Number of deceased patients 57 (50 %)
tion predicted short TTT for one CpG site (cg07395110), Del: deletion; M-CLL: chonic lymphocytic leukemia with mutated IGHV; U-CLL: chonic
while hypermethylation was associated with short TTT lymphocytic leukemia with unmutated IGHV; * ZAP70 expression is considered to be
for the remaining CpG (Online Supplementary Figure S1). high when >20% cells are positive; †CD38 expression is considered to be high when
>30% cells are positive; ‡including TP53 mutation and del(17p); **germline homolo-
We then compared the HR of the individual CpG sites. gy >98 % is considered U-CLL.

D. Hussmann et al.
Figure. 1. Identification of methylation changes that independently predict short time to treatment. (A) Test of the association between the methylation level (as b-
value) with the IGHV mutation load (as percentage identity to germline sequence) using a linear regression model for 114 patients and 671 684 CpG sites. Two scat-
terplots display a non-significant association for cg17698174 (left) and a significant association for cg08090385 (right) between the b-values on the y-axis and IGHV
mutation load on the x-axis. A total of 4 518 CpG showed significant association between b-values and IGHV mutation load using a significance threshold of P<10-8.
(B) Selection of CpG with qualitative methylation changes. The boxplots display the distribution of methylation at cg08090385 (left) and cg00029031 (right), where
each black dot represents a patient (n=114) with the b-value for the specific CpG indicated on the y-axis. The box displays the 25-, 50- and 75-percentiles. An
interquartile range (IQR) >0.80 was defined as a qualitative methylation change (Online Supplementary Methods, Section 1), and CpG with an IQR >0.80 were select-
ed for further analyses (n=147). (C) Univariate Cox regression analysis of the association between methylation level (as a continuous b-value) and time to treatment
(TTT) in 114 patients for 147 CpG. The Manhattan plot displays the significance to predict TTT for each of the 147 CpG (dots), with the -log10(P value) from Cox regres-
sion analysis on the y-axis against the chromosomal location of the CpG on the x-axis. A total of 44 CpG (red dots) showed a significance level below the threshold
of P<10-7. (D) Results from the multivariable Cox regression of 44 CpG to identify CpG sites that independently predict TTT performed using backward elimination. A
final set of nine CpG sites was identified with a statistical significance of P<0.05.

Overall, this data modeling showed that the combina- poor prognosis for 14 M-CLL cases. Those patients, how-
tion of the information from all nine CpG had a consider- ever, experienced a significantly shorter median TTT of
ably stronger prognostic power to predict TTT than had 16.2 months (95% CI: 3.9-37.9), than the median TTT of
information from individual CpG sites. Specifically, the the remaining M-CLL patients (n=58) who did not reach
stratification for patients displaying two or more CpG the median TTT (P<0.0001) (Figure 3C, dotted curves).
sites with methylation status indicating short TTT (poor Similarly, the median TTT for the three U-CLL patients
prognosis) versus patients with none or one CpG site predicted to have a favorable prognosis according to the
(favorable prognosis) identified patients experiencing methylation-based classification was 70.0 months (95%
short TTT with a HR of 8.34 (95% CI: 4.54-15.30; CI: 64.7-not reached), and significantly longer than the
P<0.001) (Online Supplementary Figure S5b-f). This HR was median TTT of the remaining U-CLL patients (n=39),
a clear improvement, as the power to identify patients which was 8.0 months (95% CI: 1.9-20.1; P=0.0188)
with short TTT for the individual CpG sites stratified (Figure 3C, dashed curves). These Kaplan-Meier curves
patients with HR ranging from 4.10 (95% CI: 2.46-6.85; clearly indicate that the methylation-based classification
P<0.001) to 6.60 (95% CI: 3.76-11.58; P<0.001) (Online predicted TTT more accurately for the discrepantly clas-
Supplementary Figure S3A-I). The overview of the devel- sified patients. We further analyzed the IGHV mutation
oped classification procedure is shown in Figure 2. load of the discrepant cases, and found that they dis-
played an intermediate level of IGHV mutations; this was
Methylation-based classification predicts time to significantly different and closer to the 98% cutoff than
treatment with significantly higher accuracy than that of the remaining patients with similar IGHV status
does IGHV mutation status (Online Supplementary Figure S7). This may indicate a lim-
Next, we compared the power to predict TTT of the itation of the IGHV mutation-based stratification of these
methylation-based classification with stratification using cases.
IGHV mutation status (using the most frequent cutoff at
98% germline identity16). In our cohort, the methylation- Accuracy of methylation-based classification to predict
based classification identified 53 patients with a poor overall survival
prognosis and median TTT of 13.1 months (95% CI: 4.1- We then compared the accuracy of the two classifica-
20.1), and 61 patients with a favorable prognosis for tions to predict OS. Overall, 57 out of 114 patients in our
whom the median TTT was not reached. At the same study cohort experienced events and the median follow-
time, stratification based on IGHV status identified 42 U- up time was 98.9 months (95% CI: 94.4-117.6). Cox
CLL patients with a median TTT of 10.1 months (95% CI: regression and Kaplan-Meier analyses showed that
3.4-21.1) and 72 M-CLL patients for whom the median patients stratified using the methylation-based classifica-
TTT was not reached. Cox regression analyses showed tion had significantly different OS (Cox regression:
that the methylation-based classification was significantly HR=1.82; 95% CI: 1.07-3.09; P=0.027; Kaplan-Meier:
more accurate in predicting the need for treatment as P=0.0246) (Figure 3D). At the same time, IGHV status-
described by a HR of 8.34 (95% CI: 4.54-15.30; P<0.001), based stratification was not able to identify patients with
compared to a HR of 4.35 (95% CI: 2.60-7.28; P<0.001) for different OS in our cohort (Cox regression: HR=1.35;
IGHV status. This was further corroborated by the 95% CI: 0.80-2.28; P=0.263; Kaplan-Meier: P=0.2608)
Kaplan-Meier analyses shown in Figure 3A, B. (Figure 3E). We did not find significant differences in OS
The two stratification methods provided discrepant for the 17 patients with discrepant classification between
classifications for 17 patients (Online Supplementary Figure the IGHV status- and methylation-based classifications
S6). The methylation-based classification predicted a (Figure 3F and Online Supplementary Figure S6). However,
A B C
Figure. 2. The methylation classification procedure based on the nine selected CpG sites. The final procedure for methylation-based classification of patients into having
a favorable or poor prognosis. (A) Methylation levels (b-values) at nine selected CpG sites obtained from the EPIC array for two random patients. (B) Classification of the
b-value according to the cutoff as 1 if the b-values predict short time to treatment (TTT). (C) The number of CpG predicting short TTT is counted for each patient, and the
patient is classified as having a favorable prognosis if 0-1 CpG predicts a short TTT, or as having a poor prognosis if 2-9 CpG predict a short TTT.

D. Hussmann et al.
the follow-up time in our cohort was relatively short and preliminary assessment of the power of methylation-
an increased number of events is likely needed to increase based classification to predict relapse. Even with the lim-
the power of this analysis. ited data available for this cohort, our methylation-based
classification procedure was able to stratify patients into
Methylation-based stratification of patients from two groups with different TTT (Online Supplementary
450K array data Figure S8A) with a similar strength to that observed in our
The cohort size available in this study did not allow us cohort: HR=8.41 (95% CI: 3.74-18.89; P<0.001). The
to divide patients into discovery and validation cohorts, methylation classification also stratified patients with dif-
which would be the most accurate way of assessing the ferent OS with HR=6.03 (95% CI: 2.65-13.73; P<0.001),
prognostic power of a proposed procedure for stratifying and a different likelihood of relapse with HR=2.38 (95%
CLL patients. Furthermore, we were not able to identify CI: 1.33-4.25; P=0.003) (Online Supplementary Figure S8B,
a publicly-available EPIC array dataset from a similar CLL C).
cohort that could be used to validate our findings. The In this cohort, we also compared the performance of
majority of genome-wide methylation profiling studies in the methylation-based classification with that of stratifi-
CLL have, so far, been performed using the 450K array; a cation using IGHV status. The methylation-based classi-
previous generation of the methylomic microarray. We fication stratified patients (96 patients/13 events) with
assessed whether limited data obtained using the 450K different TTT with HR=5.20 (95% CI: 1.53-17.71;
BeadChip, which contained only three of the nine CpG P=0.008) (Figure 4A; P=0.0038), as opposed to IGHV sta-
sites we used to classify patients (cg00395579, tus which only stratified patients with borderline statisti-
cg12032915, and cg21394039), allow for the accurate cal significance: HR=2.97 (95% CI: 0.96-9.17; P=0.059)
stratification of patients according to the classification (Figure 4B; P<0.0001). The analysis of OS for patients in
procedure we developed. The data we used here have this cohort (97 patients/14 events) showed similar results
been previously published and came from 159 CLL to those observed in our CLL cohort, among whom only
patients with TTT data available for 138 patients (34 the methylation-based classification was able to stratify
events), and OS data for 139 patients (33 events).18,21 patients with different OS (HR=5.18; 95% CI: 1.62-16.53;
Relapse data were available for a subset of the patients in P=0.006) (Figure 4D; P=0.0022), and IGHV status was not
this cohort (74 patients/74 events), allowing us to make a informative (HR) 2.46; 95% CI: 0.84-7.24; P=0.102)
A B C
D E F
Figure 3. Kaplan-Meier analyses of time to treatment and overall survival for the methylation-based classification and IGHV status stratifications. (A-C) Kaplan
Meier curves describing time to treatment , and (D-F) Kaplan-Meier curves describing overall survival for stratification of patients using methylation-based classifi-
cation (A and D), IGHV status (B and E), or both stratification methods (C and F) in our cohort of patients. In (C and F), the curves represent patients classified accord-
ing to both stratification procedures: patients with mutated IGHV (dotted line) and unmutated IGHV (dashed lines) are represented by different colors according to
the prediction by the methylation classification into favorable (green) or poor (red) prognosis. The log-rank test for equality was performed between all groups in (C
and F), and all P values are listed below: green/dotted curve versus red/dotted curve (C: P<0.0001; F: P=0.1601); green/dotted curve versus green/dashed curve
(C: P=0.3698; F: P=0.2236); green/dotted curve versus red/dashed curve (C: P<0.0001; F: P=0.0675); red/dotted curve versus green/dashed curve (C: P=0.0719;
F: P=0.0945); red/dotted curve versus red/dashed curve (C: P=0.5284; F: P=0.9315); green/dashed curve versus red/dashed curve (C: P=0.0188; F: P=0.1048).
M-CLL: patients with chronic lymphocytic leukemia with mutated IGHV; U-CLL: patients with chronic lymphocytic leukemia with unmutated IGHV.

(Figure 4E; P=0.0916). Moreover, despite a limited num- Ten patients were classified discrepantly by the two
ber of patients with available IGHV status and relapse classification procedures. The statistical analyses of data
data (39 patients/39 events), the methylation-based clas- for those patients were of very limited power. However,
sification still stratified patients experiencing different three U-CLL patients classified as likely to have a favor-
times to relapse with a HR=3.55 (95% CI: 1.54-8.18; able prognosis according to methylation signature did not
P=0.003) (Figure 4G; P=0.0018), whereas IGHV status experience an event but participated long enough in the
was not informative (HR=1.05; 95% CI: 0.55-2.01; study to speculate that they did indeed have both a favor-
P=0.872) (Figure 4B; P=0.8721). able TTT and OS, as indicated by the dashed green
A B C
D E F
G H I
Figure 4. Kaplan-Meier analyses of time to treatment and overall survival for methylation-based classification and IGHV status stratifications in the cohort for which
450K data were available. (A-C) Kaplan-Meier curves describing time to treatment, (D-F) Kaplan-Meier curves describing overall survival, and (G-I) Kaplan-Meier
curves describing relapse for patients stratified using the methylation-based classification (A, D, and G), IGHV status (B, E, and H), or both stratification methods (C,
F, and I) in an independent cohort of patients (450K data). In (C, F, and I) the curves represent patients classified according to both stratification procedures: chronic
lymphocytic leukemia patients with mutated IGHV (dotted) and unmutated IGHV (dashed) were colored according to the prediction by the methylation classification
into favorable (green) or poor (red) prognosis. In (C), the case with mutated IGHV with a favorable prognosis (dotted/red curve) is visually difficult to spot behind the
other curves in the top left of the Kaplan-Meier plot. The log-rank test for equality was performed between all groups in (C, F, and I), and all P are listed below:
green/dotted curve versus red/dotted curve (C: P=0.7598; F: P=0.0323; I: P=0.0070); green/dotted curve versus green/dashed curve (C: P=0.4285; F: P=0.5530;
I: P=0.0880); green/dotted curve versus red/dashed curve (C: P=0.0033; F: P=0.0134; I: P=0.0306); red/dotted curve versus green/dashed curve (C: P=not avail-
able because there were no events; F: P=0.1415; I: P=0.0405); red/dotted curve versus red/dashed curve (C: P=0.5550; F: P=0.3584; I: P=0.0251); green/dashed
curve versus red/dashed curve (C: P=0.1034; F: P=0.1854; I: P=0.0111). M-CLL: patients with chronic lymphocytic leukemia with mutated IGHV; U-CLL: patients with
chronic lymphocytic leukemia with unmutated IGHV.

D. Hussmann et al.
Table 2. Multivariable Cox regression analysis for time to treatment and overall survival according to the methylation-based classification adjusted
for clinicobiological biomarkers.
Time to treatment Overall survival
Variable HR (95% CI) P Variable HR (95% CI) P
Methylation-based Favorable Age at diagnosis ≤65 years
classification Poor 8.33 (4.28-16.19) <0.001 >65 years 3.11 (1.59-6.09) 0.001
Binet stage A Methylation-based Favorable
classification
B+C 4.87 (2.68-8.85) <0.001 Poor 1.96 (1.15-3.35) 0.013
Del(13q) Absent
Present 0.52 (0.30-0.91) 0.021
Del(11q) Absent
Present 0.30 (0.14-0.65) 0.002
The table displays the final model with variables showing independent prognostic potential with hazard ratios indicating the likelihood of an event given the biomarker status
in the specific row. The time to treatment (TTT) model was built on data from 113 patients with chronic lymphocytic leukemia (CLL) among whom 63 started treatment and the
overall survival model was built on data from 114 CLL patients of whom 57 died. Adjustment for confounding variables was performed using backward elimination, and P values
<0.05 were considered statistically significant. The models were built using all standard clinicobiological biomarkers available, including: age at diagnosis (≤65 vs. >65 years),
Binet stage (A vs. B+C), sex, ZAP70 expression, CD38 expression, trisomy 12, del(11q), del(13q), NOTCH1 mutation, and TP53 aberrations. HR: hazard ratio; CI: confidence interval.
OS, overall survival; TTT, time to treatment.
Kaplan-Meier curves in Figure 4C and 4F, respectively. accurately among all standard clinicobiological CLL
Similarly, the dotted red Kaplan-Meier curves in Figure biomarkers, and only age predicted OS more accurately
4C and 4F for seven M-CLL patients classified by methy- than did the methylation-based classification (Online
lation signatures as likely to have a poor prognosis sug- Supplementary Table 3). The multivariable models that
gest short TTT and OS. Furthermore, the relapse data for included all the above biomarkers and were developed
seven discrepant patients confirmed that the two U-CLL using the backward elimination procedure confirmed an
patients classified as having a favorable prognosis had a independent power of methylation-based classification to
significantly different time to relapse than that of the predict TTT with a HR=8.33 (95% CI: 4.28-16.19;
remaining U-CLL patients (Figure 4I, dashed curves; P<0.001) along with Binet stage, del(13q) and del(11q),
P=0.0111), and likewise, the five M-CLL patients classi- and OS with a HR=1.96 (95% CI: 1.15-3.35; P=0.013)
fied as having a poor prognosis had a significantly differ- along with age (Table 2). In an identical modeling proce-
ent time to relapse compared to that of the remaining M- dure, IGHV mutation status independently predicted
CLL patients (Figure 4I, dotted curves; P=0.0070). The TTT with HR=2.35 (95% CI: 1.34-4.15; P=0.003) along
IGHV mutation load was not available for this cohort and with Binet stage, NOTCH1 mutation, and ZAP70 expres-
we were not able to assess whether the mutation loads of sion, but was not informative regarding OS (Table 3). As
the discrepantly classified patients were close to the the CpG sites in our stratification procedure were select-
IGHV mutation cutoff, suggesting a difficulty in classify- ed based on the association between the methylation lev-
ing those patients similar to those in our cohort. els and IGHV mutation load (Figure 1A), the multivariable
modeling was performed separately for those variables
Association of IGHV status and methylation-based due to the expected intercorrelation.
classification with standard clinicobiological We also compared the prognosis of patients classified
biomarkers of chronic lymphocytic leukemia with our procedure with that of the biological subgroups
In our cohort, we also analyzed the association of stan- identified by classification procedure recently described
dard clinicobiological biomarkers used in CLL prognosti- by Duran-Ferrer et al.34 This procedure identified: 35 n-
cation with both methylation-based classification and CLL, 20 i-CLL and 59 m-CLL with distinct TTT in our
IGHV status. The analysis was based on all variables cohort (Online Supplementary Figure S9A). All 35 n-CLL
available for this cohort, including: sex, age, Binet stage, predicted to experience poor prognosis were also classi-
ZAP70 expression, CD38 expression, del(11q), del(13q), fied as likely to have a poor prognosis with our classifier.
trisomy 12, NOTCH1 mutation, and TP53 locus aberra- However, our classification procedure stratified 59 m-
tions (Online Supplementary Table S1, Online Supplementary CLL cases into six with a poor prognosis and 53 with a
Figure S6). Advanced Binet stage, ZAP70 expression, favorable prognosis. Similarly, 20 i-CLL patients were
CD38 expression, del(11q), del(13q), and NOTCH1 muta- stratified into 12 with a poor prognosis and eight with a
tion were significantly associated with both U-CLL favorable prognosis. The groups identified by our proce-
patients (for IGHV status stratification) and with poor dure did indeed experience, statistically, significantly dif-
prognosis patients, according to the methylation-based ferent outcomes, as illustrated by the Kaplan-Meier
classification. Furthermore, classification of patients as U- curves in Online Supplementary Figure S9B, C. In the cohort
CLL was significantly associated with sex; other of patients for whom 450k data were available, all 66 n-
biomarkers did not show statistically significant associa- CLL cases were predicted to experience a poor prognosis
tions with any of the subgroups of patients. The frequen- according to our classification, and out of 64 m-CLL
cy of the biomarkers in the discrepantly stratified patients cases, one was predicted to have a poor prognosis. Of the
were too low for definite conclusions to be drawn (Online 29 i-CLL cases, 14 were predicted to have a favorable
Supplementary Table S2); however, most of the discrepant- prognosis while 15 were predicted to have a poor progno-
ly stratified patients had early-stage disease (Binet stage sis. The comparison of clinical data for the discrepant
A: 13/17). In univariate Cox regression analyses, the cases was not possible as most of their time data were
methylation-based classification predicted TTT most censored.

Table 3. Multivariable Cox regression analysis for time to treatment and overall survival according to IGHV status adjusted for clinicobiological
biomarkers.
Time to treatment Overall survival
Variable HR (95% CI) P Variable HR (95% CI) P
Binet stage A Age at diagnosis ≤65 years
B+C 4.49 (2.55-7.91) <0.001 >65 years 3.28 (1.66-6.45) 0.001
NOTCH1 mutation Absent ZAP70 expression Low
Present 3.57 (1.20-10.66) 0.022 High 2.04 (1.20-3.47) 0.008
IGHV status M-CLL
U-CLL 2.35 (1.34-4.15) 0.003
ZAP70 expression Low
High 2.07 (1.19-3.59) 0.009
The table displays the final model with variables showing independent prognostic potential with hazard ratios indicating the likelihood of an event given the biomarker status
in the specific row.The model was built on data from 114 patients with chronic lymphocytic leukemia (CLL) with 64 events. Adjustment for confounding variables was performed
using backward elimination, and P values <0.05 were considered statistically significant. The models were built using all standard clinicobiological biomarkers available, includ-
ing: age at diagnosis (≤65 vs. >65 years), Binet stage (A vs. B+C), sex, ZAP70 expression, CD38 expression, trisomy 12, del(11q), del(13q), NOTCH1 mutation, and TP53 aberrations.
HR: hazard ratio; CI: confidence interval. M-CLL: chronic lymphocytic leukemia with mutated IGHV; U-CLL: chronic lymphocytic leukemia with unmutated IGHV.
Polymerase chain reaction validation of microarray data that in our cohort (HR=1.82; 95% CI: 1.07-3.09). At the
To follow good laboratory practice, we performed a same time, we did not see an improvement of the prog-
technical validation of methylation measurements nostic value of IGHV status regarding OS with the
obtained from the microarray analysis in our cohort with longer follow-up, which was not informative in either
methylation-sensitive high-resolution melting. The cohort. However, the fact that OS was not informative
results obtained corroborated the microarray data (Online may be attributed to the specificity of the patients in
Supplementary Figure S10). these two cohorts because IGHV status predicted OS in
other studies.35
Due to limited data availability, we were not able to
Discussion evaluate our findings in the context of an already pro-
posed methylation signature-based stratification21 and
The initiation of treatment of CLL patients is still CLL-classification indices (such as the CLL-IPI and IPS-
based on progression according to clinical symptoms. E).11,12 Nevertheless, our data indicate that it is plausible
However, as a substantial group of CLL patients pro- that the performance of biomarker indices that use IGHV
gresses shortly after diagnosis, or rapidly experiences mutation status will improve with the implementation
relapse, it is generally acknowledged that some patients of the proposed patients’ classification procedure based
may benefit from earlier intervention. IGHV mutation on methylation changes. It is also important to note that
load, together with TP53 aberrations, are currently the the genome-wide methylation screening technology
most widely adopted prognostic markers in CLL diag- used in this project has already been proposed for diag-
nostics; however, molecular biomarkers are not consid- nostic use in glioblastoma,36,37 indicating that, despite its
ered in the decision to treat, and the most clinically rele- still high cost, this technology is worth considering given
vant cutoff for IGHV status is still debated.29 Moreover, the data quality and the amount of data obtained from
some studies have indicated that TP53 locus aberrations single experiments.
may not to be informative for patients with early-stage In summary, our results show that IGHV-associated
disease.11,12 methylation signatures may be more accurate than IGHV
The prognostic value of methylation changes in CLL mutation status in predicting CLL patients’ outcomes,
has been described;18,21 however, the clinical utility of including the identification of patients with aggressive
methylation signatures directly associated with IGHV disease at diagnosis as well as treatment outcomes. Our
mutation load has not yet been studied. Here we inves- results also indicate that the prognostic power of
tigated the prognostic power of the IGHV-associated biomarker indices including IGHV mutation status can,
methylation changes in CLL and developed a procedure potentially, be improved with the addition of methyla-
for classifying patients based on those signatures. tion markers, but this needs to be addressed in further
We then evaluated the prognostic accuracy of the studies.
developed classification procedure and found that it pro-
vided a significantly more accurate prediction of TTT Disclosures
and OS than the stratification based on IGHV status No conflicts of interest to disclose.
alone. Furthermore, we assessed the prognostic validity
of classification in an independent cohort of patients;18,21 Contributions
despite the fact that the methylation data for this cohort DH and TKW designed the study; TEK, ML, and KDJ man-
were limited, compared to IGHV status the methylation aged the preparation of patients’ samples and quality control;
signatures in the independent CLL cohort also displayed JBG performed bisulfite treatment and microarray analysis; DH
significantly higher prognostic accuracy to predict TTT, performed the bioinformatic and statistical analyses assisted by
OS and relapse. Moreover, the analysis of clinical out- TKW and AS; DH, TKW, AS, and LLH interpreted results;
comes is this cohort indicated that considerably longer DH designed the methylation-sensitive high-resolution melting
follow-up (138.0 months vs. 98.9 months in our cohort) assays, and AT performed them; DH, TKW, and LLH wrote the
further improved the accuracy of the methylation classi- manuscript draft; TKW and LLH supervised the study; LK, ID,
fication (HR=6.03; 95% CI: 2.65-13.74) compared to TK, TSL, MBM and CGN participated in the collection of clin-

D. Hussmann et al.
ical samples and clinical data, and standard biomarker analy- Eriksens Mindefond and Arvid Nilsson Fonden. The Graduate
ses. All authors critically reviewed the manuscript and approved School of Health, Aarhus University, Denmark, funded the PhD
the final version. fellowship for DH. TKW was supported by the ‘Polish Returns’
program from the Polish National Agency for Academic
Acknowledgements Exchange and the individual Maria Skłodowska-Curie fellow-
We thank the clinical contributors and the data producers for ship CONFUND at Aarhus Institute of Advanced Studies. AS
the publicly available dataset through ICGC used for indepen- was supported by the Lundbeck Foundation.
dent validation.
Data-sharing statement
Funding The raw data upon which we built the classification procedure
This work was supported by Harboefonden, Knud og Edith are available in the Online Supplementary File.
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Hematopoiesis ARTICLE
Perturbed hematopoiesis in individuals with Ferrata Storti Foundation

germline DNMT3A overgrowth
Tatton-Brown-Rahman syndrome
Ayala Tovy,1,2 Carina Rosas,1,2 Amos S. Gaikwad,3 Geraldo Medrano,3
Linda Zhang,1,2,4 Jaime M. Reyes,1,2,5 Yung-Hsin Huang,1 Tatsuhiko Arakawa,1,2
Kristen Kurtz,3 Shannon E. Conneely,3 Anna G. Guzman,1,2 Rogelio Aguilar,3
Anne Gao,3 Chun-Wei Chen,1,2,4 Jean J. Kim,1,2,6 Melissa T. Carter,7
Amaia Lasa-Aranzasti,8 Irene Valenzuela,8 Lionel Van Maldergem,9
Lorenzo Brunetti,310 M. John Hicks,11 Andrea N. Marcogliese,3 Haematologica 2022
Margaret A. Goodell1,2,3,4,5,6# and Rachel E. Rau1,3# Volume 107(4):887-898
1
Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston,
TX, USA; 2Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX, USA; 3Department of Pediatrics, Baylor College of Medicine and Texas
Children's Hospital, Houston, TX, USA; 4Graduate Program in Translational Biology and
Molecular Medicine, Baylor College of Medicine, Houston, TX, USA; 5Department of
Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA;
6
Department of Education, Innovation and Technology, Baylor College of Medicine,
Houston, TX, USA; 7Department of Genetics, Children’s Hospital of Eastern Ontario,
Ottawa, Ontario, Canada; 8Department of Clinical and Molecular Genetics, Vall d´Hebron
University Hospital and Medicine Genetics Group, Vall d´Hebron Research Institute,
Barcelona, Spain; 9Centre de Génétique Humaine and Integrative and Cognitive
Neuroscience Research Unit EA481, University of Franche-Comté, Besancon, France;
10
Department of Medicine and Surgery, University of Perugia, Perugia, Italy and
11
Department of Pathology Texas Children’s Hospital and Department of Pathology
and Immunology, Baylor College of Medicine, Houston, TX, USA
#
MAG and RER contributed equally as co-senior authors.
ABSTRACT
T
atton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder
caused by germline heterozygous mutations in the DNA methyl-
transferase DNMT3A. DNMT3A is a critical regulator of hematopoi-
etic stem cell (HSC) differentiation and somatic DNMT3A mutations are Correspondence:
frequent in hematologic malignancies and clonal hematopoiesis. Yet, the RACHEL E. RAU
impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is rachel.rau@bcm.edu
undefined. In order to establish how constitutive mutation of DNMT3A
MARGARET A. GOODELL
impacts blood development in TBRS we gathered clinical data and ana- goodell@bcm.edu
lyzed blood parameters in 18 individuals with TBRS. We also determined
the distribution of major peripheral blood cell lineages by flow cytometric
analyses. Our analyses revealed non-anemic macrocytosis, a relative Received: April 14, 2021.
decrease in lymphocytes and increase in neutrophils in TBRS individuals Accepted: May 21, 2021.
compared to unaffected controls. We were able to recapitulate these hema- Pre-published: June 3, 2021.
tologic phenotypes in multiple murine models of TBRS and identified rare
hematological and non-hematological malignancies associated with con-
stitutive Dnmt3a mutation. We further show that loss of DNMT3A in https://doi.org/10.3324/haematol.2021.278990
TBRS is associated with an altered DNA methylation landscape in
hematopoietic cells affecting regions critical to stem cell function and ©2022 Ferrata Storti Foundation
tumorigenesis. Overall, our data identify key hematopoietic effects driven Material published in Haematologica is covered by copyright.
by DNMT3A mutation with clinical implications for individuals with All rights are reserved to the Ferrata Storti Foundation. Use of
TBRS and DNMT3A-associated clonal hematopoiesis or malignancies. published material is allowed under the following terms and
conditions:
Introduction poses is subject to the following conditions:
Tatton-Brown-Rahman syndrome (TBRS; OMIM: 615879) is a germline dominant sect. 3. Reproducing and sharing published material for com-
disorder due to constitutive heterozygous mutations in the de novo DNA methyltrans- mercial purposes is not allowed without permission in writing
ferase DNMT3A. Individuals with TBRS are typically tall, obese, macrocephalic and from the publisher.
have varying degrees of intellectual disability.1,2 Thus far, approximately 60 TBRS indi-
viduals have been described in the literature. However, the number of diagnosed indi-

A. Tovy et al.
viduals is steadily increasing with growing awareness of assent where appropriate were obtained from each participant
DNMT3A mutations as a cause of overgrowth and develop- and/or his or her guardian in accordance with the declaration of
mental disorders. As the full extent of TBRS clinical features Helsinki prior to any study procedures. Peripheral blood sam-
is not yet defined, ongoing efforts to characterize the effects ples from individuals with germline DNMT3A mutations or
of constitutive DNMT3A loss will be critical to facilitate deletions and unaffected controls were collected in EDTA-coat-
identification of affected individuals and improve their care ed tubes. Available medical records for enrolled patients were
and quality of life. reviewed for growth parameters, laboratory studies and hema-
DNMT3A is perhaps best known as a regulator of blood tologic diagnoses.
development governing the balance between hematopoietic
stem cell (HSC) self-renewal and differentiation.3 Loss-of- Mouse models
function DNMT3A mutations are highly prevalent across a All mice were housed in AAALAC-accredited, specific-
spectrum of adult hematologic malignancies, further con- pathogen-free animal care facilities at Baylor College of
firming its indispensable role in hematopoiesis.4-6 In addi- Medicine, and all procedures were approved by the
tion, DNMT3A is the most commonly mutated gene in clon- Institutional Animal Care and Use Committee. C57BL/6 mice
al hematopoiesis (CH),7,8 a phenomenon of aging associated of both sexes were used unless stated otherwise, and experi-
with mutant hematopoietic stem and progenitor cell (HSPC) mental mice were separated by sex and housed with four mice
expansion.9 Individuals with CH have a significantly per cage. All mice were immune-competent and healthy prior
increased risk for hematologic malignancy development. to the experiments described. Mice were bred and maintained
This increased risk is likely due to a competitive growth at regular housing temperatures (23°C) and 12-hour (h) light/12-
advantage that leads to an accumulation of DNMT3A- h dark cycle. In order to generate germline Dnmt3a heterozy-
mutant HSPC in the bone marrow over time,3 with a pro- gous haploinsufficient mice we crossed Dnmt3+/fl mice with Ella-
portional increase in the likelihood of acquiring collaborat- cre transgenic mice obtained from The Jackson Laboratories
ing leukemogenic mutations. Consistent with this time- (Bar Harbor, ME). The DNMT3A p.293 deletion and DNMT3A
dependent model of clonal expansion and malignant trans- p.W577R mutation were generated in our laboratory utilizing
formation is the fact that, while common in adult hemato- CRISPR strategies with a single guide RNA and single strand
logic malignancies, DNMT3A mutations are exceedingly DNA template injected into mouse blastocysts.16 Guide
rare in pediatric leukemias.10-13 sequence and DNA template are described in the Online
The mutation spectrum reported for TBRS individuals Supplementary Table S3. For all assays mouse data was obtained
covers all the functional domains of DNMT3A and overlaps at 12 or 15 months of age.
with CH and hematologic malignancies.1 However, in con-
trast to the somatic mutations in CH and cancer, in TBRS Lymphoblastoid cells generation
individuals, the DNMT3A mutation is constitutive, arising Peripheral blood mononuclear cells were isolated from whole
before embryogenesis. This timing raises the concern that blood by density gradient centrifugation using Lymphoprep
the negative hematopoietic sequelae associated with somat- density gradient medium (STEMCELL Technologies,
ic DNMT3A mutations in older individuals could have an Vancouver, Canada) and then incubated with concentrated
early onset in TBRS. Reports of recurrent infections, bleed- Epstein–Barr virus in a total of 200 mL of RPMI medium for 30
ing tendencies, and leukemias in individuals with TBRS sug- minutes. The cells were then plated in a flat-bottomed 96-well
gest that there may be a correlation between constitutive plate with 1 mg/mL cyclosporin A (Sandoz Pharmaceuticals,
DNMT3A loss and abnormal hematopoiesis.1,14,15 Despite Washington, DC). Cells were fed bi-weekly until lymphoblas-
these concerns and anecdotal observations, how constitu- toid cell lines (LCL) were established.
tive loss of DNMT3A impacts blood formation and
leukemia predisposition in TBRS remains unknown. Whole genome bisulfite sequencing
In order to define basal hematopoiesis in TBRS, we exten- DNA was extracted from LCL using the DNeasy kit (Qiagen,
sively characterized the hematological parameters and Hilden, Germany). DNA was fragmented prior to bisulfite con-
peripheral blood cellular composition of TBRS individuals versation using the NEBNext fragmentase kit, following the
with a spectrum of DNMT3A mutations and deletions. We manufacturer’s protocol (New England BioLabs, Inc., Ipswich,
found that germline DNMT3A mutations impact multilin- MA). We used 200 ng of DNA to prepare whole genome bisul-
eage blood development, resulting in clinically relevant phe- fite sequencing (WGBS) libraries with the Swift-whole genome
notypes. We further identified DNA methylation changes as bisulfite kit according to the manufacturer’s instruction (Swift
one mechanism through which constitutive loss of Biosciences, Ann Arbor, MI). Libraries were sequenced and
DNMT3A alters blood development in TBRS. In order to processed as previously described.17 The Wildcard ALignment
confirm the observed hematopoietic effects, we extensively tool (WALT) pipeline was used for duplicate removal and
investigated hematopoiesis in multiple mouse models of hypomethylated regions (HMR) calling.18 A minimum of five
TBRS. These murine models recapitulated the major hema- reads per CpG was used to calculate methylation values.
tologic phenotypes identified in humans. Longitudinal stud- Coverage per base for mutant or control cells was >7x. For iden-
ies of the TBRS mouse models demonstrated that while the tification of differentially methylated regions, we used the
risk of hematologic malignancies is increased in TBRS, dis- WALT pipeline default settings.
ease penetrance overall is low.
Statistical analyses
Statistical analyses of the clinical data were performed with
Methods the GraphPad Prism 8 software (GraphPad Software, San
Diego, CA). P-values were interpreted as statistically significant
Human samples if less than 0.05, unless otherwise stated. Throughout the man-
This study was approved by the Institutional Review Board uscript data are expressed as the mean +/- standard error of the
of Baylor College of Medicine. Written informed consent and mean, unless otherwise stated. The statistical significance of the

DNMT3A mutations in TBRS
Figure 1. DNMT3A mutations and deletions identified in patients with Tatton-Brown-Rahman syndrome. Map of DNMT3A variants identified in individuals with
Tatton-Brown-Rahman syndrome (TBRS) based on clinical sequencing data from our patient cohort, published data, and information obtained from the TBRS com-
munity. AA: amino acid; PWWP: Pro-Trp-Trp-Pro motif domain; ADD: ATRX-DNMT3L-DNMT3L domain; Mtase: methyltransferase domain.
differences between two groups was calculated using unpaired ferent between the groups, in TBRS individuals the per-
Student’s t-test (two-sided, without assuming equal standard centage of neutrophils was significantly increased, and
deviations), Fisher’s exact test or the nonparametric Mann- percentage of monocytes and lymphocytes significantly
Whitney test where appropriate. Statistical details are also decreased relative to controls (Figure 2A to D). Also,
described in the figure legends, including the number of repli- while hemoglobin and red cell counts (RBC) were not
cates, animals or human samples per group (denoted by “n”). significantly different (Figure 2E and F) compared to
unaffected individuals, those with TBRS had a signifi-
cantly increased mean corpuscular volumes (MCV) and
Results mean corpuscular hemoglobin (MCH) levels, with no
difference in mean corpuscular hemoglobin concentra-
Characteristics of germline DNMT3A mutations tion (MCHC) (Figure 2G; Online Supplementary Figure 1;
in Tatton-Brown-Rahman syndrome Figure 2H, respectively). As MCV varies by age and sex,
In order to identify the current spectrum of TBRS vari- we plotted the MCV of our enrolled TBRS individuals by
ants, we gathered mutational information on 48 individ- age/sex, including past CBC from medical records where
uals with TBRS utilizing the TBRS organization data- available. We found that overall, the vast majority of
base. These individuals harbored mutations (missense, TBRS individuals had MCV above the 50th percentile at
frameshift, deletions) in the PWWP, ADD and methyl- all ages examined including ten of 14 with an MCV ≥90th
transferase domains, including ~30 not previously percentile and eight of 14 >97th percentile for age/sex at
reported in TBRS (Figure 1, Table 1; Online Supplementary one or more timepoints (Figure 2I and J). All other CBC
Table S1). We also identified two TBRS individuals with parameters were similar between TBRS and unaffected
deletion of the entire DNMT3A gene. These data show individuals (Online Supplementary Figure 1). These finding
that, as previously suggested, DNMT3A mutations in indicate that heterozygous germline DNMT3A lesions
TBRS can occur across all the DNMT3A functional impact multilineage hematopoiesis in the absence of
domains and are shared between TBRS, CH and overt clinical blood disorders.
leukemia.
Analytical characterization of blood populations in
White blood cell differential of Tatton-Brown-Rahman Tatton-Brown-Rahman syndrome individuals
syndrome individuals is characterized by increased We next conducted immunophenotypic analyses of
neutrophils and decreased lymphocytes the peripheral blood from 15 TBRS individuals and ten
In order to assess the composition of the blood of indi- controls. The age of TBRS versus control individuals with
viduals with TBRS, we enrolled 18 individuals with immunophenotypic data was not significantly different
TBRS, seven unaffected siblings, and four unrelated unaf- (median 13.5 years and 10 years, respectively). Our find-
fected individuals (Table 1; Online Supplementary Table ings confirmed neutrophil expansion in the blood of
S2). The clinical and genetic information for the included TBRS individuals (Figure 3A). Given reports of recurrent
TBRS individuals is shown in Table 1. One individual in infections and hypogammaglobulinemia in individuals
the TBRS cohort had severe iron deficiency anemia and with TBRS as well as mouse model data suggesting a role
one had hypogammaglobulinemia requiring intravenous for DNMT3A in T-cell development,19 we conducted a
immunoglobulin (IVIG) replacement. No other blood or detailed immunophenotypic analysis of T- and B-cell
immune defects were reported in either cohort. subsets. We found a relative reduction in CD19+, CD20+,
We performed complete blood cell counts (CBC) with and CD22+ B cells in TBRS individuals compared to unaf-
white blood cell (WBC) differential on the enrolled indi- fected controls (Figure 3B; Online Supplementary Figure
viduals, excluding from the analysis the TBRS patient S2A and B). We also identified a trend towards reduced
with severe iron deficiency anemia. An additional four total CD3+ T cells that did not reach statistical signifi-
TBRS individuals and one control did not have a CBC cance but found significant differences in the relative
performed, thus 13 TBRS individuals and nine controls populations of CD4+ and CD8+ expressing T cell subsets
were included in our analysis. The age of TBRS individ- with a higher CD4 to CD8 ratio in TBRS compared to
uals (median 15.2 years, range, 3.5-35.8 years) with CBC unaffected individuals (Figure 3C; Online Supplementary
was not significantly different than controls (median 10.0 Figure 2C). These results confirm a relative increase in
years, range, 3.5-40 years). neutrophils in individuals with TBRS and identify signif-
We found that while the total WBC count was not dif- icant changes in B- and T-cell populations.

A. Tovy et al.
Table 1. Table of Tatton-Brown-Rahman syndrome individuals’ clinical and mutation information.

ID Sex Age Height Height Weight Weight Mutation DNMT3A coding DNMT3A Flow Y/N CBC
(years) (cm) percentile (kg) percentile type sequence variant variant protein
RR01 M 3.8 110.4 96 19.9 99 Missense c.901C>T p.R301W Y Y
RR04 M 21.8 183 83 89 55 Frameshift deletion c.297del p.M99fs N N
RR07 M 2.6 104.8 >99 26.2 >99 Gene deletion 2p23.3 del Ex Ex
RR16 M 17.6 183 84 79.5 84 Missense c.1748 G>A p.C583Y Y Y
RR17 M 34.5 196 >99 79.4 35 Frameshift duplication c.1238dupG p.F414fs*7 Y Y
RR18 M 20.8 196 >99 74.4 23 Missense c.2309 C>T p.S770L Y Y
RR19 F 35.8 194 >99 77.5 30 Missense c.2207 G>A p.R736H Y Y
RR20 F 16* 172 93 68.5 88 Missense c.2063 G>A p.A688H Y Y
RR22 M 3.5 99 47 17.7 87 Missense c.1978 T>C p.Y660H Y Y
RR24 M 5.7 130 >99 33.1 >99 Gene deletion 2p23.3 del Y Y
RR25 M 13.5 168 78 83.9 99 Missense c.2645 G>A p.R882H Y Y
RR26 F 16.0 180.3 >99 115.5 99 Missense c.929 T>A p.I310N Y Y
RR28 F 18* 168 77 64 75 Missense c.2114T>C p.V704A N N
RR34 M 5.9 n/k n/k n/k n/k Missense c.2129G p.C710Y Y Y
RR38 F 7.0 137 >99 30 91 Missense c.919C>T p.P307S Y Y
RR40 M 15.2 193 >99 74.5 89 Frameshift deletion/ c.2432_2434delinsC Y Y
insertion
RR43 F 2.0 93 99 15 97 Missense c.1627 G>T p.G543C Y N
RR45 M 3.7 107 92 21 98 Missense c.2645 G>A p.R882H Y N
Blood and medical records we collected from 18 Tatton-Brown-Rahman syndrome (TBRS) individuals. Presented is mutation data and the type of analyses that were done on
each sample. *: approximate; n/k: not known; Flow: flow cytometric analysis of peripheral blood; CBC: complete blood cell count with white blood cell differential; Y: yes; N: no;
Ex: excluded due to severe iron deficiency anemia.
Myeloid cells expand in the peripheral blood of mice Supplementary Figure S4A and B, respectively) consistent
with germline Dnmt3a mutations with findings in our TBRS cohort. Like TBRS individuals,
In order to validate the findings of our human cohort, the percentage of Ly6G+ neutrophils in HET293 mice was
we examined hematopoiesis in three mouse models with significantly increased relative to littermate controls with
constitutive Dnmt3a lesions. We first evaluated mice with no significant difference in the percentage of Ly6C+
a heterozygous germline deletion affecting amino acid 293 monocytes (Figure 4G). Together, these results demon-
(HET293) (mouse equivalent of human amino acid 297) in strate that multiple constitutive Dnmt3a mutant mouse
the PWWP domain of DNMT3A, a lesion previously models recapitulate the hematopoietic phenotypes
reported in TBRS.1,2 Consistent with our human data, observed in TBRS.
CBC revealed significantly increased percentage of neu- Previous publications have shown that the key inflam-
trophils and decreased lymphocytes compared to wild- matory cytokine, interleukin 6 (IL6), is abnormally elevated
type littermate controls (WT) without a significant differ- in DNMT3A-mediated CH.21 We therefore postulated that
ence in the overall WBC count (Figure 4A to C; Online the neutrophil expansion identified in TBRS and our
Supplementary Figure S3A). murine models may be in response to inflammation driven
In order to evaluate for potential phenotype-modifying by mutation or deletion of Dnmt3a. In order to explore this
effects of differing Dnmt3a mutations on hematopoiesis, possibility, we measured IL6 levels in the serum of HET293
we also analyzed the peripheral blood of mice with het- mice and WT littermates at 12 months and in the TBRS
erozygous germline mutations affecting the DNMT3A individuals. Although in the young TBRS individuals IL6
ADD domain amino acid W577R (HET577) (mouse equiv- was not altered, in HET293 mice we measured significant-
alent of human W581R), a mutation which has been ly elevated levels of IL6 (Online Supplementary Figure 4C and
reported in CH,20 and mice with heterozygous deletion of D). Our findings suggest that germline Dnmt3a mutations
Dnmt3a (HET), recapitulating a complete loss of function are consistently associated with neutrophil expansion,
or deletion of one DNMT3A copy. At 1 year of age, we potentially in response to heightened inflammation.
compared the peripheral blood of HET577 and HET mice
to their WT littermates and found that the percentage of Stem and myeloid cells expand in bone marrow of
neutrophils was significantly increased whereas the per- aged germline Dnmt3a-mutant mice
centage of lymphocytes was significantly decreased Knockout (KO) of Dnmt3a in hematopoietic stem cells
(Online Supplementary Figure 3B and C). HET577 mice also has been widely investigated in bone marrow transplanta-
had significantly decreased total WBC and decreased tion experiments, in which transplant of Dnmt3a-KO HSC
platelets, unlike the HET293 and HET mice and the TBRS leads to expansion of the stem cell pool and increased
cohort. repopulation capability.22 The impact of DNMT3A muta-
Flow cytometric immunophenotypic analysis of periph- tion or loss on hematopoietic stem and progenitor cell
eral blood leukocytes at 12 and 15 months demonstrated populations in an unperturbed model such as TBRS has
significant myeloid expansion and B-cell reduction in the not been fully investigated. In order to address this we
HET293 mice and HET577 (Figure 4D to F; Online next analyzed the composition of hematopoietic cells in

A B C D
E F G H
I J
Figure 2. Blood of Tatton-Brown-Rahman syndrome individuals is characterized by relative increase in neutrophils, decrease in lymphocytes and non-anemic
macrocytosis. Complete blood cell counts were performed on peripheral blood from Tatton-Brown-Rahman syndrome (TBRS) individuals (n=13) and unaffected con-
trols (n=9) including (A) total white blood cell (WBC) count and WBC differential with percent of (B) neutrophils, (C) monocytes, and (D) lymphocytes. Red blood cell
(RBC) indices were also compared including (E) hemoglobin, (F) total RBC number, (G) mean corpuscular volume (MCV), and (H) mean corpuscular hemoglobin con-
centration (MCHC). One TBRS individual had no available RBC count. MCV plotted by age for (I) TBRS males and (J) TBRS females including data from study CBC and
from CBC data extracted from medical records for some individuals over multiple timepoints.
the bone marrow of HET293 mice compared to WT litter- These finding support that the germline mutation of
mate controls. Like the blood, the bone marrow of Dnmt3a, like somatic loss of Dnmt3a in the hematopoietic
HET293 mice showed relative myeloid expansion and compartment, results in stem cell expansion with aging
reduced frequency of B-cells (Figure 4 H to J). Comparison and leads to myeloid expansion in the bone marrow.3
of the stem/progenitor cell compartment (gating scheme
in the Online Supplementary Figure S4E and F) in 15-month- Germline Dnmt3a-mutant mice have defects in
old mice without any overt hematologic malignancies, lymphocytes production
showed a moderate but significant expansion of Evaluation of T- and B-cell subsets in the blood of TBRS
hematopoietic stem cells and multipotent progenitor cells individuals revealed significantly reduced CD19+ B cells
(Figure 4K and L). The relative frequency of other and an altered ratio of CD4/CD8 T cells in those with
stem/progenitor populations, including common myeloid TBRS. We therefore performed flow cytometric analysis
progenitors, common lymphoid progenitors, granulo- of B- and T-cell subsets in the peripheral blood of
cytes/monocyte progenitors, megakaryocyte/erythroid Dnmt3a-mutant mice. We found that unlike TBRS individ-
progenitors did not differ significantly (Online uals, in HET293 and HET577 mice the CD4/CD8 T-cell
Supplementary Figure 4G and H). ratio was significantly lower than WT controls (Online

A. Tovy et al.
A B
C D
Figure 3. Immunophenotypic analysis of Tatton-Brown-Rahman syndrome individuals identifies neutrophil expansion and deficiencies in specific T- and B- cell sub-
sets. Flow cytometry analysis was performed on peripheral blood from Tatton-Brown-Rahman syndrome (TBRS) individuals (n=15) and unaffected controls (n=10).
Percent of leukocytes categorized by immunophenotype as (A) neutrophils, (B) B cells expressing CD10, CD19, CD20 and/or CD22, (C) total CD3+ T cells and the per-
centage of CD3+ T cells that are CD4+ and CD8+. (D) Quantification of the CD4/CD8 ratio of controls versus TBRS individuals.
Supplementary Figure S5A). Quantification of B-cell subsets decreased RBC counts compared to WT controls (Figure
by flow cytometry demonstrated that although HET293 5C to E). In order to further characterize erythropoiesis in
mice had a relative decrease in total B220+ B cells, the pro- TBRS, we performed flow cytometric examination of ery-
portion of different B-cell subsets was not altered when throid development in the bone marrow of HET293
compared to WT mice (Online Supplementary Figure S5B). mice.25 Staining of bone marrow cells with CD71 and
Only mature B lymphocytes can effectively contribute to TER119 is used to identify developmental stages of ery-
the immune response, and maturation of B-cells that thropoiesis, labeled ProE (proerythroblasts), Erythroblasts
occurs in the spleen. Therefore, in order to determine if A-C (EryA, EryB and EryC), which correspond to sequen-
the B-cell reduction reflects defects in splenic B-cell matu- tial steps in erythroid development.26 Although the total
ration we analyzed the frequency of transitional immature percentage of TER119 positive cells was not significantly
B cells in the spleen by flow cytometry (T1 and T2 popu- different in HET293 mice compared to WT littermates,
lations, respectively).23 T1 B cells are bone marrow derived HET293 mice had significantly fewer large, immature ery-
immature B cells that migrate to the spleen where they throblasts (EryA) and more small mature erythroblasts
mature into T2 B cells which are the progenitors for (EryC) (Figure 5F and G). Thus, loss of DNMT3A in our
mature B cells.24 We identified a significant decrease in the murine model leads to a subtle, but significant effect on
frequency of T1 B cells but not of T2 B cells in the spleen early erythroid development and differentiation.
of HET293 mice compared to WT controls (Figure 5A and
B). These data suggest that DNMT3A loss leads to a reduc- Dnmt3a-mutant mice develop hematologic
tion in B-cell frequency via decreased B-cell progenitors in malignancies at higher rates than controls
the bone marrow. While reduced in numbers, these pro- Given the frequency of somatic DNMT3A mutations in
genitors are capable of normal maturation. adult hematologic malignancies, as well as case reports
documenting hematologic malignancies in individuals
Germline Dnmt3a-mutant mice have defects in with TBRS,1,14 we looked for malignancies in cohorts of
erythropoiesis HET293 mice at the age of 15 months. At this age, eight of
Evaluation of RBC indices in our TBRS cohort revealed 36 (22%) HET293 mice had malignancies compared to one
increased average MCV and MCH relative to unaffected of 35 (3%) WT littermate controls (P=0.028). Of the eight
individuals. This finding was recapitulated in the HET293, malignancies in the HET293 mice, seven were hematologic
HET577, and HET murine models (Figure 5D and E; including both myeloid and lymphoid diseases (Online
Online Supplementary Figure S3), although unlike the TBRS Supplementary Table S7; Online Supplementary Figure 6 A and
individuals, HET293 mice additionally had significantly B) consistent with results from mice with heterozygous

A B C
D E F
G H
I J K L
Figure 4. Tatton-Brown-Rahman syndrome mouse model characterized by myeloid expansion and increased frequency of hematopoietic stem and progenitor cells
in the marrow. Complete blood cell counts performed on the blood of a representative cohort of mice with heterozygous in frame deletion of amino acid 293 of
DNMT3A (HET293) (n=23) and wild-type (WT) littermate controls (n=31). All mice included in the analyses did not display hematologic malignancies. Displayed com-
parisons of (A) total white blood cell (WBC) count, (B) percentage of neutrophils and (C) percentage of lymphocytes. Flow cytometric analysis of peripheral blood
CD45+ leukocytes depicting (D) relative distribution of myeloid (defined as cells expressing CD11b and/or Ly6G), T cells (defined as CD3 and CD4+ and/or CD8+ cells)
and B cells (defined as B220+ cells) in the HET293 mice compared to WT. Quantification of the percentage of (E) myeloid and (F) B cells in the HET293 mice and WT
mice as determined by flow cytometry from (D). (G) Analysis of the different subtypes of CD11b+ myeloid cells into neutrophils (Ly6G expressing cells) or monocytes
(Ly6C expressing cells). Flow cytometric analysis of bone marrow CD45+ leukocytes depicting H) relative distribution of myeloid, T cells and B cells in the HET293
mice compared to WT. Quantification of the percentage of I) myeloid and J) B cells in the HET293 mice and WT mice as determined by flow cytometry. Bone marrow
flow cytometry assessment of HET293 and WT mice showing the percent of (K) hematopoietic stem/progenitor cells defined by SLAM markers and (L) multipotent
progenitor (MPP) cells.

A. Tovy et al.
germline deletion of Dnmt3a.27 The affected mice were had lymphomas including B- (n=3) and T-cell (n=1) malig-
moribund, with weight loss, pallor, adenopathy, tumors nancies (Figure 6B). We also identified histolytic sarcoma in
and splenomegaly (Figure 6A). Two mice had malignant two mice, both of which had an additional malignancy
myeloid neoplasms: one acute myeloid leukemia and one (one with lymphoma and one with angiosarcoma). Two
myeloproliferative neoplasm with infiltration of malignant mice had angiosarcomas, a non-hematological malignancy
cell into blood, bone marrow, liver and spleen. Four mice (Online Supplementaty Figure S7).
A B
C D E
F G
Figure 5. Differences in lymphoid and erythroid compartments in Tatton-Brown-Rahman syndrome mouse model. Quantification of the proportion of B220+ splenic
B cells that are (A) T1 B cells expressing immunglobulin (Ig) M and intermediate levels of IgD and (B) T2 B cells expressing both IgM and IgD in HET293 mice (n=19)
and wild-type (WT) littermates (n=20). From complete blood cell counts performed on peripheral blood, comparison of (C) red blood cell (RBC) number, (D) mean
corpuscular volume (MCV), and (E) mean corpuscular hemoglobin concentration (MCHC) of WT mice (n=29) and HET293 mice (n=24). (F) Flow cytometric gating
strategy for assessment of the erythroid development for representative WT and HET293 mice. Viable cells were gated based on their TER119 expression and then
TER119+ cells were plotted by forward scatter and CD71 expression levels to identify erythroblasts in different developmental stages (Ery A-C). (G) Top: the total pro-
portion of TER119 and proerythroblasts (ProE) cells. Bottom: the proportion of the indicated populations within the TER119+ fraction defined by CD71 and FSC
(n=18) and HET293 mice (n=16).

These data, combined with case reports of individuals TBRS individual with WT DNMT3A. We measured a 6%
with TBRS and hematologic cancers, suggest that while decrease in global DNA methylation in the 297del LCL
the presence of a germline variant of DNMT3A increases compared to WT LCL (60.34% and 66.77%, respectively)
the relative risk for the development of hematologic (Figure 7A). When we analyzed the distribution of DNA
malignancies, a majority will not develop a blood cancer. methylation in the WT LCL compared to the 297del LCL,
we observed a significant decrease in DNA methylation in
Constitutive loss of DNMT3A in Tatton-Brown-Rahman enhancer regions. This observation suggests that, similar
syndrome leads to significant hypomethylation in to leukemia,28 in TBRS hematopoietic cells DNMT3A loss
hematopoietic cells impacts methylation at regulatory enhancer regions
In TBRS individuals only one functional allele of (Figure 7B). We identified 1,068 differentially methylated
DNMT3A remains. In order to examine if constitutive loss regions (DMR) in 297del compared to WT LCL (Online
of DNMT3A in TBRS impacts the DNA methylation land- Supplementary Table S8). Interestingly, some of these DMR
scape, we performed whole genome bisulfite sequencing include key loci with known importance in blood devel-
on LCL derived from B cells of a TBRS individual with a opment such as the HOXA cluster, similar to the
DNMT3A-297deletion (297del) and from a sibling of a hypomethylation patterns we previously reported for
Figure 6. Development of hematologic malignancies in Tatton-Brown-Rahman syndrome mouse model. A subset of the HET293 mice had hematologic malignan-
cies at 15 months of age. These mice were noted to have (A) enlarged spleens relative to wild-type (WT) and HET293 mice without malignancies. (B) Pathologic eval-
uation of the bone marrow, spleen and liver of a WT mouse (WT #1, top row), a HET293 mouse with acute myeloid leukemia noted in the bone marrow, spleen and
liver (HET293 2b, second row), and a HET293 with T-cell lymphoma in the bone marrow, spleen and liver (HET293 #40, third row) and focal histiocytic sarcoma in
the bone marrow (HET293 #40, bottom image).

A. Tovy et al.
A B
Figure 7. Altered DNA methylation in
enhancer regions of hematopoietic
cells of a Tatton-Brown-Rahman syn-
drome individual. Whole genome
bisulfite sequencing was performed
on a lymphoblastoid cell line (LCL)
derived from a Tatton-Brown-Rahman
syndrome (TBRS) individual with a
heterozygous deletion of amino acid
297 in the PWWP domain of DNMT3A
(297 del) and an LCL derived from the
unaffected sibling of an individual
with TBRS (control). (A) Overall % CpG
methylation of control and 297 del
LCL. (B) Plot represents density of
DNA CpG methylation of enhancer
regions in control and 297 del LCL. (C)
Genome browser tracks of CpG
C methylation at the HOXA locus of the
control and 297 del LCL compared to
a previously published DNMT3A
p.R771Q mutant LCL.
LCLs generated from an individual with a constitutive viduals and controls. In our TBRS cohort, the CD4/CD8
DNMT3A 771Q mutation17 (Figure 7C). These results sup- ratio was increased, a finding that has been associated
port that the heterozygous loss of DNMT3A leads to with obesity which is noted in most of our TBRS
hematopoietic defects likely through altered DNA methy- cohort.29 Conversely, in our murine models, we found a
lation. decreased CD4/CD8 ratio in mice with Dnmt3a mutation
or deletion. There are known differences in lymphoid
development between human and mouse that may
Discussion explain this discrepancy, but further investigations will
be needed to fully understand the impact of DNMT3A
In order to examine the impact of constitutive loss of loss on T-cell development.30 Lower CD4/CD8 ratios
one copy of DNMT3A on blood production, we analyzed have been associated with altered immune responses
primary human specimens from patients with TBRS and and inflammation; perhaps this relative change might
murine models recapitulating pathogenic Dnmt3a variants explain the increased level of inflammatory cytokine IL6
identified in TBRS, CH and hematologic malignancies. in our aged mutant mice.31 Overall, the clinical conse-
Our data reveal a shift in the distribution of leukocytes quences of these immune cell phenotypes will require
with an overall increase in the myeloid compartment, additional evaluation and longitudinal follow-up of
specifically neutrophils, in individuals with TBRS com- affected individuals with a focus on how these B- and T-
pared to controls. Further, we identified a significant cell deficits impact susceptibility to infections and
reduction in the percentage of lymphocytes and specific response to vaccinations.
B- and T-cell subsets. We also noted erythropoiesis defects In our cohort, we found increased MCV in TBRS individ-
in TBRS, manifested as increased MCV. We further found uals relative to controls. This finding is similar to the non-
that our TBRS murine models developed hematologic anemic macrocytosis characteristic of Down syndrome
malignancies of low penetrance after a long latency. We (DS) and Williams-Beuren syndrome (WBS).32,33 It is likely
also identified differences in the blood parameters of mice that in TBRS, like in DS and WBS, isolated macrocytosis is
with differing Dnmt3a lesions (PWWP domain, ADD a benign variant of normal, warranting conservative obser-
domain, or complete deletion of one allele), raising the vation only. Indeed, multiple individuals in our cohort had
possibility that mutation-specific effects on DNMT3A MCV consistently at or above the 90th percentile for
function may modify blood phenotypes. This work sex/age over many years yet have not developed any
demonstrates that constitutive heterozygous loss of one hematologic disorders. It is notable that increased red cell
DNMT3A allele leads to significant multilineage perturba- distribution width (RDW), reflecting variability in RBC
tions of hematopoiesis. size, is a consistent finding in CH.9,21 Our data suggest that
Our findings have potential clinical implications for DNMT3A-mutant HSPC generate larger RBC than RBC
individuals with TBRS. In our TBRS cohort and all exam- derived from non-mutant HSPC. Therefore, in DNMT3A-
ined TBRS mouse models, we found significantly mutant CH, increased RDW may be attributable to a
reduced B cells relative to controls. Additionally, one of mixed population of WT and DNMT3A-mutant cells in the
our enrolled patients reported hypogammaglobulinemia peripheral blood. An expansion of the DNMT3A-mutant
requiring IVIG supplementation. Therefore, our observa- HSPC would therefore be expected to result in an
tions suggest that evaluation of immunoglobulin levels increased MCV due to increased representation of
may be warranted for individuals with TBRS, particular- DNMT3A-mutant RBC in the blood. Indeed, increased
ly those with recurrent infections. We also identified sig- MCV in individuals with CH has been associated with
nificant differences in T-cell subsets between TBRS indi- increased risk of hematologic malignancy development.34

Unlike the TBRS patients, the aged HET293 mice were not an inevitable consequence of having a constitutive
noted to have a modest, but significant decrease in RBC DNMT3A mutation. Indeed, our previous report demon-
count compared to littermate controls. This may reflect a strated that, although loss of DNMT3A leads to a signifi-
feature associated with aging and warrants further longitu- cant competitive advantage in the blood, this advantage is
dinal studies on TBRS individuals to definitively determine not necessarily associated with leukemogenesis.17 Natural
changes in erythropoiesis over time. history studies of TBRS individuals are needed to define
In our TBRS cohort and murine models, we also identi- the incidence of hematologic malignancy, the disease
fied neutrophil expansion, potentially in response to spectrum, and risk predictors for malignancy develop-
inflammation. CH is associated with increased risk not ment to enable tailored surveillance guidelines for
only for hematological malignancies, but also for cardio- prospective monitoring.
vascular disease, largely due to a pathologic inflammatory In conclusion, we show that constitutive loss of
state.35 Previous studies have shown that loss in DNMT3A significantly impacts multilineage blood devel-
hematopoietic cells of Dnmt3a or Tet2, another commonly opment and leads to phenotypic changes with clinical
mutated gene in CH, leads to increased expression of implications. While our findings offer important insights
inflammatory chemokines and cytokines and accelerated into blood development in individuals with TBRS, these
atherosclerosis.21,36,37 Our cohort of TBRS individuals rela- results may also have broader implications. The DNMT3A
tive to unaffected controls did not show significantly mutations in TBRS and CH largely overlap and, therefore,
increased IL6. However, it should be noted that our TBRS TBRS may offer a unique opportunity to address the
cohort included only children and young adults and it is impact of DNMT3A mutations on hematopoiesis in a
possible an increase in inflammatory cytokines detectable state mimicking accelerated CH.
by the relatively insensitive enzyme-linked immunosor-
bant assay may only be apparent with aging. If confirmed, Disclosures
increased inflammation with aging in TBRS individuals No conflicts of interest to disclose.
may elevate the risk of atherosclerotic cardiovascular dis-
ease, warranting lifetime monitoring. This monitoring Contributions
may be of particular importance given that TBRS is asso- AT, MAG and RER conceived the project, discussed and
ciated with congenital cardiac defects.1,37 designed experiments; AT performed experiments and analysis
Given the association between somatic DNMT3A with assistance of CR, GM, LZ, KK, SEC, AGG, RA and
mutations and leukemia, malignancy risk is a major con- CWC; AGS, AG and ANM performed flow cytometry analysis
cern for TBRS individuals. While there are case reports in on TBRS individuals; ANM and MJH performed all the patho-
the literature of TBRS individuals developing hematologic logical analysis in this manuscript; YHH, TA and AT generated
malignancies, the absolute risk is currently undefined. In the mouse models for all studies; JJK, MTC, ALA, IV, RS, LVM
this study we found that unperturbed HET293 mice and LB assisted with collection of blood and medical data from
developed spontaneous hematologic malignancies at TBRS patients; JMR conducted the bioinformatic analyses in the
higher rates than WT mice, consistent with constitutive manuscript; AT, MAG and RER wrote the manuscript; all
Dnmt3a-HET and previous studies using complete somat- authors interpreted the results and edited the manuscript.
ic Dnmt3a deletion.27,38,39 We also identified malignancies
which are not often associated with DNMT3A-mutations Acknowledgments
and not reported to date in TBRS; histolytic sarcoma and We thank our research participants and their families for their
angiosarcoma. Interestingly, pathologic examination of contributions and active involvement. We thank the TBRS
non-moribund aged HET293 mice revealed Community for their kind help in supporting this project. We
extramedullary hematopoiesis (EMH) involving spleen, especially wish to thank the TBRS Community board of directors,
liver and kidney in a subset of mice (Online Supplementary particularly Jill Kiernan and Kerry Grens. We also thank C.
Figure S7). EMH can be a feature of myeloproliferative Gillespie for critical review.
neoplasm.40 However, in our TBRS models this finding is
of uncertain significance as the affected HET293 mice Funding
were healthy without significant blood count abnormali- This project was supported by the Baylor College of Medicine’s
ties. Overall, our findings support a possible increased Human Stem Cell Core and Baylor College of Medicine’s
risk for hematologic malignancies in individuals with Cytometry and Cell Sorting Core, which are funded in part by
TBRS. Thus, TBRS individuals and their families should the institution and the NIH CA125123, OD028591,
be educated about the signs and symptoms of leukemia AI036211, A125123, RR024574, DK092833, CA183252,
that would warrant medical evaluation. However, the rel- K08CA201611 and the HHMI James H. Gilliam Fellowships.
atively low penetrance of malignancy in our mouse Publication costs were generously supported by the Texas
model, even at advanced ages, indicates that leukemia is Children's Hospital Young Investigators Endowed Fund.
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Hodgkin Lymphoma ARTICLE
Improved outcomes of high-risk relapsed Ferrata Storti Foundation

Hodgkin lymphoma patients after high-dose
chemotherapy: a 15-year analysis
Yago Nieto,1 Stephen Gruschkus,2 Benigno C. Valdez,1 Roy B. Jones,1
Paolo Anderlini,1 Chitra Hosing,1 Uday Popat,1 Muzaffar Qazilbash,1
Partow Kebriaei,1 Amin Alousi,1 Neeraj Saini,1 Samer Srour,1
Katayoun Rezvani,1 Jeremy Ramdial,1 Melissa Barnett,1 Alison Gulbis,3
Terri Lynn Shigle,3 Sairah Ahmed,4 Swaminathan Iyer,4 Hun Lee,4 Ranjit Nair,4
Simrit Parmar,4 Raphael Steiner,4 Bouthaina Dabaja,5 Chelsea Pinnix,5 Haematologica 2022
Jillian Gunther,5 Branko Cuglievan,6 Kris Mahadeo,6 Sajad Khazal,6 Volume 107(4):899-908
Hubert Chuang,7 Richard Champlin,1 Elizabeth J. Shpall1
and Borje S. Andersson1
1
Department of Stem Cell Transplantation and Cellular Therapy, University of Texas MD
Anderson Cancer Center; 2Biostatistics, University of Texas MD Anderson Cancer
Center; 3Pharmacy, University of Texas MD Anderson Cancer Center; 4Lymphoma and
Myeloma, University of Texas MD Anderson Cancer Center; 5Radiation Oncology,
University of Texas MD Anderson Cancer Center; 6Pediatrics, University of Texas MD
Anderson Cancer Center and 7Nuclear Medicine, University of Texas MD Anderson
Cancer Center, Houston, TX, USA
ABSTRACT
H
igh-dose chemotherapy and autologous stem-cell transplant
(HDC/ASCT) is standard treatment for chemosensitive relapsed
classical Hodgkin lymphoma, although outcomes of high-risk
relapse (HRR) patients remain suboptimal. We retrospectively analyzed
all HRR classical Hodgkin lymphoma patients treated with HDC/ASCT
at our institution between 01/01/2005 and 12/31/2019. HRR criteria
included primary refractory disease/relapse within 1 year, extranodal
extension, B symptoms, requiring more than one salvage line, or
positron emission tomography (PET)-positive disease at ASCT. All
patients met the same ASCT eligibility criteria. We treated 501 patients
with BEAM (n=146), busulphan/melphalan (BuMel) (n=38), gemc-
itabine(Gem)/BuMel (n=189) and vorinostat/Gem/BuMel (n=128). The Correspondence:
Gem/BuMel and vorinostat/Gem/BuMel cohorts had more HRR criteria YAGO NIETO
and more patients with PET-positive disease at ASCT. Treatment with ynieto@mdanderson.org
brentuximab vedotin (BV) or anti-PD1 prior to ASCT, PET-negative dis-
ease at ASCT, and maintenance BV increased over time. BEAM and
BuMel predominated in earlier years (2005-2007), GemBuMel and
BEAM in middle years (2008-2015), and vorinostat/GemBuMel and Accepted: April 22, 2021.
BEAM in later years (2016-2019). The median follow-up is 50 months Pre-published: May 6, 2021.
(range, 6-186). Outcomes improved over time, with 2-year progression-
free survival (PFS)/overall survival (OS) rates of 58%/82% (2005-2007),
59%/83% (2008-2011), 71%/94% (2012-2015) and 86%/99% (2016-
2019) (P<0.0001). Five-year PFS/OS rates were 72%/87% after vorinos-
tat/GemBuMel, 55%/75% after GemBuMel, 45%/61% after BEAM, and ©2022 Ferrata Storti Foundation
39%/57% after BuMel (PFS: P=0.0003; OS: P<0.0001). These differences Material published in Haematologica is covered by copyright.
persisted within the PET-negative and PET-positive subgroups. Prior BV All rights are reserved to the Ferrata Storti Foundation. Use of
and vorinostat/GemBuMel were independent predictors of more favor- conditions:
able outcome, whereas primary refractory disease, ≥2 salvage lines, https://creativecommons.org/licenses/by-nc/4.0/legalcode.
bulky relapse, B symptoms and PET-positivity at ASCT correlated inde- Copies of published material are allowed for personal or inter-
pendently with unfavorable outcomes. In conclusion, post-HDC/ASCT poses is subject to the following conditions:
outcomes of patients with HRR classic Hodgkin lymphoma have https://creativecommons.org/licenses/by-nc/4.0/legalcode,
improved over the last 15 years. Pre-ASCT BV treatment and optimized sect. 3. Reproducing and sharing published material for com-
mercial purposes is not allowed without permission in writing
synergistic HDC (vorinostat/GemBuMel) were associated with this from the publisher.
improvement.

Y. Nieto et al.
Introduction defined as those ≥5 cm. All demographic and tumor-related vari-

ables were captured prospectively in our departmental database.
High-dose chemotherapy (HDC) with autologous During this 15-year period we studied new HDC regimens for
stem-cell transplant (ASCT) is standard treatment of HRR cHL in sequential Institutional Review Board-approved clini-
relapsed classical Hodgkin lymphoma (cHL).1,2 Adverse cal trials: a phase II trial of BuMel (NCT00427765),14 a phase I trial
predictors of post-ASCT outcome include primary refrac- of GemBuMel (NCT00410982),15 a phase II study of GemBuMel
tory disease, short first complete remissions, extranodal (NCT01200329),16 a phase I/II trial of vorinostat/GemBuMel (NCI-
extension, bulky lesions or B symptoms at the time of 2011-02891),18 and a phase I/II trial of azacytidine/
relapse, performance status ≥1 at relapse, relapse within vorinostat/GemBuMel (NCT01983969).19 The upper age limit of
a prior radiation field, requirement for more than one line participants in these trials was 65 years and the patients had to
of salvage chemotherapy and, particularly, the persistence have had a performance status 0-2 and normal renal, pulmonary,
of metabolically active tumor on pre-HDC positron emis- cardiac and hepatic function (Online Supplementary Material).
sion tomography (PET).3-5 BuMel,14 GemBuMel,15 vorinostat/GemBuMel,18 and azacyti-
The BEAM regimen (carmustine/etoposide/cytarabine/ dine/vorinostat/GemBuMel19 were administered as previously
melphalan) has long been the standard HDC combination described (Online Supplementary Material). Since azacytidine did
for cHL despite its suboptimal results in patients with high- not improve the activity of vorinostat/GemBuMel, we included
risk relapses (HRR), whose long-term progression-free sur- those patients with the vorinostat/GemBuMel group in this analy-
vival (PFS) rate is around 50%.3,5,6 Efforts to improve ASCT sis.
outcomes have focused mainly in the pretransplant and Patients with HRR cHL who were eligible for those trials but
posttransplant settings. Pretransplant PET-guided use of who instead received standard BEAM were prospectively regis-
non-cross-resistant chemotherapy and incorporation of tered in our database. In addition, both GemBuMel and vorinos-
novel drugs, such as brentuximab vedotin (BV), seem to tat/Gem/Bu/Mel were adopted as standard regimens at our insti-
improve results.7,8 In the post-ASCT setting, a randomized tution upon publication of their trials. Reasons for treating patients
trial of maintenance BV after BEAM for HRR cHL showed off study included their declining trial participation, patients’ lack
improvement of 5-year PFS from 41% to 59% as compared of clinical trial insurance benefits or periods when no trial was
to that with placebo.9,10 open to enrollment. The choice of HDC regimen off study was at
In contrast, despite clearly suboptimal results obtained the treating physician’s discretion.
with BEAM, little effort has gone into developing a more Institutional guidelines for supportive care and follow-up visits
efficacious HDC program, save for the notable exceptions were followed (Online Supplementary Material). All patients in this
of attempts to deliver HDC in a tandem fashion.11-13 We analysis had undergone pre-ASCT PET/computed tomography
have systematically sought to develop more effective HDC scans, prospectively interpreted as positive (active tumor) or nega-
regimens based on investigations of synergistic interactions tive (no active tumor) using the International Harmonization
between its components. We started with a combination of Project in Lymphoma (IHPL) criteria up to 2013 (with mediastinal
pharmacokinetically-guided intravenous busulfan with blood pool activity as the reference background),20 and the
melphalan (BuMel), which was as safe as BEAM but Deauville score from 2014 thereafter (with a score 1-3 considered
appeared not to be more effective.14 Following the demon- a complete remission).21
stration of marked preclinical synergy between gemcitabine Post-transplant radiotherapy, delivered at 30-41.4 Gy, was con-
and BuMel, we next investigated clinically the GemBuMel sidered for bulky relapses and/or PET-positive lesions at ASCT.
combination.15,16 Finally, our preclinical work on epigenetic Maintenance BV was considered for all patients after the results of
modulation, the synergistic interactions between nucleo- the AETHERA study became available.9
side analogs and bifunctional DNA-alkylating agents used
in HDC17 led us to clinically test vorinostat/GemBuMel18 Statistical analyses
and azacytidine/vorinostat/ GemBuMel.19 Differences in variables by cohort were assessed with Wilcoxon
We herein report our experience with HDC with ASCT rank-sum and c2 tests for continuous and categorical variables,
for HRR cHL over the last 15 years, analyzing patient-, respectively.22 PFS and overall survival (OS) were measured from
tumor-, and treatment-related factors (pre-ASCT, HDC reg- the initiation of HDC to relapse or death, respectively, or last fol-
imens, and post-ASCT) associated with outcome. low-up visit. The Kaplan-Meier method estimated 12-, 24-, and
60-month PFS and OS,23 and differences in outcomes were
assessed using the log-rank test.24 Univariable and multivariable
Methods Cox regression analyses identified factors associated with PFS and
OS. Statistical significance was defined by a=0.05 and all analyses
We retrospectively analyzed all patients with HRR cHL treated used SAS v.9.4 (Cary, NC, USA).
at MD Anderson Cancer Center with HDC and ASCT between
01/01/2005 and 12/31/2019. This analysis was approved by the
Institutional Review Board. As in our sequential GemBuMel trials, Results
HRR was defined for this analysis by one or more of the following
criteria: relapse within 1 year or refractoriness to frontline therapy, A total of 501 patients with HRR cHL were treated with
extranodal extension at relapse, B symptoms at relapse, failure to HDC and ASCT between 01/01/2005 and 12/31/2019 and
achieve a complete remission in response to the most recent ther- all are included in this analysis: 189 received GemBuMel
apy, or requiring two or more lines of salvage therapy. Lines of sal- (159 on two clinical trials and 30 off study), 128 received
vage chemotherapy were defined as different regimens used to vorinostat-GemBuMel (± azacytidine) (41 on trial, 87 off
treat persistent/progressive disease and did not include a different trial), 146 received BEAM (all standard of care), and 38
regimen given to mobilize peripheral blood progenitor cells fol- BuMel (all on study). Thirty-seven patients (7.3%) received
lowing a complete remission. Patients not meeting any HRR crite- maintenance BV.
ria were excluded from this analysis. Bulky lesions at relapse were Over a median follow-up of 50 months (range, 6-186), a

ASCT for poor-risk relapsed HL in the last 15 years
Figure 1. Progression-free survival and overall

survival of all patients. PFS: progression-free
survival; OS: overall survival; ASCT: autologous
stem-cell transplantation.
total of 205 patients (40.9%) experienced relapse and 130 high-risk criteria (P=0.0006), as well as more patients
patients (25.9%) died. Treatment-related mortality from with PET-positive disease at ASCT (P=0.0002), as com-
HDC/ASCT was the cause of death of two patients, aged pared to patients treated with BEAM or BuMel.
40 and 45, who died from infectious complications, both Patient- and tumor-related variables did not change
around 3 months after BEAM therapy. Other causes of substantially over time but there was an increase in the
death were progressive disease (n=96), second primary use of pre-ASCT BV (P<0.0001) and anti-PD1 (P<0.0001),
malignancies (n=8), toxicity from post-ASCT salvage thera- a decrease in PET-positive disease at ASCT (P=0.0008),
pies (n=21, 19 after allogeneic stem cell transplantation, 2 and an increase in post-transplant BV (P<0.0001) (Table
after BV), unrelated late events (n=2), and unknown (n=1). 2). BEAM and BuMel predominated in earlier years
The causes of death did not vary across the different time (2005-2007), GemBuMel and BEAM in middle years
periods (Online Supplementary Table S1). (2008-2015), and vorinostat/GemBuMel and BEAM in
The overall 1-year, 2-year and 5-year PFS rates for the the last 4 years (2016-2019) (P<0.0001). Consequently,
whole population were 67% (95% confidence interval the use of post-ASCT maintenance BV was largely
[95% CI]: 63-71%), 60% (95% CI: 56-64%), and 55% restricted to the vorinostat/GemBuMel and BEAM
(95% CI: 50-59%), respectively. The 1-year, 2-year and 5- cohorts (P<0.0001). These two cohorts, in particular the
year OS rates were 92% (95% CI: 89-94%), 84% (95% CI: one treated with vorinostat/GemBuMel, also received
81-87%), and 73%, respectively (Figure 1). There was a more prior BV (P<0.0001) and anti-PD1 (P=0.0001).
gradual improvement in PFS and OS over time (Figures 2 To discern a possible confounding effect of having fol-
and 3). The 2-year PFS rates were 48% for those transplant- lowed two different sets of criteria for interpretation of
ed between 2005-2007, 50.6% for those transplanted PET scans (IHPL from 2005-2013 and the Deauville score
between 2008-2010, 64.3% for those transplanted between from 2014-2019) we retrospectively reviewed all patients
2011-2015 and 78.7% for those transplanted between 2016- from the earlier period who had a positive PET at ASCT
2019 (P<0.0001) (Figure 2A). Their respective 2-year OS by IHPL criteria. Thus, those whose PET showed uptake
rates were 74.6%, 76.8%, 89.7% and 96.2% (P<0.0001) greater than mediastinum but not than liver were reas-
(Figure 2B). signed as negative (Deauville score 3). Of 115 patients
Seven BEAM patients were ineligible for the clinical trials with a positive PET by IHPL, 23 were reassigned as PET-
due to age older than 65 years. Three of them are alive in negative: 15 in the GemBuMel cohort (21.7% of PET-
complete remission at 15 months, 3 years and 5 years after positive patients by IHPL in this cohort), two in the
ASCT. The other four relapsed at a median of 17 months vorinostat/GemBuMel cohort (18.1%), four in the BEAM
after ASCT (range, 4-39 months), and three died from cohort (16.6%), and two in the BuMel cohort (18.1%).
tumor progression. We excluded these seven patients from There were significant differences among the four
the cohort and prognostic analyses described below, for cohorts in PFS (P=0.0003) (Figure 3A) and OS (P<0.0001)
which all patients met the same eligibility criteria. (Figure 3B), with patients receiving vorinostat/
GemBuMel having the best outcomes, followed by
Cohort analyses those treated with GemBuMel, BEAM and BuMel. The
There were significant differences in disease character- respective 2-year and 5-year PFS rates were 73.2% and
istics among the four cohorts (Table 1). The GemBuMel 71.9% (vorinostat/GemBuMel), 57.3% and 55%
and vorinostat/GemBuMel groups included more (GemBuMel), 56.3% and 45% (BEAM), and 47.4% and
patients with primary refractory disease (P=0.001), bulky 38.9% (BuMel) (Figure 4). Likewise, the respective 2-
relapse (P<0.0001) and more patients with three or more year and 5-year OS rates were 93.8% and 87.3%

Y. Nieto et al.
(vorinostat/GemBuMel), 85.5% and 75.5% 88.3%, and vorinostat/GemBuMel 88.6% (P=0.48).

(GemBuMel), 75.2% and 60.8% (BEAM), and 78.9% However, complete remission rates were higher after
and 57.2% (BuMel) (Figure 5). The differences among vorinostat/GemBuMel (82.8%) than after GemBuMel
regimens persisted within the subgroups with PET-neg- (70.1%), BuMel (63.6%) or BEAM (50%) (P=0.03).
ative (PFS: P=0.0002; OS: P<0.0001) (Figure 4A) and The median follow-up times of the four cohorts were
PET-positive disease at ASCT (PFS: P=0.002; OS: 97 months (range, 3-189) for those transplanted between
P<0.0001) (Figure 4B). Likewise, these differences were 2005-2007, 93 months (range, 6-138) for those trans-
also seen when patients were analyzed by number of planted between 2008-2011), 57 months (range, 6-91) for
risk factors (Online Supplementary Figures S1-3). those transplanted between 2012-2015, and 26 months
Overall responses to HDC, determined around day (range, 3-55) for those transplanted between 2016-2019.
+30 after ASCT in patients with measurable active dis-
ease at the time of transplantation, did not vary among Prognostic analyses
the cohorts: BEAM 76.9%; BuMel 72.7%, GemBuMel Univariate analyses of PFS showed that primary refracto-
Table 1. Patient and clinical features of the matched cohorts of patients (n=494).
All BEAM BuMel GemBuMel Vorinostat/GemBuMel P
(N=494) (N=139) (N=38) (N=189) (N=128)
Age in years, median (range) 34.5(8-65) 36 (10-65) 36 (20-63) 34 (13-65) 33 (8-62) 0.31
Gender, male/female, % 57.5 / 42.5 58.2 / 41.8 65.8 / 34.2 56.1 / 43.9 56.3 / 43.8 0.72
ASCT year interval
2005-2007 97 (19.6%) 57 (41%) 38 (100%) 2 (1.1%) 0 (0%) <0.0001
2008-2011 138 (27.9%) 37 (27.2%) 0 (0%) 97 (51.3%) 4 (3.1%)
2012-2015 157 (31.7%) 25 (17.9%) 0 (0%) 88 (46.6%) 44 (34.4%)
2016-2019 102 (20.6%) 20 (14.7%) 0 (0%) 2 (1.1%) 80 (62.5%)
N. of modified AETHERA criteria
Median (range) 2 (1-4) 1 (1-4) 2 (1-4) 2 (1-4) 2 (1-4) 0.001
1 198 (40%) 72 (51.8%) 13 (34.2%) 72 (38%) 41 (32%) 0.0006
2 196 (39.6%) 48 (34.5%) 19 (50%) 77 (40.7%) 52 (40.6%)
3 84 (17%) 16 (11.5%) 5 (13.1%) 35 (18.5%) 28 (21.9%)
4 16 (3.2%) 3 (2.1%) 1 (2.6%) 5 (2.6%) 7 (5.5%)
Primary refractory disease 218 (44.1%) 45 (34.5%) 12 (31.5%) 100 (53.4%) 61 (50%) 0.001
Prior disease-free interval#
Median (range) 2 (3-242) 4 (3-115) 1 (3-98) 0 (3-166) 0 (3-242) 0.02
<12 months 186 (67.4%) 65 (69.1%) 24 (92.3%) 59 (66.3%) 38 (56.7%) 0.01
≥12 months 90 (32.6%) 29 (30.9%) 2 (76.9%) 30 (33.7%) 29 (43.2%)
PS ≥1 at relapse 179 (36.2%) 55 (37.6%) 11 (28.9%) 64 (33.8%) 49 (38.2%) 0.55
Extranodal extension at relapse 205 (41.4%) 44 (31.6%) 13 (34.2%) 84 (44.4%) 57 (44.5%) 0.21
B symptoms at relapse 79 (16%) 21 (15.1%) 5 (13.1%) 26 (13.7%) 27 (21.1%) 0.29
Prior radiotherapy 133 (26.9%) 42 (30.2%) 11 (28.9%) 47 (24.9%) 33 (25.8%) 0.85
Relapse within prior RT field 55 (11.1%) 12 (8.6%) 4 (10.5%) 19 (10%) 21 (16.4%) 0.16
Bulky relapse 150 (30.3%) 22 (15.8%) 7 (18.4%) 74 (39.1%) 47 (36.7%) <0.0001
Prior BV 120 (24.3%) 25 (18%) 0 (0%) 24 (12.7%) 71 (55.4%) <0.0001
Prior anti-PD1 19 (3.8%) 3 (2.1%) 0 (0%) 2 (1%) 14 (10.9%) 0.0001
N. of prior lines of therapy
Median (range) 2 (2-8) 2 (2-8) 2 (2-6) 2 (2-6) 3 (2-7) <0.0001
>2 206 (41.7%) 44 (31.6%) 16 (42.1%) 72 (38.1%) 74 (57.8%) <0.0001
N. of prior relapses
Median (range) 1 (1-7) 1 (1-7) 1 (1-4) 1 (1-5) 1 (1-6) 0.10
>1 165 (33.4%) 41 (29.4%) 10 (26.3%) 63 (33.3%) 51 (39.8%) 0.19
Positive PET at ASCT 141 (28.5%) * 25 (18%) * 11 (28.9%) * 75 (39.7%) * 37 (28.9%) * 0.0002 *
118 (23.9%) ** 21 (15.1%) ** 9 (23.6%) ** 60 (31.7%) ** 35 (27.3%) ** 0.007 **
Progressive disease at ASCT 38 (7.6%) 4 (2.8%) 1 (2.6%) 24 (12.7%) 9 (7%) 0.004
Post-ASCT radiotherapy 69 (14%) 11 (7.9%) 4 (10.5%) 28 (14.8%) 27 (21.1%) 0.02
Post-ASCT BV 37 (7.4%) 14 (10.3%) 0 (0%) 1 (0.5%) 20 (15.6%) <0.001
Values are numbers (percentages) unless otherwise stated. #Disease-free interval excludes patients with primary refractory disease. *PET interpreted per International
Harmonization Project in Lymphoma (2005-2013) and Deauville criteria (2014-2019). **All PET interpreted per Deauville criteria. BEAM: carmustine/ etoposide/cytarabine/mel-
phalan; BuMel: busulphan/melphalan; GemBuMel: gemcitabine/busulphan/melphalan; ASCT: autologous stem-cell transplant; PS: performance status; RT: radiotherapy; BV: bren-
tuximab vedotin; PET: positron emission tomography.

ry disease (P=0.005), B symptoms at relapse (P=0.009), per- PFS. In contrast, prior BV treatment (P=0.01), prior anti-PD1
formance status ≥1 at relapse (P=0.01), more than one prior treatment (P=0.03), the use of vorinostat/GemBuMel
relapse (P=0.0001), more than two prior lines of therapy (P=0.0004), and post-ASCT maintenance therapy with BV
(P=0.0004), positive PET at ASCT (P<0.0001), and progres- (P=0.01) were associated with a favorable PFS (Table 3).
sive disease at ASCT (P<0.0001) correlated with an adverse In multivariable analyses of PFS, primary refractory dis-
Table 2. Patient and clinical characteristics by treatment year interval (entire population, n=501).
Treatment year interval
2005-2007 2008-2011 2012-2015 2016-2019 P
(N=98) (N=138) (N=158) (N=107)
Age in years, median (range) 30 (18-72) 35 (10-69) 32 (13-70) 34 (8-71) 0.58
Gender, male/female (%) 62 (63%) / 36 (37%) 80 (58%) / 58 (42%) 87 (55%) /71 (45%) 59 (55%) /48 (45%) 0.57
HDC regimen
BEAM 58 (59.2%) 37 (26.8%) 26 (16.5%) 25 (23.4%) <0.0001
BuMel 38 (100%) 0 (0%) 0 (0%) 0 (0%)
GemBuMel 2 (2%) 97 (70.3%) 88 (55.7%) 2 (1.9%)
Vorinostat/GemBuMel 0 (0%) 4 (2.9%) 44 (27.8%) 80 (74.8%)
Primary refractory disease 41 (41.8%) 69 (50%) 74 (46.8%) 43 (40.2%) 0.39
Prior disease-free interval *
Median (range) 1 (3-108) 0 (3-166) 3 (3-145) 3 (3-242) 0.11
<12 months 43 (75.4%) 45 (65.2%) 45 (53.5%) 83 (62.5%) 0.16
PS ≥1 at relapse 37 (37.7%) 48 (34.4%) 61 (38.6%) 33 (30.8%) 0.59
Bulky relapse 19 (19.4%) 45 (32.6%) 60 (38%) 27 (25.2%) 0.008
Extranodal extension at relapse 33 (33.7%) 64 (46.4%) 68 (43%) 40 (37.4%) 0.19
B symptoms at relapse 14 (14.3%) 19 (13.8%) 24 (15.2%) 22 (20.6%) 0.48
N. of prior relapses
Median (range) 1 (1-4) 1 (1-6) 1 (1-7) 1 (1-5) 0.24
>1 27 (27.6%) 54 (39.1%) 51 (32.3%) 34 (31.8%) 0.28
N. of prior lines of therapy
Median (range) 2 (2-6) 2 (2-7) 2 (2-8) 2 (2-10) 0.29
>2 34 (34.7%) 54 (39.1%) 72 (45.6%) 47 (43.9%) 0.31
Prior BV 0 (0%) 4 (2.9%) 60 (38%) 58 (54.2%) <0.0001
Prior anti-PD1 0 (0%) 0 (0%) 2 (1.3%) 19 (17.8%) <0.0001
Positive PET at ASCT 29 (29.6%) 58 (42%) 40 (25.3%) 21 (19.6%) 0.0008
Progressive disease at ASCT 5 (5.1%) 23 (16.7%) 7 (4.4%) 3 (2.8%) <0.0001
Post-ASCT radiotherapy 7 (7.1%) 28 (20.3%) 16 (10.1%) 19 (17.8%) 0.0089
Post-ASCT BV 0 (0%) 0 (0%) 2 (1.3%) 35 (32.7%) <0.0001
Values are numbers (percentages) unless otherwise stated. *Disease-free interval excludes patients with primary refractory disease. HDC: high-dose chemotherapy; BEAM: car-
mustine/etoposide/cytarabine/melphalan; BuMel: busulphan/melphalan; GemBuMel: gemcitabine/busulphan/melphalan; PS: performance status; BV: brentuximab vedotin; PET:
positron emission tomography; ASCT: autologous stem-cell transplant.
A B
Figure 2. Outcomes by treatment year. (A) Progression-free survival, (B) overall survival.

Y. Nieto et al.
Table 3. Cox regression univariable and multivariable analyses of progression-free survival of the matched cohorts of patients.
Univariable Multivariable
95% CI 95% CI
HR LL UL P HR LL UL P
Age >35 years 1.17 0.89 1.54 0.26 1.13 0.85 1.50 0.39
Female gender 0.92 0.70 1.22 0.57 1.02 0.76 1.36 0.9
Treatment year
2005-2007 3.85 2.27 6.54 <0.0001 - - - -
2008-2011 3.14 1.87 5.28 <0.0001 - - - -
2012-2015 2.07 1.22 3.51 0.006 - - - -
2016-2019 1 (ref)
Primary refractory disease 1.49 1.14 1.96 0.003 1.41 1.01 1.97 0.04
Prior disease-free interval <1 year 0.76 0.53 1.09 0.14 0.92 0.60 1.40 0.69
N. of prior relapses >1 1.72 1.30 2.27 0.0001 1.19 0.79 1.78 0.4
N. of prior lines of therapy >2 1.65 1.26 2.17 0.0003 1.60 1.08 2.12 0.01
PS ≥1 at relapse 1.28 1.19 1.92 0.01 1.15 0.78 1.52 0.7
Bulky relapse 1.33 1.00 1.77 0.047 1.56 1.15 2.12 0.004
Extranodal extension at relapse 0.97 0.73 1.28 0.81 0.98 0.73 1.31 0.88
B symptoms at relapse 1.58 1.13 2.22 0.008 1.68 1.19 2.37 0.003
Positive PET at ASCT 2.90 2.20 3.83 <0.0001 2.60 1.83 3.69 <0.0001
Progressive disease at ASCT 2.64 1.77 3.94 <0.0001 1.06 0.64 1.76 0.83
Prior BV 0.65 0.45 0.93 0.01 0.58 0.36 0.93 0.02
Prior anti-PD1 0.23 0.06 0.94 0.04 0.35 0.08 1.48 0.15
HDC regimen global global
P=0.0006 P=0.001
Vorinostat/GemBuMel 1 (ref) 1 (ref)
GemBuMel 1.74 1.16 2.61 0.007 1.33 0.83 2.13 0.23
BEAM 2.10 1.38 3.19 0.0005 2.19 1.35 3.55 0.001
BuMel 2.74 1.63 4.62 0.0002 2.29 1.24 4.23 0.007
Post-ASCT BV 0.23 0.08 0.73 0.01 0.37 0.12 1.20 0.09
Post-ASCT radiotherapy 0.94 0.63 1.39 0.75 0.66 0.43 1.02 0.06
HR: hazard ratio. 95% CI: 95% confidence interval. LL: lower limit. UL: upper limit; ref: reference; PS: performance status; PET: positron emission tomography; ASCT: autologous stem-
cell transplant. BV: brentuximab vedotin; HDC: high-dose chemotherapy; GemBuMel: gemcitabine/busulphan/melphalan; BEAM: carmustine/etoposide/cytarabine/melphalan;
BuMel: busulphan/ melphalan.
ease (hazard ratio [HR]=1.41 [95% CI: 1.01-1.97], P=0.04), prior lines of therapy (HR=2.11 [95% CI: 1.26-3.53],
more than two prior lines of therapy (HR=1.60 [95% CI: P=0.004), and positive PET at ASCT (HR=1.88 [95% CI:
1.08-2.36], P=0.01), bulky relapse (HR=1.56 [95% CI: 1.15- 1.16-3.04], P=0.01) as independent adverse prognostic fac-
2.12], P=0.004), B symptoms (HR=1.68 [95% CI: 1.19-2.37], tors. On the contrary, prior BV treatment (HR=0.46 [95%
P=0.003) and a positive PET at ASCT (HR=2.60 [95% CI: CI: 0.23-0.90], P=0.02) and vorinostat/GemBuMel
1.83-3.69], P<0.0001) were independent adverse prognostic (P<0.0001) were independently associated with better OS.
factors, whereas prior BV (HR=0.58 [95% CI: 0.36-0.93], The hazard ratios for the other three HDC regimens com-
P=0.02) and vorinostat/GemBuMel (P<0.0001) were inde- pared to vorinostat/GemBuMel were: GemBuMel: 1.63
pendently associated with improved PFS. The hazard ratios (95% CI: 0.75-3.57), BEAM: 5.06 (95% CI: 2.30-11.10), and
for the other three HDC regimens compared to vorinos- BuMel 5.17 (95% CI: 2.13-12.54) (Table 4).
tat/GemBuMel were: GemBuMel: 1.33 (95% CI: 0.83-2.13), Of note, evaluation of all PET according to the Deauville
BEAM: 2.19 (95% CI: 1.35-3.55), and BuMel 2.29 (95% CI: score did not change the prognostic effect of this variate in
1.24-4.23) (Table 3). univariate analyses or the results for any variable in the
The following were unfavorably associated with OS in multivariate analyses.
univariate analyses: age >35 years (P=0.006), B symptoms
(P=0.006), performance status ≥1 (P=0.002), more than one Treatment for post-ASCT relapse
prior relapse (P=0.0001), more than two prior lines of ther- Patients received a median of two (range, 0-12) lines of
apy (P<0.0001), positive PET at ASCT (P<0.0001), and pro- salvage therapy for post-ASCT recurrence. Salvage thera-
gressive disease at ASCT (P<0.0001). In contrast, prior BV pies included BV (n=85), conventional chemotherapy
treatment (P=0.01) and vorinostat/GemBuMel (P<0.0001) (n=72), clinical trials of experimental agents (n=67), allo-
were associated with a more favorable OS (Table 4). geneic stem cell transplantation (n=64), anti-PD1 (n=37),
Multivariable OS analyses identified age >35 years radiotherapy (n=27), and unknown (n=14). No salvage
(HR=1.80 [95% CI: 1.24-2.60], P=0.002), B symptoms therapy was administered to 13 patients. Of the 205
(HR=1.74 [95% CI: 1.13-2.68], P=0.01), more than two patients who relapsed, 53 are currently in a new clinical

complete remission after allogeneic stem cell transplanta- Second primary malignancies
tion (n=26), anti-PD1 (n=13), BV (n=9), radiotherapy (n=3) Out of the entire population (n=501) eight patients
and chemotherapy (n=2). developed therapy-related myelodysplastic syndrome and
A B
Figure 3. Outcomes by high-dose chemotherapy regimen. (A) Progression-free survival, (B) overall survival. GemBuMel: gemcitabine/busulphan/melphalan; BEAM:
carmustine/etoposide /cytarabine/melphalan; BuMel: busulphan/melphalan.
Table 4. Cox regression univariable and multivariable analyses of overall survival of the matched cohorts of patients.
Univariable Multivariable
95% CI 95% CI
HR LL UL P HR LL UL P
Age >35 years 1.63 1.15 2.32 0.006 1.80 1.24 2.60 0.002
Female gender 0.99 0.69 1.42 0.96 1.18 0.81 1.73 0.39
Treatment year
2005-2007 11.87 3.69 38.23 <0.0001 - - - -
2008-2011 7.21 2.23 23.31 0.001 - - - -
2012-2015 3.41 1.03 11.31 0.04 - - - -
2016-2019 1 (ref)
Primary refractory disease 1.38 0.97 1.97 0.07 1.27 0.82 1.96 0.28
Prior disease-free interval <1 year 0.70 0.42 1.15 0.15 0.78 0.44 1.39 0.39
N. of prior relapses >1 2.03 1.42 2.89 0.0001 1.25 0.74 2.13 0.40
N. of prior lines of therapy >2 2.07 1.45 2.96 <0.0001 2.11 1.26 3.53 0.004
PS ≥1 at relapse 1.37 1.21 1.98 0.002 1.21 0.79 1.85 0.44
Bulky relapse 1.22 0.84 1.77 0.29 1.51 1.00 2.29 0.05
Extranodal extension at relapse 1.00 0.70 1.42 0.98 1.08 0.74 1.57 0.69
B symptoms at relapse 1.86 1.22 2.84 0.004 1.74 1.13 2.68 0.01
Positive PET at ASCT 2.80 1.96 4.00 <0.0001 1.88 1.16 3.04 0.01
Progressive disease at ASCT 3.38 2.14 5.32 <0.0001 1.92 1.02 3.58 0.04
Prior BV 0.52 0.30 0.90 0.01 0.46 0.23 0.90 0.02
Prior anti-PD1 0.34 0.05 2.42 0.28 0.72 0.09 5.64 0.75
HDC regimen global global
P<0.0001 P<0.0001
Vorinostat/GemBuMel 1 (ref) 1 (ref)
GemBuMel 2.27 1.14 4.51 0.01 1.63 0.75 3.57 0.22
BEAM 4.24 2.14 8.39 <0.0001 5.06 2.30 11.10 <0.0001
BuMel 5.53 2.59 11.80 <0.0001 5.17 2.13 12.54 0.0003
Post-ASCT BV 0.21 0.03 1.54 0.12 0.41 0.06 3.05 0.38
Post-ASCT radiotherapy 1.18 0.74 1.89 0.48 1.06 0.62 1.82 0.83
HR: hazard ratio. 95% CI: 95% confidence interval. LL: lower limit. UL: upper limit; ref: reference; PS: performance status; PET: positron emission tomography; ASCT: autologous
stem-cell transplant. BV: brentuximab vedotin; HDC: high-dose chemotherapy; GemBuMel: gemcitabine/busulphan/melphalan; BEAM: carmustine/etoposide/cytarabine/mel-
phalan; BuMel: busulphan/ melphalan.

Y. Nieto et al.
A B
Figure 4. Progression-free survival by high-dose chemotherapy regimen according to positron emission tomography status. (A) Progression-free survival in patients
with (A) positron emission tomography (PET)-negative disease and with (B) PET-positive disease.
five patients developed therapy-related acute myeloblastic itabine, busulfan and melphalan, to DNA, which
leukemia: seven after BEAM (4.8%), three after increased DNA damage, apoptosis and cytotoxicity in
GemBuMel (1.5%), two after BuMel (5.2%) and one after refractory lymphoma cell lines.17 Those preclinical obser-
vorinostat/GemBuMel (0.07%), at a median 31 months vations, tested in subsequent clinical trials, are confirmed
(range, 5-133) after ASCT. The incidence of therapy-relat- in the present analysis, and notably did not increase the
ed myelodysplastic syndrome/acute myeloid leukemia risk of treatment-related mortality.
did not vary significantly among the cohorts (P=0.13). The other major favorable factor was the use of BV
Cytogenetic findings in these patients included complex before ASCT. BV has revolutionized the treatment of cHL
abnormalities with -7/del(7q) ± -5/del(5q) (n=7), -7 alone in the last decade. Following its favorable results and
(n=3), 11q+ (n=1), and other abnormalities (n=2). These approval in 2011 for post-ASCT relapses,29 BV was moved
patients were older (median age 54, range 22-72) than all up to the first or second line of salvage therapy before
other patients (n=493) who did not develop therapy-relat- ASCT,8,30 which allows more patients to receive HDC in a
ed myelodysplastic syndrome/acute myeloid leukemia PET-negative complete remission. Lastly, BV was success-
(median age 32; range, 8-71) (P=0.0005). fully tested in the post-ASCT maintenance setting, in which
Other second primary malignancies were renal-cell car- the randomized AETHERA trial compared 16 cycles of BV
cinoma (2 BEAM patients), Müllerian adenocarcinoma with placebo in 329 patients with HRR cHL, defined by the
and epithelioid hemangioendothelioma (1 GemBuMel same criteria as in our analysis. The use of BV resulted in
patient each), and diffuse large B-cell lymphoma (1 BuMel improved 2-year PFS (63% vs. 51%) and 5-year PFS (59%
patient, 1 vorinostat/GemBuMel patient). vs. 41%), albeit with no OS benefit.9,10 In contrast to our
population, this trial did not allow prior BV and more than
60% of patients received BEAM. Despite these differences,
Discussion we also saw a correlation of maintenance BV with improve-
ment of PFS but not OS in our univariate analyses.
Our analysis of patients with HRR cHL treated with Maintenance BV was restricted to patients we treated in
HDC and ASCT shows a gradual and significant improve- later years, which likely resulted in a loss of power and sig-
ment of outcomes over the last 15 years. Improved tumor nificance in the multivariable analysis.
control with BV before ASCT and the use of more active We saw that the pre-ASCT use of the anti-PD1 antibod-
HDC regimens, particularly vorinostat/GemBuMel, ies nivolumab and pembrolizumab was associated with
emerged independently as favorable prognostic factors in improved PFS, although this did not hold significance in
multivariable analysis. multivariable analysis, probably due to the small number
The clinical development of vorinostat/GemBuMel was of patients who received them. This class of drugs has
based on two important preclinical observations. The first produced another breakthrough in the treatment of
one was the synergistic inhibition by gemcitabine of the Hodgkin lymphoma. Besides their efficacy in post-ASCT
repair of DNA damage caused by busulfan and relapses,31,32 these drugs can serve as a successful bridge to
melphalan.15 The use of ASCT enables the infusion of ASCT by inducing responses in refractory relapses.33 In
gemcitabine at its optimal rate of 10 mg/m2/min, previ- addition, anti-PD1 might chemosensitize refractory
ously shown to avoid saturation of its intracellular enzy- tumors and improve results of HDC.34
matic activation,25 which results in greater activity and The strengths of our analysis include the homogeneity
myelotoxicity than shorter infusions of this drug,26,27 and of the population of patients and of the treatments admin-
in turn optimizes the synergy with the bifunctional DNA- istered in the four cohorts and the large sample size,
alkylating agents.28 Our second major in vitro observation which allowed us to independently dissect the prognostic
was that relaxation of chromatin after increased histone value of the patient-, tumor-, and treatment-related vari-
acetylation with vorinostat facilitated access of gemc- ables. On the other hand, our study has several limita-

tions. First, we only intended to analyze those patients vorinostat/GemBuMel over BEAM will require a random-
who ultimately received HDC and ASCT after HDC, and ized trial.
our analyses exclude patients who failed to successfully Other novel strategies developed to improve the out-
undergo salvage chemotherapy, e.g., due to morbidity or come of HRR cHL patients undergoing ASCT include new
highly refractory disease. Thus, our population does not maintenance therapies, such as anti-PD-1 alone35 or anti-
represent an unselected real-world cohort of HRR cHL PD-1 plus BV,36 which have shown very promising results.
patients. Second, the comparison of the different HDC Anti-PD-1 can be easily used after ASCT with vorinos-
regimens is nonrandomized. While all of our HRR patients tat/GemBuMel. Tandem ASCT based on BEAM has been
met the eligibility criteria of the prospective trials, physi- studied,11-13 but it is unclear, in the absence of a prospec-
cian biases in assigning patients with more aggressive tive randomized trial, how this approach might compare
tumors who were perceived to be fitter to a clinical trial to a single ASCT using vorinostat/GemBuMel.
instead of standard HDC likely played a role, as was In conclusion, the outcome of HRR cHL patients treat-
reflected in the higher proportion of patients with positive ed with HDC and ASCT has improved substantially over
PET at ASCT or other HRR criteria in the cohorts treat- the last 15 years. The incorporation of BV into pre-ASCT
ment with GemBuMel with or without vorinostat, com- salvage therapy and the use of pharmacologically opti-
pared with the BEAM group. Third, while all patients’ mized, more active HDC regimens, particularly vorinos-
data were captured prospectively, this study is retrospec- tat/GemBuMel, were associated with these improved
tive in nature, and thus, fraught with the usual limitations results.
of these analyses, including the possibility of reporting
biases or underreporting of second primary malignancies Disclosures
and other long-term events. Fourth, our analysis, which YN has provided consultancy services for Affimed and Novo
encompasses a 15-year period, is subject to the changes in Nordisk; and has received research funding from Affimed,
ASCT supportive care during this time span. However, Novartis, Takeda, Astra-Zeneca, and Biosecura. BA holds a
refinement of supportive measures does not appear to be patent for intravenous busulfan. None of the other authors has
the cause of the improvement in results over time, as the any conflicts of interest to disclose.
treatment-related mortality was minimal. Fifth, the
weight in our analysis of some major new treatments of Contributions
Hodgkin lymphoma incorporated more recently, such as YN designed research, collected data, treated patients, and
post-ASCT maintenance or the pre-ASCT use of anti-PD1, wrote the manuscript; SG and HC: analyzed the data and wrote
is limited by smaller numbers of patients. Lastly, since the manuscript; BCV, RBJ, PA, CH, UP, MQ, PK, AA, NS, SS,
patients in the vorinostat/GemBuMel cohort had worse KR, JR, MB, AG, TLS, SA, SI, HL, RN, SP, RS, BD, CP, JG,
prognostic features and since this was the HDC regimen BC, KM, SK, RC, EJS and BSA treated patients and reviewed
most used in the last period (2016-2019), this cohort had the manuscript.
the highest use of pre-ASCT and post-ASCT BV, which
likely contributed to its better results. Nevertheless, this Data sharing statement
regimen clearly stands out as an independent favorable All clinical trial protocols (NCT00427765, NCT00410982,
factor for both PFS and OS after adjusting for all other NCT01200329, NCI-2011-02891, NCT01983969) and
variables. However, definite proof of superiority of treatment orders will be made available upon request.
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Hodgkin Lymphoma ARTICLE
Inhibitors of ADAM10 reduce Hodgkin Ferrata Storti Foundation

lymphoma cell growth in 3D microenviron-
ments and enhance brentuximab-vedotin
effect
Roberta Pece,1* Sara Tavella,1* Delfina Costa,2* Serena Varesano,2* Caterina
Camodeca,3 Doretta Cuffaro,3 Elisa Nuti,3 Armando Rossello,3 Massimo
Alfano,4 Cristina D’Arrigo,5 Denise Galante,5° Jean-Louis Ravetti,6 Marco
Gobbi,7 Francesca Tosetti,2 Alessandro Poggi2# and Maria Raffaella Zocchi8#
Haematologica 2022
1
Cellular Oncology Unit, IRCCS Ospedale Policlinico San Martino and Department of Volume 107(4):909-920
Experimental Medicine, University of Genoa, Genoa; 2Molecular Oncology and
Angiogenesis Unit, IRCCS Ospedale Policlinico San Martino, Genoa; 3Department of
Pharmacy, University of Pisa, Pisa; 4Division of Experimental Oncology/Unit of Urology,
URI, IRCCS Ospedale San Raffaele, Milan; 5SCITEC-CNR, Genoa; 6Patology Unit, IRCCS
Ospedale Policlinico San Martino, Genoa; 7Clinical Hematology, University of Genoa,
Genoa and 8Division of Immunology, Transplants and Infectious Diseases, IRCCS San
Raffaele Scientific Institute, Milan, Italy.
*
RP ST DC and SV contributed equally as co-first authors.
#
AP and MRZ contributed equally as co-senior authors.
°DG current address: ARPAL (Regional Agency for Environmental Protection, Liguria), Genoa, Italy.
ABSTRACT
S
hedding of ADAM10 substrates, like TNFa or CD30, can affect
both anti-tumor immune response and antibody-drug-conjugate
(ADC)-based immunotherapy. We have published two new
ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in
Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influ-
ences the outcome of anti-cancer treatments, we set up a new three-
dimensional (3D) culture systems to verify whether ADAM10 inhibitors
can contribute to, or enhance, the anti-lymphoma effects of the ADC
brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of
lymphoma structure and architecture, we assembled two 3D culture
Correspondence:
models: mixed spheroids made of HL lymph node (LN) mesenchymal
stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL MARIA RAFFAELLA ZOCCHI
cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In zocchi.maria@hsr.it
these 3D systems we found that: i) the ADAM10 inhibitors LT4 and
MN8 reduce ATP content or glucose consumption, related to cell prolif- Received: February 8, 2021.
eration, increasing lactate dehydrogenase release as a cell damage hall- Accepted: May 28, 2021.
mark; ii) these events are paralleled by mixed spheroids size reduction
Pre-published: June 10, 2021.
and inhibition of CD30 and TNFa shedding; iii) the effects observed can
be reproduced in repopulated HL LN-derived matrix or collagen scaf-
folds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the https://doi.org/10.3324/haematol.2021.278469
anti-CD30 ADC BtxVed both in conventional cultures and in repopulat-
ed scaffolds. Thus, we provide evidence for a direct and combined anti-
lymphoma effect of ADAM10 inhibitors with BtxVed, leading to the
improvement of ADC effects; this is documented in 3D models recapit- All rights are reserved to the Ferrata Storti Foundation. Use of
ulating features of the LN microenvironment, that can be proposed as a published material is allowed under the following terms and
reliable tool for anti-lymphoma drug testing. conditions:
Introduction https://creativecommons.org/licenses/by-nc/4.0/legalcode,
sect. 3. Reproducing and sharing published material for com-
ADAM (A Disintegrin And Metalloproteinases) are transmembrane proteins with mercial purposes is not allowed without permission in writing
protease activity exerted on several substrates, including growth factors, cytokines, from the publisher.
receptors and their ligands, leading to the release of soluble bioactive molecules.1,2
Some of them, such as tumor necrosis factor (TNF)a, are involved in the develop-

R. Pece et al.
ment of different cancers.3-6 Moreover, since overexpression Methods

of ADAM10 relates with parameters of tumor progres-
sion,6,7 ADAM have been proposed as both biomarkers and ADAM10 inhibitors
therapeutic targets for cancer,7,8 and ADAM10 inhibitors LT4, MN8, and cyanine 5.5-conjugated MN8 (CAM36)
with anti-tumor effects have been developed.9-11 were synthesized as previously published,16,17,30 compared to
ADAM10 expression and increased enzymatic activity GI254023X (GIX, Sigma-Aldrich) and used at 10 mM to 1
has been documented in many tumors including chronic mM, alone or with BtxVed ((20-2 mg/mL, Pharmacy Unit,
lymphocytic leukemia, acute myeloid leukemia, non- IRCCS Policlinico San Martino).
Hodgkin and Hodgkin lymphomas (HL).12-14 In particular,
we described the overexpression of ADAM10 in the lymph Spheroids
node (LN) microenvironment in HL, together with impaired Mixed spheroids of LN-derived MSC and HL cells were
stimulation of T lymphocytes with anti-tumor activity.14 prepared as previously published.14,31 L428 or L540 cell lines
Likewise, CD30 shedding due to ADAM10 activity has (DSMZ GmbH), RS773, LN-MSC773, LN-MSC16412 or
been reported to decrease the efficiency of targeted lym- LN-MSC23274 primary cell lines were stabilized and cul-
phoma cell killing obtained with anti-CD30 monoclonal tured as previously reported.14 Spheroid dimension was
antibodies (mAb) in vitro and this effect can be prevented by analyzed at 48 hours (h), 72 h and 96 h of culture without
the use of the inhibitor GI254023X (abbreviated to GIX).15 or with 10 mM LT4 or MN8, by the CellSens 1.12 software
We have developed the inhibitors LT4 and MN8 with high (Olympus).31 Conventional co-cultures were performed
specificity for ADAM10 with the aim to enhance efficiency with HL cell lines and LN-MSC at the ratio of 10:1 in flat
and selectivity of action and to reduce the shedding of bottom 96-multiwell plates.
NKG2D ligands (NKG2DL) and CD30 by HL cell lines. The
exposure of HL cells to these compounds significantly Scaffolds
increased their sensitivity to lymphocyte-mediated killing Extracellular matrix (ECM) from LN of HL patients
due to NKG2D/NKG2DL interaction or due to the anti- (Pathology Unit, IRCCS Policlinico San Martino; IRB
body-dependent cellular cytotoxicity in the presence of the approvals 0026910/07, 03/2009, 14/09/15) was prepared as
anti-CD30 mAb Iratumumab.16,17 Thus, the ADAM10 described.32LN-MSC were co-cultured on ECM or
inhibitors, maintaining the expression of surface CD30, AviteneTMmicrofibrillar collagen sponge (Davol Inc.) scaf-
could further sensitize HL cells to the anti-lymphoma activ- folds with L428 or L540 cells, in the presence or absence of
ity of the anti-CD30-drug conjugated (ADC) humanized 10 mM LT4 or MN8, either alone or in combination with 20-
mAb brentuximab-vedotin (BtxVed) used for HL 2 mg/mL BtxVed, and analyzed at 48 h, 72 h, 72 h, 96 h and
treatment.18 120 h. Culture supernatants (SN) were recovered for TNFa
All the mentioned reported findings rely on in vitro exper- or soluble CD30 detection.
iments performed with conventional culture systems that
do neither consider the importance of tissue architecture nor Confocal microscopy
the complexity of cell populations at the tumor site. The tis- Mixed spheroids were incubated with 10 mM CAM36 for
sue microenvironment deeply contributes to determine both 1 h at 37°C, followed by 1 mM Syto16 (ThermoFisher
cancer progression and the outcome of anti-cancer treat- Scientific) and analyzed in sequence mode with a FV500
ments;19-22 thus, newly designed models for the definition of confocal microscope (Olympus). Z-stack sections were
drug safety and efficacy are rapidly developing in the field. taken every 2 mm and data was analyzed with FluoView
In turn, there is increasing evidence that tumor development 4.3b software.
in humans is not always reproducible and predictable in ani-
mal models, mostly used in preclinical studies.23-25 Scanning electron microscopy
Furthermore, they are very expensive and require a long After fixation with 4% paraformaldehyde and 1% osmi-
time to be set up and defined for each tumor. As an alterna- um tetroxide post-fixation, 10 mm sections from paraffin-
tive, several three-dimensional (3D) culture systems, includ- embedded empty scaffolds or 3D cultures, were collected
ing spheroids and scaffolds, have been validated by the on glass coverslips, mounted on aluminum stubs and sput-
European Union Reference Laboratories for Alternatives to ter-coated with gold-palladium. The ultrastructure was ana-
Animal Testing (EURL ECVAM) as preclinical models, to lyzed on a Hitachi TM3000 Benchtop scanning electron
overcome these inconveniences at least in part.26-29 microscopy (SEM) instrument operating at 15 kV accelera-
In this work we demonstrate the anti-lymphoma effect of tion voltage.
ADAM10 inhibitors in HL in two different 3D culture sys-
tems: mixed spheroids made of LN mesenchymal stromal Immunohistochemistry and immunofluorescence
cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells Five mm serial sections from 3D cultures fixed in
(from now on HL cells) and collagen sponges repopulated Histochoice (Amresco) were stained with rabbit anti-Ki67
with both LN-MSC and HL cells. In these 3D models we antiserum (1:100, Ventana Hoffman-La Roche), anti-CD30
found that: i) the ADAM10 inhibitors LT4 and MN8 reduced Ber-H2 mAb (2 mg/mL,Ventana), rabbit anti-TGII antiserum
HL cell ATP content and glucose consumption related to (1:100, ThermoScientific), rabbit anti-caspase-3 mAb
proliferation, while increasing lactate dehydrogenase (LDH) (1:1,000, Cell Signaling) or an isotypic unrelated antibody
release as a cell damage hallmark; ii) these events are paral- (Dako Cytomation). For immunohistochemistry (IHC),
leled by mixed spheroids size reduction and inhibition of biotinylated goat anti-mouse (Biot-GAM) or goat anti-rab-
soluble CD30 and TNFa shedding; iii) the effects due to bit antiserum (Biot-GAR, BioOptica) was added, followed
ADAM10 inhibitors can be reproduced in LN-derived by horseradish peroxidase (HRP)-conjugated avidin (HRP-
matrix or collagen scaffolds repopulated with LN-MSC and Av, ThermoScientific) and 3,3′-diaminobenzidine (DAB,
HL cells; iv) ADAM10 inhibitors exerted a direct anti-lym- Sigma). Immunofluorescence (IF) was performed with anti-
phoma effect and enhanced the effect of BtxVed. CD30 mAb, anti-Ki67 antiserum and DAPI, using the auto-

ADAM10 inhibitors in Hodgkin lymphoma 3D models
mated stainer BOND-Rxm. Slides were observed under a incubated with 1 µM Syto16 (blue) to stain nuclei and 10
Leica DM-MB2 microscope with a CCD camera (Olympus µM CAM36 (Cy5.5-MN8, red) documenting that the
DP70) or the AperioVERSA or AperioAT2 Scanner and data inhibitor reaches the inner part of the 3D structure. The z-
was analyzed with theAperio Cellular IF Algorithm (Leica stack images, taken every 2 mm, depicted in the Online
Biosystems).33 IHC images were analyzed with the Genie Supplementary Figure S2A and B, confirm this result. Thus,
software and the nuclear-count V9 macro (Leica 10 µM (Figure 1C and D) or dilutions (Figure 1E) of LT4 or
Biosystems).33 MN8 were added to the mixed spheroids and cultured for
ATP, LDH, soluble CD30, TNFa and glucose measure- 48 h, 72 h or 96 h. Spheroid dimensions (area in Figure 1C
ment ATP content was tested using the CellTiter-Glo® and volume in D) were analyzed in each culture well as
Luminescent Kit (Promega Italia).34 LDH was determined described in the Online Supplementary Appendix. In D, the
using the CytoTox96 Kit (Promega). Soluble CD30 and volume of spheroids made of LN-MSC16412 alone is indi-
TNFa were measured by the Picokine ELISA kit (Boster cated as well. Of note, both LT4 and MN8 could significant-
Bio) and the specific cytokine detection kit (PeproTech).34 ly reduce the size of mixed spheroids, and this effect was
Glucose was evaluated with the D-glucose Assay particularly evident after 96 h (Figure 1C, right, and D). No
(Megazyme), referred to a standard curve. effect was observed on LN-MSC16412 spheroids (Figure 1
D). In parallel samples, mixed spheroids were exposed to
Statistical analysis serial dilutions (10-0 mM) of LT4, MN8, GIX or DMSO
Data are presented as mean ± standard error of the mean 1:1,000 for 96 h; then supernatants were recovered for LDH
(SEM) or ± standard deviation (SD). Statistical analysis was detection and cells lysed for ATP measurement. All
performed by two-tailed unpaired Student’s t-test, with ADAM10 inhibitors induced a decrease in ATP cellular con-
Welch correction, using the Graph Pad Prism software 5.0. tent (Figure 1E, left) and an increase in LDH release (Figure
1E, right), with LT4 and MN8 displaying a more efficient
effect than GIX.
Results Of note, LT4 or MN8 were effective also in autologous
mixed spheroids of LN-MSC773 (2x105) and RS773 cells
ADAM10 inhibitors reduce ATP content and the size (4x105), isolated from the same LN and prepared as previ-
of Hodgkin lymphoma-mesenchymal stromal cell ously described.16 Both inhibitors (10 mM) could reduce the
spheroids ATP content at 72 h, and this was more evident at 96 h
In experiments performed under conventional culture (Figure 2Aa); at this time point, LT4 and MN8 were more
conditions, the HL cells RS773 or L428 or L540 (4x105) were effective than GIX also at 5 mM concentration (Figure 2A
cultured for 96 h with medium alone or the vehicle and B and the decrease in intracellular ATP was paralleled
dimethyl sulfoxide (DMSO) (1:1,000), or LT4, MN8 or the by a rise in LDH release (Figure 2B) and by a reduction in
commercial inhibitor GIX (10-2.5 mM). ATP content at this the secretion of TNFa (Figure 2C). Interestingly, spheroid
time point was consistently lower in the presence of the dimension was significantly lower in the cultures exposed
inhibitors than in the absence (0 mM, culture medium alone) to LT4 at 48h (Figure 2D, left), or MN8 at 72 h (central), at
or in the solvent (DMSO) and both LT4 and MN8 were variance to GIX (not shown).
more efficient than GIX, even at low (2.5 mM) concentra- In order to verify that the effects of ADAM10 inhibitors
tions (Online Supplementary Figure S1A). The effect on ATP on spheroid size and ATP content were mainly directed
cell content paralleled an impairment in cell proliferation, as against HL cells, spheroids made of LN-MSC16412 only
documented by the reduced cell number in the cultures were prepared.30 ATP intracellular content, LDH release and
containing ADAM10 inhibitors (Online Supplementary Figure size were measured at different time points, in the cultures
S1B). Online Supplementary Table S1 shows the effect of without (Online Supplementary Figure S3A to C) or with 10
other three sulfonamido-based hydroxamate compounds, mM LT4 or MN8 (Online Supplementary Figure S3D and E).
synthesized in our lab, on the ATP intracellular content and First, LDH release (A), ATP intracellular content (B) and
TNFa shedding by L428 and L540 cell lines. In the same spheroids size (C) were stable over time. Second, the two
table, the half maximal inhibitory concentration (IC ) (nM)
50 ADAM10 inhibitors did neither affect LN-MSC16412 sphe-
on ADAM10 or ADAM17 for each compound is also roid dimension (D) nor ATP (E), nor LDH release (not
shown to better compare the distinct effects of the shown). Altogether, these data support the hypothesis that
inhibitors related to their specificity. In particular, FC410, blocking of ADAM10 with specific inhibitors can interfere
FC143 and FC130, which are more specific for ADAM17, with HL cell growth in a 3D microenvironment composed
display IC of 0.1-2.5 mM on TNFa shedding, and IC of 10-
50 50 of stromal and lymphoma cells.
50 mM on intracellular ATP, while LT4 and MN8 IC 5-10 50
mM both on TNFa shedding and on intracellular ATP. The ADAM10 inhibitors decrease CD30 and TNFa shedding
IC of GIX (that shows an in vitro IC on the purified ADAM
50 50 in 3D cultures of Hodgkin lymphoma-mesenchymal
similar to LT4 but, at variance with LT4, has also nM activ- stromal cells on extracellular matrix scaffolds
ity on some MMP)17 is 5-10 mM on TNFa shedding, super- In order to resemble the architecture of HL more closely,
imposable to that of MN8, but higher (15-20 mM) on intra- decellularized ECM derived from patient LN were repopu-
cellular ATP modulation. lated with LN-MSC16412 (2x105) for 2 days, followed by
As a first 3D culture model to test ADAM10 inhibitors in the addition of 4x105 L428 for further 3 days. Figure 3
HL microenvironment, mixed spheroids of LN-MSC16412 shows an example of a repopulated ECM scaffold, with
(2x105) and L540 cells (4x105) were prepared as previously L428 cell identified in IHC with anti-CD30 mAb (A) and
described.31 Figure 1A shows confocal microscopy images LN-MSC16412 stained with anti-TGII antiserum (B). SN
of an exemplar mixed spheroid in bright field (left) or after were harvested 96 h after addition of 10 mM LT4 or MN8 or
staining with anti-CD30 mAb to identify HL cells (right). GIX for TNFa and soluble CD30 measurement. It is of note
Figure 1B shows the confocal analysis of a mixed spheroid that the inhibitors reduced the shedding of TNFa (Figure

R. Pece et al.
3C) and CD30 (Figure 3D). Superimposable results were These sponges, analyzed by SEM display a 3D structure
obtained with decellularized LN-derived ECM cultured (Figure 4D to F) similar to that of LN-derived ECM (Figure
with MSC16412 and L540 (not shown), confirming that test- 4A to C), with a network of round shaped niches, of
ing ADAM10 inhibitors on 3D cultures of stromal and HL approximately 10-50 mm of diameter, defined by collagen
cells on matrix scaffolds from human ECM is feasible. fibre bundles (arrows). AviteneTM sponges were cut in equal-
However, ECM obtainable from every LN specimen can sized scaffolds, and LN-MSC16412 were co-cultured with
barely allow a single/double experiment in triplicate (i.e., L540 or L428 HL cells as above. These scaffolds were effi-
three 3D replicates with MSC co-cultered with one HL cell ciently repopulated by LN-MSC16412 (TGII+, Figure 5A)
line only). Moreover, this 3D system is difficult to standard- and L428 cells (CD30+, Figure 5B) or L540 (not shown) cells
ize in terms of scaffold size, shape and structure. Thus, we after 96 h. Scaffold repopulation was also documented by
introduced commercial sponges made of microfibrillar col- SEM, where both cell types could be distiguished by mor-
lagen (AviteneTM Sponges), used as hemostats in surgery. phology (Figure 5C), suggesting that the model was feasible
A B
D E
Figure 1. Effects of ADAM10 inhibitors on lymph node-mesenchymal stromal/Hodgkin lymphoma cell spheroids. (A) Confocal microscopy of mixed spheroids, made
of LN-MSC16412 (2x105) and L540 cells (4x105), in bright field (left) or after staining with anti-CD30 monoclonal antibody (mAb), to identify Hodgkin lymphoma (HL)
cells (arrows), followed by FITC-GAM (right). (B) Confocal analysis of a mixed spheroid incubated with 1 µM Syto16 (blue) to stain nuclei and 10 µM CAM36 (red):
single pseudocolor or merged images and bright field as indicated (FV500 confocal LSM, Olympus, PlanApo 40x NA1.00 oil objective). Images were taken in
sequence mode to avoid cross-contribution of each fluorochrome, analyzed with the FluoView4.3b software (Olympus) and shown in pseudocolor or bright field. Scale
bar: 10 mm. (C to E) 10 mM (C and D) or dilutions (E) of LT4 or MN8 were added to the mixed spheroids and the cultures were kept at 37°C, for further 48 hours (h)
(C, left), 72 h (C, central) or 96 h (C, right, D and E). (C and D) Mixed spheroid dimension (C: area, D: volume) analyzed in each culture well as previously described.31
In (D), also the volume of spheroids made of LN-MSC16412 alone is indicated. Mean ± standard error of the mean (SEM) of triplicates analyzed for each culture con-
dition with a minimum of 50 single spheroids for each sample in three independent experiment. Nil: no drug added. *P<0.01 and **P<0.001 vs. nil. (E) Mixed sphe-
roids exposed to serial dilutions (10-0 mM) of LT4, MN8, GIX or the solvent dimethyl sulpfoxide (DMSO) (1:1,000) for 96 h at 37°C. Left graph: intracellular ATP content
(luminescence a.u./104 L540 cells); right graph: lactate dehydrogenase (LDH) detection (O.D.490/104 L540 cells) in the supernatant.*P<0.01 and **P<0.001 vs.
dimethyl sulfoxide (DMSO).

and reproducible. Also in this 3D system, 10 mM LT4 and dent at 72 h (left), while MN8 inhibition was maintained
MN8 could significantly reduce the shedding of CD30 also at 96 h (right). The L540 cell line was less sensitive to
(Figure 5D) and TNFa (Figure 5E) by both L428 (left) and the inhibition exerted by the two ADAM10 blockers;
L540 (right) HL cells. The anti-shedding effect was evident nevertheless, the reduction of proliferating HL cells by
at 72 h for L428 and at 96 h for L540 cells. MN8 was significant at 96 h (Figure 6C).
These results indicate that ADAM10 inhibitors are func-
tional in 3D cultures recapitulating some features of the HL BtxVed, LT4 and MN8 reduce ATP content, glucose
lymphoma microenvironment, such as ECM and MSC. consumption and induce caspase-3 in Hodgkin
Furthermore, microfibrillar collagen sponges can substitute lymphoma-mesenchymal stromal cell co-cultures
LN-derived ECM to allow larger sampling. Given the reduction of CD30 shedding due to ADAM10
inhibitors, and considering the reported antagonistic effect
LT4 and MN8 lower the number of Ki67+ Hodkin of soluble CD30 on the therapeutic efficacy of the anti-
lymphoma cells in 3D repopulated scaffolds body-drug conjugate (ADC) BtxVed,35 we asked whether in
In order to analyze HL cell proliferation, AviteneTM the presence of LT4 or MN8, BtxVed could enhance its anti-
sponges repopulated with LN-MSC16412 and L428 or L540 lymphoma effect. First, we set up this experiment in con-
cells and exposed to 10 mM LT4 or MN8, for 72 h and 96 h ventional 2D co-cultures of HL and LN-MSC cells. To this
were paraffin embedded and 5 µm sections were prepared aim, L428 or L540 cells were added to LN-MSC16412 and
for IF with the anti-Ki67 polyclonal antibody that identifies co-cultures were performed in the presence of BtxVed (10
cycling cells.34 or 1 mg/mL), alone or in combination with 10 mM LT4 or
Figure 6A shows representative images of repopulated MN8. After 96 h, HL cells were harvested (free of LN-MSC
scaffolds (LN-MSC16412 and L428 cells), cultured for 96 that remained adherent) and counted at the MACS Quant
h in medium alone: the anti-CD30 mAb identifies HL Analyzer 10, while parallel samples were analyzed for ATP
cells (red membrane staining) and the anti-Ki67 polyclon- content. The two ADAM10 inhibitors, reduced L428 and
al antibody (green nuclear staining) identifies cycling L540 cell growth by about 50%; this effect was similar to
cells. Images from untreated and treated samples were that exerted by 1 mg/mL BtxVed; moreover, LT4 and MN8
automatically analyzed with the Aperio Cellular IF could enhance the inhibitory effect of BtxVed (10 µg/mL) by
Algorithm (Leica Biosystems) and the number of 25% (Figure 7A). Accordingly, the combinatory effect of
CD30+/Ki67+ cells was calculated as described in the LT4 or MN8 and BtxVed was detectable in decreasing the
Online Supplementary Figure S4. The number of content of ATP in HL cells, also with BtxVed used at 1
CD30+Ki67+L428 cells was significantly lower in the pres- mg/mL (Figure 7B). Indeed, the inhibitory effect of LT4 or
ence of LT4 or MN8 (Figure 6B); the effect of LT4 was evi- MN8 in combination with BtxVed was 2- or 3-fold that of
A B
i ii
C D
Figure 2. Effects of ADAM10 inhibitors on ATP content, lactate dehydrogenase or TNFa release and size of autologous lymph node-mesenchymal stromal/Hodgkin
lymphoma cell spheroids. Autologous mixed spheroids of LN-MSC773 (2x105) and RS773 cells (4x105) were prepared as previously described.31 (A to C) 10 mM (panel
Ai) or dilutions (panel Aii), B and C) of LT4 or MN8 or GIX were added to the mixed spheroids for 48 hours (h) (Ai) and C), 72 h (Ai)) or 96 h (Ai), Aii) and B). At the
indicated time points ATP (Ai) and Aii)), lactate dehydrogenase (LDH) (B) or TNFa (C) were measured by specific assays. Results are expressed as luminescence arbi-
trary units (a.u./104cells, A) or O.D (a.u./10mcells, B) or pg/mL/104cells (C) and are referred to one representative experiment out of three in triplicate. (D) Spheroid
.490
area was measured as previously described31 in each culture well at 48 h (left), 72 h (central), 96 h (right). At least triplicates were analyzed for each culture condition
and a minimum of 50 single spheroids for each of three independent experiment. Nil: no drug added (medium with dimethyl sulfoxide 1:1,000). Mean ± standard
deviation from three experiments is indicated. *P<0.01 and **P<0.001 vs. nil.

R. Pece et al.
A B Figure 3. Decellularized lymph node

matrices repopulated with lymph node-
mesenchymal stromal and Hodgkin
lymphoma cells as 3D culture model to
test ADAM10 inhibitors. (A and B)
Immunohistochemistry (IHC) of 3D cul-
tures performed on decellularized
Hodgkin lymphoma (HL) lymph node
(LN) matrices (extracellular matrix
[ECM], one reprentative experiment, LN
I-19032-16) with 2x105 LN-MSC16412
for 3 days, followed by the addition of
4x105 L428 cells to each ECM/LN-
MSC16412 scaffold for a further 2
days. (A) 4 mm sections stained with the
anti-CD30 monoclonal antibody (mAb),
to identify HL (L428) cells, followed by
Biot-GAM, HRP-Av and developed with
DAB; (B) sections stained with the anti-
TGII rabbit polyclonal antiserum, to
C D identify LN-MSC16412, followed by Biot-
GAR, HRP-Av and developed with DAB.
Inset in (A) negative control with Biot-
GAM alone. Slides were counterstained
with hematoxylin and analyzed under a
Leica DM MB2 microscope (40x objec-
tive). (C to D) TNFa C) or soluble CD30
(D) content (pg/mL/106 cells) in the
supernatant (SN) recovered after 48
hour (h) from the addition of 10 mM LT4
or MN8 or GIX ADAM10 inhibitors to the
3D cultures, measured by specific
enzyme-linked immunosorbant assay.
Nil: solvent (dimethylsulfoxide 1:1,000).
Results are the mean ± standard devia-
tion from three experiments performed
in duplicate with ECM from three HL
patients.*P<0.005 vs. nil; **P<0.001
vs. nil.
Figure 4. Structural analysis of lymph node matrices and collagen scaffolds for 3D culture models. Scanning electron microscopy (SEM) images of lymph node (LN)
matrix (extracellular matrix [ECM]) specimens obtained, as previously described,32 from one Hodgkin lymphoma (HL) patient (I-19032-16, A and B) or a non-neoplas-
tic LN (I-19273-16, C). SEM of AviteneTMUltrafoam Collagen sponges, at the indicated magnifications, embedded and processed as LN-ECM specimens (D and F).
Arrows indicate matrix branches surrounding empty spaces. Magnifications and scale bars are reported in each panel.

A
D
Figure 5. AviteneTM microfibrillar collagen scaffolds reconstituted with lymph node-mesenchymal stromal and Hodgkin lymphoma cells as 3D culture model to test
ADAM10 inhibitors. AviteneTM scaffolds were cultured with 2x105 LN-MSC16412 for 3 days, followed by 4x105 L428 (A to C and D to E left histograms) or L540 cells
(D to E right histograms) for further 2 days, before addition of 10 mM LT4 or MN8 for 72 hours (h) or 96 h. (A) 4 mm sections of repopulated AviteneTM scaffolds stained
with anti-TGII polyclonal antiserum followed by Biot-GAR, HRP-Av and developed with DAB; (B) sections stained with anti-CD30 monoclonal antibody (mAb) followed
by Biot-GAM, HRP-Av and developed with DAB. Inset in the left pictures: images of the whole repopulated scaffold. Inset in the right pictures: negative control (Nil)
with Biot-GAR(A) or Biot-GAM (B) alone. Slides were counterstained with hematoxylin and analyzed under a Leica DM MB2 microscope (left: 20x enlargement, right:
40x enlargement). (C) Scanning electron microscopy (SEM) images of AviteneTM scaffolds, repopulated with LN-MSC16412 and L428 cells (arrows). Magnifications
and scale bar are reported in each panel. Inset in the left picture: the whole scaffold with a white square indicating the area enlarged. (D and E) Soluble CD30 (D)
or TNFa (E) content (pg/mL/106 cells), measured by specific enzyme-linked immunosorbant assay, in the supernatant (SN) recovered after 72 h or 96 h from addition
of ADAM10 inhibitors to the scaffolds repopulated with LN-MSC16412 and L428 (left histograms) orL540 (right histograms). Results are the mean the mean ± stan-
dard deviation of quadruplicates from three independent experiments.**P<0.005 vs. nil; ***P<0.001 vs. nil.
BtxVed alone in L428 cells (Figure 7B, left); this effect was dilutions is also shown (Online Supplementary Figure S5F and
still detectable in L540 cells despite its higher sensitivity to G; Figure 8E and F).
BtxVed (Figure 7B, right). In order to test the hypothesis of a synergic or additive
In order to closely reproduce the cellularity of a LN node effect of ADAM10 inhibitors and BtxVed on HL cells, we
microenvironment, we tried to improve scaffold repopula- chose the IHC analysis of caspase-3 activation, rather than
tion by simultaneously seeding a mixture of LN-MSC and Ki67 expression, since both the ADC and the inhibitors act
HL cells onto the scaffold. This system allowed HL cells to mainly by inducing programmed cell death.19,35,36 Moreover,
fill the niches between the collagen branches of the scaffold as Ki67 is detectable in the nucleus not only in the mitotic
(Online Supplementary Figure S5B), compared to the sequen- phase but also in the interphase,34 it is possible to miscount
tial seeding where empty spaces are still evident (Online Ki67+ cells as proliferating, even though they are in other
Supplementary Figure S5A). Cells maintained their metabolic cell cycle phases.
activity as documented by glucose consumption (Online Figure 8 shows scanned images of repopulated scaffolds
Supplementary Figure S5C). In this 3D setting, we decided to (A: untreated scaffold, B: scaffold exposed to 20 mg/mL
use BtxVed at 20 mg/mL to maximize its effect on HL cells, BtxVed) stained with the anti-caspase-3 antibody (subpan-
compared with one tenth of the concentration (2 mg/mL). In els b) and automatically analyzed with the Genie software,
order to avoid sudden cell starvation due to total glucose combined with the nuclear count V9 macro of Image-Scope
consumption by 48-72 h (Online Supplementary Figure S5C), software (subpanels c: HL cells in blue and caspase-3+ cells
half of the medium was replaced at 48 h, after recovering in red). As reported in panel C, the percentage of caspase-
the SN for glucose measurement. Glucose consumption by 3+L428 HL cells increased significantly upon treatment with
either L428 (Online Supplementary Figure S5D) or L540 20 mg/mL BtxVed or 10 mM LT4 or MN8, with a slight addi-
(Online Supplementary Figure S5E) cells rapidly decreased tional effect using BtxVed at 20 mg/mL and 10 mM LT4
over time in the presence of 20 mg/mL BtxVed, while the together. In turn, the percentage of L540 HL cells expressing
effect was less evident with the lower dose of the ADC (2 caspase-3 increased especially upon treatment with BtxVed
µg/mL). Glucose consumption in the presence of DMSO at 2 mg/mL; of note, both LT4 and MN8 could significantly

R. Pece et al.
A B
Figure 6. Effects of ADAM10 inhibitors on Hodgkin lymphoma cell growth in 3D scaffolds repopulated with lymph node-mesenchymal stromal and Hodgkin lym-
phoma cells. (A) A representative AviteneTM scaffold repopulated by LN-MSC16412 (2x105) and L428 cells (4x105). Sections (5 mm) from paraffin-embedded repop-
ulated scaffolds were stained with DAPI for nuclei (blue), anti-CD30 monoclonal antibody (mAb) followed by anti-mouse Alexa Fluor594 (red) for Hodgkin lymphoma
(HL) cells and anti-Ki67 polyclonal antibody followed by anti-rabbit Alexa Fluor488 (green) to identify cycling cells. Images were taken with the Aperio VERSA Digital
Pathology Scanner (Leica Biosystems) with a 10x objective. Subpanel b: enlargement of the blue rectangle in subpanel a. Scale bars as indicated. (B and C) AviteneTM
scaffolds repopulated with 2x105 MSC16412 and 4x105 L428 (B) or L540 cells (C) and exposed to 10 mM LT4 or MN8 ADAM10 inhibitors for 72 hours (h) or 96 h
as indicated. Nil: solvent (dimethyl sulfoxide 1:1,000) were subjected to immunofluorescence (IF) as in (A). At least three sections/scaffold, cut at 15 µm distance,
were acquired. Image data were analyzed with the Aperio Cellular IF Algorithm (Leica Biosystems) and the percentage of CD30+/Ki67+ cells was calculated as
described in the Online Supplementary Figure S1. Results are the mean ± standard error of the mean (SEM) from three independent experiments performed in dupli-
cate (two scaffolds). *P<0.05 vs. nil; **P<0.005 vs. nil.
enhance the number of caspase-3+ cells when used in com- Discussion

bination with 2 mg/mL BtxVed (from about 7-10% with
BtxVed alone to 12-16%, Figure 8D). In this work we demonstrate the efficiency of ADAM10
In addition, glucose consumption was measured every 24 inhibitors in HL growth control in two different 3D culture
h in the supernatant of the 3D cultures, referred to the glu- systems: mixed spheroids made of LN-MSC and HL cells
cose content in fresh culture medium. In these experiments, and scaffolds repopulated with both LN-MSC and HL cells.
BtxVed was used at low doses (2 mg/mL) to emphasize any In these 3D models we found that: i) in HL cells LT4 and
potential additive effect of ADAM10 inhibitors at 10 mM. MN8 reduce ATP content, related to proliferation, and glu-
Glucose depletion in the medium of LN-MSC23274+L428 cose consumption, while increasing LDH release as a mark-
repopulated scaffolds increased during time; of note, con- er of cell damage; ii) the two inhibitors lead to mixed sphe-
sumption was significantly reduced at 72 h by BtxVed and roids size reduction and inhibition of soluble CD30 and
at 72 h to 96 h by LT4 and MN8, with an additive effect of TNFa shedding; iii) both effects can be reproduced in 3D
BtxVed and LT4 used together already evident at 24 h to 48 culture systems based on patients LN matrix or AviteneTM
h (Figure 8E). In the case of 3D cultures of LN- collagen scaffolds repopulated with LN-MSC and HL cells;
MSC23274+L540 cells (Figure 8F), glucose depletion iv) LT4/MN8 enhance the anti-lymphoma effect of BtxVed,
reached a steady state at 48 h, while the pharmacological evaluated as reduction of proliferation and induction of
effects of ADAM10 inhibitors, were evident at 96 h to 120 apoptosis, both in conventional co-cultures and in repopu-
h. At these time points, LT4 and MN8 were effective in lated scaffolds.
reducing glucose consumption, at variance with BtxVed; of The mixed spheroid 3D system, like other spheroids
note, additive effects were observed using combinations of approved as animal-free preclinical models,25-27 displays
LT4 or MN8 and BtxVed (Figure 8E and F). Glucose con- some advantages such as feasibility, low cost, reproducibil-
sumption did not vary in the presence of DMSO at the ity analysis of high sample number at a time. On the other
same dilution used as solvent, supporting a direct anti-lym- hand, the presence of LN-MSC as a core guarantees a
phoma action of the drugs (Figure 8E and F). These data rounded shape of the spheroid, allowing their measure-
indicate a direct effect of ADAM10 inhibitors on HL cell ment. Indeed, HL cells alone grow in culture as cell suspen-
growth in 3D microenvironment and, more importantly, an sion forming little clumps without a real spherical spatial
additive effect to the anti-lymphoma action of BtxVed organization. It is of note that in this system, LT4 and MN8
ADC. could enter the spheroid and proved to be highly effective

A Figure 7. ADAM10 inhibitors reduce

Hodgkin lymphoma cell growth and
ATP content and enhance brentux-
imab-vedotin effect. (A) L428 (left) or
L540 (right) cells (104) were added to
LN-MSC16412 (103) previously seed-
ed into 96 microwell plates in the
presence of brentuximab-vedotin
(BtxVed) (10 mg/mL), alone or in com-
bination with 10 mM LT4 or MN8. After
96 hours (h), 200 ml cell suspension
were collected and cells counted at
the MACS Quant Analyzer 10 (Miltenyi
Biotech GmbH). Results are shown as
percentage of cell growth inhibition
calculated as the number of cells/well
in cultures with the indicated drugs
referred to cultures in the solvent
(dimethyl sufoxide [DMSO] 1:1,000),
as described in the Online
Supplementary Appendix. Mean ±
B standard deviation (SD) of quadrupli-
cates from three independent experi-
ments.*P<0.01 and **P<0.001 vs.
BtxVed or LT4 or MN8 alone. (B)
Cultures were performed as in (A),
with BtxVed used at 10 mg/mL and 1
mg/mL. After 96 h, ATP content meas-
ured by specific assay. Results are
expressed as percent inhibition of
luminescence in cultures exposed to
the indicated drugs referred to cul-
tures in the solvent (DMSO 1:1000),
as described in the Online
Supplementary Appendix. Mean ± SD
of quadruplicates from three inde-
pendent experiments.*P<0.001 vs.
BtxVed or LT4 or MN8 alone
**P<0.0001 vs. BtxVed alone.
not only in their anti-sheddase activity, but also in the inter- The second 3D system is based on LN extracellular
ference with HL cell viability, in terms of ATP content, and matrix and collagen scaffolds repopulated with LN-derived
cell damage documented by LDH release. These effects MSC and HL cells. As a first model, matrices obtained from
were accompanied by a reduction of spheroid size, meas- patient LN specimens were used to test ADAM10
ured with a tailored image analysis procedure reported in a inhibitors. This model is fairly physiological, as the decellu-
previous paper,30 as a proof of the limited HL cell growth. larization process allows the removal of cells while main-
The inhibition of soluble CD30 and TNFa shedding was taining the biochemical composition and tridimensional
considerable as well; indeed, on one side the release of organization of the tissue of origin32,39 and allows MSC and
CD30 would impair the effect of anti-CD30 therapeutic HL cells to repopulate the structure creating a bona fide, lym-
mAb,15,33 on the other side released TNFa would function as phoma microenvironment. In these LN-derived and repop-
a growth factor for HL cells.37,38 It is of note that metabolic ulated ECM, LT4 and MN8 displayed the same anti-shed-
impairment, shedding inhibition and spheroid size reduc- dase activity observed in the spheroid system, i.e., blocking
tion were obtained with all the three HL cell lines tested, of CD30 and TNFa release. However, the reduced number
i.e., L428 (derived from pleural effusion of a HL patient), of replicates and the difficult standardization of the scaffold
L540 (from bone marrow of a different patient) and RS773 size, due to the paucity of bioptic samples and to various
(from a LN of a distinct patient), thus proving that the sys- shapes of LN-derived ECM, represent limitations of this 3D
tem can be applied to multiple subjects and the pharmaco- culture system. Therefore, the microfibrillar collagen
logic effect of ADAM10 inhibitors can be elicited in HL cells sponge AviteneTM, used for hemostatic purposes in surgery,
regardless the tumor site from which they derive. With was chosen for further studies. This system allows the
regard to the anti-lymphoma action of ADAM10 inhibitors, preparation of a high number of replicates with homoge-
it has to be noted that exposure to LT4 and MN8 leads to neous and reproducible sampling. Interestingly, ultrastruc-
ADAM10 compartmentalization in endolysosomes possi- ture analyses evidenced that the architecture of these
bly interfering with ADAM10 stability due to retention in sponges was very similar to that of decellularized matrices
the degradative pathway while decreasing membrane local- obtained from LN biopsies, thus representing a good alter-
ization.35 Following their intracellular pathways the native to test anti-lymphoma drugs, including ADAM10
inhibitors may encounter different substrates involved in inhibitors. AviteneTM scaffolds could be actively repopulat-
tumor cell growth, not only TNFa in HL, but also mediators ed by LN-MSC and HL cells, that migrate through the col-
linked to cell proliferation, such as Notch1 or receptor asso- lagen branches into the empty spaces, as shown by SEM.
ciated kinases, limiting their function.1,3,5 We are aware of the complex LN cellular composition in

R. Pece et al.
HL, including multiple types of immunocompetent and reported that fibroblasts from HL lymph node suspen-
inflammatory cells that influence anti-tumor and drug sions protect lymphoma cells from BtxVed effects,41 sup-
response,38 that cannot be fully recapitulated by LN-MSC. porting that LN-MSC might represent leading actors in HL
In particular, this has been documented by a multi-center response to this ADC.
phase II trial with immune checkpoint inhibitors in classi- In AviteneTM scaffolds repopulated by LN-MSC and HL
cal HL that failed both autologous stem-cell transplanta- cells, the anti-sheddase effect of LT4 and MN8 on CD30
tion and BtxVed therapy.40 Nevertheless, it has been and TNFa was documented using two different HL cell
A B
C D
E F
Figure 8. ADAM10 inhibitors and brentuximab-vedotin induce caspase-3 activation in Hodgkin lymphoma cells and reduce glucose consumption in repopulated
scaffolds. AviteneTM repopulated scaffolds (LN-MSC23274+L428 cells), were either untreated (A) and nil (C to F) or exposed to brentuximab-vedotin (BtxVed) (20
mg/mL or 2 mg/mL as indicated) (B to D) or 10 mM LT4 or MN8 (C to F) or BtxVed plus one of the inhibitors as indicated (C to F). (A and B) After 96 hours (h), scaffolds
were fixed and 5 mm serial sections were stained in immunohistochemistry (IHC) with the rabbit monoclonal anti-caspase-3 antibody. Images were acquired with the
Aperio AT2 Digital Pathology Scanner and data analyzed with the Genie software to identify and count caspase-3+cells. Subpanel a: sections of the whole scaffold;
subpanel b: enlargements of squares in subpanel a; subpanel c: Hodgkin lymphoma (HL) cells, identified by morphology (blue), and caspase-3+ cells recognized by
the Genie software (red) (C and D) percentage of caspase-3+ cells (C: L428, D: L540) counted in serial sections (3 every 15 mm/each scaffold) by the Genie software,
are reported as the mean ± standard deviation (SD) of three serial sections analyzed from three different experiments in duplicate (2 scaffolds/experiment). (C)
**P<0.0005 and *P<0.05 vs. nil; (D) **P<0.05 vs. BtxVed and *P<0.05 vs. nil. (E and F) Glucose evaluation in the supernatant (SN) recovered from LN-
MSC23274+L428 (E) or LN-MSC23274+L540 (F) repopulated scaffolds exposed for the indicated time periods to either 2 mg/mL BtxVed, 10 mM LT4 or MN8, or
LT4+BtxVed or MN8+BtxVed. Green symbols: medium containing dimethyl sulfoxide (DMSO) 1:1,000. Glucose was measured with the specific kit (Megazyme) in cul-
ture SN recovered every 24 h and data are expressed as percentage glucose consumpion referred to the content in fresh culture medium; mean ± SD from three
experiments performed in duplicate (2 scaffolds/experiment). (E) *P<0.02 and **P<0.001 vs. nil; ***P<0.0001 vs. nil and vs. BtxVed. (F) *P<0.01, **P<0.001
and ***P<0.0001 vs. nil and vs. BtxVed.

lines, with a slightly different time course. Actually, the Moreover, as MMAE is eliminated through liver and kid-
response to ADAM10 inhibitors show a time-dependent ney, patients with hepatic diseases or renal failure, who
biphasic kinetics, at least in the time frame we selected develop severe adverse reaction to full-dose BtxVed,
for our observations, while the morphological and biolog- require ADC administration at reduced doses. Also,
ical global response led us to conclude they were effective BtxVed can induce peripheral neuropathy, severe anemia
in reshaping our 3D cultures with an overall, persistent and neutropenia in a fraction of patients regardless of renal
antitumor effect. A possible explanation might be based and hepatic impairment.36 From this viewpoint, ADAM10
on the different fusion gene expression involving the inhibitors could help to contain the ADC dosage in suit-
hyperactivation of different kinases (ELMO1-SCLO3A1 able ranges to this purpose. Given the overall efficiency at
in L428) related to NFkB regulation. In fact biphasic non toxic doses (5-10 mM) of LT4 and MN8 in eliciting an
changes in NFkB as well as TNFa signaling, both inducing additive effect on BtxVed anti-lymphoma action in 3D cul-
either damage and repair like other inflammatory regula- tures, these compounds might be proposed for a com-
tors, has been reported to possibly influence the observed bined therapy. Potential toxicity to normal cells should be
effects.42 Another possibility is a different location of considered, as ADAM10 expression is not confined to
ADAM10 in intracellular compartments, that may influ- cancer cells, although it is upregulated in tumors com-
ence the speed of action of the inhibitors in different cell pared to healthy tissues.35 Another limitation to overcome
lines and cell types. We reported that ADAM10 intracel- is represented by the need of DMSO for the first solubi-
lular distribution changes after exposure to LT4 or MN8 lization step. However, ADC-based anti-HL therapy is
functionalized with a linker.35 LT4 and more effectively usually based on short repeated cycles of drug administra-
MN8 induced a substantial reorganization of the intracel- tion in order to reduce any potential side effect and this
lular vesicular network, with secretion of extracellular should be useful to limit ADAM10 inhibitors undesired
vesicles (EV) carrying the inhibitors.35 It is then possible effects as well. In any case, at present the potential thera-
that LT4 and MN8 can act on CD30 early after uptake, peutic use of these compounds is only a suggestion since
while at a later time point the effect is temporary lost due their pharmacodynamics is still to be defined and deserves
to extracellular release in EV. Since EV-bound inhibitors further studies.
are then taken up by tumor and bystander cells, we can- In conclusion, our data point toward three main pieces of
not exclude the possibility that extended biological information: first, a direct anti-lymphoma effect exerted by
effects occur later than 96 h. ADAM10 inhibitors, more efficient than the known com-
Both inhibitors could reduce the number of cycling HL mercial inhibitor GIX. Second, the enhancement of BtxVed
cells, evaluated by automatic cell count and computerized anti-lymphoma effect, due to a combinatory action of the
imaging as the number of CD30+ cells co-expressing the ADC and the inhibitors, detectable at low and ineffective
Ki67 marker, in repopulated scaffolds. Also, HL cell doses of the ADC. Last, the evidence of these effects in 3D
metabolism evaluated by glucose consumption, was systems, very similar to those approved as preclinical mod-
impaired by ADAM10 inhibitors. Epigenetic effects of els. Repopulated scaffolds may also represent a starting
DMSO, used for the first solubilization step of LT4 and point to reconstitute the whole LN cellular composition,
MN8, on genes mainly controlling glucose metabolism including inflammatory or endothelial cells that can con-
have been reported in human cardiac fibroblasts and pri- tribute to HL pathogenesis, influencing drug response as
mary hepatocytes.43 Nevertheless, in our 2D and 3D cul- well. Nevertheless, our 3D model based on ECM, stromal
ture models glucose consumption did not vary in the pres- cells and lymphoma cells, recapitulates the main aspects of
ence of DMSO at the same dilution used as solvent, sup- the lymphoma microenvironment architecture, represent-
porting a direct anti-lymphoma action of the drugs. Of ing a reliable tool for anti-lymphoma drug testing.
note, LT4 and MN8 could enhance the anti-tumor action
of BtxVed, rescuing the effect of this ADC at low doses, Disclosures
both in 2D cultures and in the 3D scaffold system, and No conflicts of interest to disclose.
leading to an increase in the number of caspase-3+ apop-
totic HL cells. These effects are conceivably due to the Contributions
double action of the inhibitors as anti-sheddase on CD30, RP and ST performed scaffold repopulation, IHC and IF assays;
that is the target of BtxVed,15,16 and TNFa that represents a DC performed IHC and image analysis; SV performed experi-
lymphoma growth factor.37,44 ments with mixed spheroids; CC, DC, EN and AR designed and
On its own, BtxVed targets CD30 mainly expressed on produced ADAM10 inhibitors; MA performed decelluarization
lymphoma cells, although minimally present also on nor- matrix experiments; CD’A and DG performed SEM preparation
mal cells, predominantly activated B and T lymphocytes.38 of samples and analysis; JLR provided LN specimens; MG provid-
The internalization of the ADC in cell lysosomes results in ed LN specimens and clinical patient information; FT performed
proteolytic cleavage of the microtubule-disrupting agent ELISA experiments for TNF and soluble CD30 quantitation; AP
monomethyl auristatin E (MMAE), with consequent performed LN-MSC isolation and culture, repopulation of scaf-
apoptosis. Nevertheless, the first effect of microtubule dis- folds, confocal microscope analysis and planned some experiments;
ruption may be the impairment of cell proliferation before MRZ planned, designed and scheduled the experiments. All the
accelerating apoptosis45 and this might explain why cer- authors read and revised the manuscript.
tain HL cell lines, such as L540, display a lower caspase-3
activation in response to the ADC. As a possible undesired Funding
effect, MMAE released into the surrounding extracellular This study has been supported by the AIRC IG-17074 grant to
matrix might exert toxicity on adjacent normal cells. MRZ.

R. Pece et al.
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Plasma Cell Disorders ARTICLE
DIS3 mutations in multiple myeloma impact Ferrata Storti Foundation

the transcriptional signature and clinical
outcome
Katia Todoerti,1,2* Domenica Ronchetti,2* Vanessa Favasuli,2
Francesco Maura,3 Fortunato Morabito,4,5 Niccolò Bolli,1,2 Elisa Taiana1,2#
and Antonino Neri1,2#
1
Hematology, Fondazione Cà Granda IRCCS Policlinico, Milan, Italy; 2Department of
Oncology and Hemato-oncology, University of Milan, Milan, Italy; 3Myeloma Program,
Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA; Haematologica 2022
4
Biotechnology Research Unit, Aprigliano, A.O./ASP, Cosenza, Italy and 5Department of Volume 107(4):921-932
Hematology and Bone Marrow Transplant Unit, Augusta Victoria Hospital, Jerusalem,
Israel.
*KT and DR contributed equally as co-first authors.
#
ET and AN contributed equally as co-senior authors.
ABSTRACT
D
IS3 gene mutations occur in roughly 10% of patients with multi-
ple myeloma (MM); furthermore, DIS3 expression can be affect-
ed by monosomy 13 and del(13q), which occur in approximately
40% of MM cases. Despite several reports on the prevalence of DIS3
mutations, their contribution to the pathobiology of MM remains largely
unknown. We took advantage of the large public CoMMpass dataset to
investigate the spectrum of DIS3 mutations in MM and its impact on the
transcriptome and clinical outcome. We found that the clinical relevance
of DIS3 mutations strictly depended on the co-occurrence of del(13q). In
particular, bi-allelic DIS3 lesions significantly affected progression-free
survival, independently of other predictors of poor clinical outcome,
while mono-allelic events mostly affected overall survival. As expected,
DIS3 mutations affect the MM transcriptome involving cellular process-
es and signaling pathways associated with RNA metabolism, and the
deregulation of a large number of long non-coding RNA, among which
we identified five distinct transcripts as independent predictors of poorer Correspondence:
overall survival and nine of worse progression-free survival, with two
(AC015982.2 and AL445228.3) predicting both unfavorable outcomes. ANTONINO NERI
antonino.neri@unimi.it
These findings strongly prompt further studies investigating the rele-
vance of these long non-coding RNA in MM.
Accepted: April 22, 2021.
Introduction
Pre-published: May 6, 2021.
Multiple myeloma (MM) is a hematologic malignancy that is still incurable
despite the remarkable improvements in treatment and patients’ care.1 MM is char-
acterized by a profound genomic instability involving ploidy, structural rearrange- https://doi.org/10.3324/haematol.2021.278342
ments, and a wide array of mutations affecting both putative oncogenes and tumor
suppressor genes, such as KRAS, NRAS, TP53, BRAF, TRAF3, FAM46C, and DIS3.2-7
These abnormalities are predicted to influence the biological and clinical behavior of
the tumor and yet, despite a clear driver role, only a few carry prognostic value.8-10 Material published in Haematologica is covered by copyright.
Among mutated genes, DIS3 deserves great attention; this gene maps to 13q22.1 All rights are reserved to the Ferrata Storti Foundation. Use of
and encodes for a highly conserved ribonuclease indispensable for survival in ver- conditions:
tebrates.11 DIS3 is a multidomain protein with two different catalytic activities: a https://creativecommons.org/licenses/by-nc/4.0/legalcode.
3’–5’ exonucleolytic activity via the RNase II/R (RNB) domain and an Copies of published material are allowed for personal or inter-
endonucleolytic activity via the PilT N-terminal (PIN) domain at the N-terminal use. Sharing published material for non-commercial pur-
nus.11,12 DIS3 provides catalytic activity to the exosome, a multi-subunit complex https://creativecommons.org/licenses/by-nc/4.0/legalcode,
involved in RNA degradation and metabolism, including mRNA quality control, sect. 3. Reproducing and sharing published material for com-
gene expression regulation, and small RNA processing.12-15 mercial purposes is not allowed without permission in writing
DIS3 mutations and altered expression have been reported in roughly 10% of from the publisher.
MM patients.6,16,17 Moreover, DIS3 expression can be affected by monosomy 13
and del(13q), which occur in approximately 40% of MM patients.18,19 DIS3 muta-

K. Todoerti et al.
tions are mainly located within the major ribonuclease lar and clinical data of the CoMMpass cohort selected for the pres-
domains of the protein, probably impairing DIS3 catalytic ent study are described in the Online Supplementary Methods.
activity, as has been demonstrated to occur for some
mutated residues.11 Recently, the analysis of a large cohort Statistical and survival analyses
of MM patients enrolled in the Multiple Myeloma Fisher exact test was applied to verify the association between
Research Foundation (MMRF) CoMMpass study pointed genomic alterations in stratified MM cases. The two-tailed P-value
out that DIS3 mutations are almost exclusively missense, was corrected using the Benjamini-Hochberg method, and adjust-
with no nonsense or frameshift mutations, copy neutral ed P-values <0.05 were considered statistically significant. Survival
loss of heterozygosity, or bi-allelic deletions. In addition, a analyses were performed using survival and survminer packages
third of the mutations occur in three codons (D479, D488, in R Bioconductor (version 3.5.1) as reported in the Online
and R780) within the RNB domain, almost never associat- Supplementary Methods.
ed with del(13q); in contrast, the remaining two-thirds of
DIS3 mutations are distributed across the various exons, Differential expression analysis of the CoMMpass
almost always associated with del(13q).20 cohort
Despite the detailed overview of DIS3 mutations, their A global dataset from 655 MM cases was stratified on the basis
functional consequences on MM pathogenesis remain of DIS3 mutation RNA frequency and 56 DIS3-mutated cases
largely unknown, to the point that it is not clear whether with at least 20% RNA mutational load were considered in the
DIS3 acts as an oncogene or a tumor suppressor gene.21,22 A differential expression analysis. Further steps of analysis are
crucial role for the DIS3 ribonuclease has been demonstrat- described in the Online Supplementary Methods. Principal compo-
ed in human MM cell lines, in which it promotes the mat- nent analysis was applied on differentially expressed (DE) anno-
uration of the let-7 microRNA tumor suppressor family; tated transcripts by means of the prcomp function in R. Volcano
indeed, through the reduction of mature let-7, DIS3 inacti- plots were used in R to represent significantly up- or down-regu-
vation enhances the translation of let-7 targets such as MYC lated transcripts. The heatmap of the expression levels of the top
and RAS, leading to enhanced tumorigenesis.23 However, 100 DE annotated transcripts, according to the limma B statistics
the extent to which this pathway is affected in MM value, was created using dChip software.25
patients harboring DIS3 mutations and how it may con-
tribute to myelomagenesis need to be investigated further. Functional enrichment analysis on differentially
The clinical relevance of DIS3 mutations in MM was expressed protein-coding genes
initially investigated by Weissbach et al;22 despite the cau- Gene set enrichment analysis (GSEA)26 was performed on the
tion due to the small size of the cohort investigated, their pre-ranked DE protein-coding gene lists based on the fold change
data suggested that response to therapy was affected by values by computing 1,000 permutations and using default analy-
DIS3 mutations depending on the presence of such muta- sis conditions. Further details about GSEA are provided in the
tions in minor rather than major subclones. Recently, the Online Supplementary Methods.
analysis of a larger cohort of MM cases at diagnosis
revealed that DIS3 mutations were significantly associat- Long non-coding RNA expression validation
ed with shorter event-free survival, showing an even We investigated long non-coding (lnc)RNA expression in a pro-
worse outcome when in association with 13q deletion. prietary dataset that includes 43 MM patients (Online
However, overall survival (OS) was not affected.16 Supplementary Table S1), upon written informed consent (Ethical
Notably, DIS3 mutations retained their significance for Committee approval n. 575, 03/29/2018, Fondazione IRCCS Ca’
event-free survival in a multivariate analysis including Granda Ospedale Maggiore Policlinico). Further details about this
t(4;14) and the high-risk “double hit” group, i.e. patients analysis are reported in the Online Supplementary Methods.
with bi-allelic TP53 inactivation and amplification of
1q21.10 However, it is becoming a well-established notion
that, given the complexity of the genetic background in Results
MM, it is mandatory to assess DIS3 mutations in associa-
tion with other oncogenic events, and their transcriptomic Assessment of DIS3 mutations in myeloma patients
consequences, to establish their impact on MM We focused on a CoMMpass cohort of 930 bone marrow
prognosis.10,22,24 plasma cell samples from newly diagnosed MM patients
Based on these considerations, our study mined genom- for whom non-synonymous somatic mutation data,
ic and transcriptomic data from cases included in the gained by whole exome sequencing, were available (Online
MMRF CoMMpass dataset to dissect the impact of DIS3 Supplementary Table S2), identifying 103 DIS3 mutations in
mutations in the context of the MM genomic landscape in 94 of the 930 cases (Figure 1A, Online Supplementary Table
order to improve the definition of relevant clinical subsets. S3). The variant allelic frequency ranged between 5.3%
In addition, we investigated the transcriptomic profile and 100% (mean: 48%; median: 43%). The majority of
related to DIS3 mutations to elucidate its role in myeloma mutations were missense variants (100/103) in the coding
cells and identify relevant pathways in MM pathobiology. region (95/103) or within the region of the splice site
(2/103), whereas in a minority of cases they consisted in
start-lost (3/103). Three mutations were classified as splice
Methods acceptor or donor variants involving intronic sequences
(3/103), all by means of SnpEff&SnpSift tools
Multi-omics data in the CoMMpass study (http://snpeff.sourceforge.net/). Strikingly, DIS3 mutations
Multi-omics data regarding bone marrow MM samples at base- mainly occurred in the active domains of the protein; in
line (BM_1) were freely accessible from the MMRF CoMMpass detail, 70/103 mutations fell within the RNB domain and
study (https://research.themmrf.org/) and retrieved from the Interim ten in the PIN domain. With regard to the main mutational
Analysis 12a (MMRF_CoMMpass_IA12a). Details about molecu- hotspots reported to occur within the RNB domain, R780

Transcriptomic impact of DIS3 mutations in MM
Figure 1. Distribution of DIS3 non-synonymous somatic variants in 94 patients with multiple myeloma of the CoMMpass cohort and the correlation of the variants
with other genetic alterations. (A) Frequency of the 100 missense single nucleotide variants in DIS3 protein domains schematized below the histogram; the splice
acceptor or donor variants occurring in intronic regions are indicated by red arrows. (B) Plot of co-occurrence of main copy number alterations, IGH translocations and
non-synonymous somatic mutations in 651 cases of multiple myeloma in the CoMMpass dataset entirely profiled by whole exome sequencing and RNA-sequencing.

K. Todoerti et al.
Table 1. Cox regression univariate analysis in 630 patients with multiple myeloma for whom all data were available.
OS PFS
Univariate Cox Analysis Univariate Cox Analysis
Variable N (%) adj.P-value HR (95% CI) adj.P-value HR (95% CI)
ISS I 223 (35.4%) 2.59E-06 0.26 (0.15-0.43) 2.00E-07 0.42 (0.31-0.57)
ISS II 226 (35.9%) 7.13E-01 1.09 (0.76-1.57) 6.08E-01 1.09 (0.85-1.42)
ISS III 181 (28.7%) 1.92E-06 2.60 (1.83-3.70) 2.00E-07 2.08 (1.62-2.68)
del(13q) 281 (44.6%) 9.80E-02 1.46 (1.03 -2.07) 2.77E-01 1.21 (0.94 -1.55 )
DIS3mut 17 (2.7%) 9.80E-02 2.41 (1.06 - 5.49) 2.94E-01 1.67 (0.83 -3.39)
del(13q)/DIS3mut 298 (47.3%) 3.71E-02 1.63 (1.14 -2.32) 1.52E-01 1.27 (0.99 -1.63)
del(13q) + DIS3mut 50 (7.9%) 1.45E-01 1.60 (0.93-2.74) 4.03E-02 1.71 (1.15-2.54)
TP53.alterations (del(17p)/TP53 or TP53mt) 53 (8.4%) 9.42E-01 0.96 (0.50 -1.84) 7.63E-01 0.93 (0.58 -1.49)
1q21 gain/amp 207 (32.9%) 9.80E-02 1.45 (1.02 - 2.07) 5.63E-02 1.38 (1.07-1.78)
TP53.alterations + 1q21 gain/amp 24 (3.8%) 1.60E-04 3.64 (2.00 -6.62) 4.80E-03 2.56 (1.49-4.4)
N-RAS mut 142 (22.5%) 9.42E-01 1.02 (0.65-1.50) 6.31E-01 0.91 (0.68-1.23)
K-RAS mut 155 (24.6%) 4.82E-01 1.18 (0.80-1.74) 5.79E-01 1.12 (0.84-1.48)
BRAF mut 49 (7.8%) 2.64E-01 1.49 (0.84-2.65) 4.49E-01 1.26 (0.81-1.96)
RAS/BRAF mut 310 (49.2%) 3.60E-01 1.22 (0.86-1.74) 6.48E-01 1.07 (0.83-1.37)
TRAF3 mut 48 (7.6%) 1.10E-01 0.39 (0.14-1.05) 6.48E-01 0.88 (0.54-1.44)
FAM46C mut 64 (10.2%) 4.82E-01 1.27 (0.73-2.21) 4.49E-01 1.22 (0.82-1.83)
del(1p)/CDKN2C 190 (30.2%) 7.43E-02 1.55 (1.08 -2.23) 3.27E-01 1.20 (0.92-1.56)
HD 359 (57.0%) 9.80E-02 0.70 (0.50-0.99) 6.60E-02 0.75 (0.58-0.96)
WHSC1-FGFR3.trx 86 (13.7%) 3.60E-01 1.29 (0.82-2.05) 6.60E-02 1.47 (1.06-2.03)
MAF.trx 40 (6.3%) 1.03E-01 1.78 (1.00-3.16) 3.83E-01 1.32 (0.83-2.09)
MYC.trx 26 (4.1%) 1.10E-01 1.91 (0.97-3.78) 2.64E-01 1.55 (0.90-2.66)
Number (N) and percentage (%) of positive cases are indicated for each variable with the hazard ratio and 95% confidence interval. P values that were statistically significant
after Benjamini-Hochberg adjustment are reported in bold. OS: overall survival; PFS: progression-free survival; HR: hazard ratio; 95% CI: 95% confidence interval; ISS: International
Staging System; del: deletion; mut: mutation; amp: amplification; HD: hyperdiploid; trx: translocation.
was involved in 12 MM cases, whereas D488 and D479 of the 86 cases harboring DIS3 mutations (Online
residues were affected in 11 MM samples each (Figure 1, Supplementary Table S4).
Online Supplementary Table S3). We then looked at the co-occurrence of DIS3 mutations
We evaluated the association of DIS3 mutations with with the most frequently mutated genes in MM, i.e.,
the presence of the main IGH chromosomal translocations KRAS, NRAS, BRAF, FAM46C, TP53, and TRAF3 (Online
in 724 MM cases (Online Supplementary Table S2). A signif- Supplementary Table S2). A statistically significant associa-
icant co-occurrence was observed both with t(4;14) and tion with DIS3 mutations was only observed for BRAF
MAF (MAF, MAFB, or MAFA) translocations (P=0.0035). mutations (P=0.0126) (Online Supplementary Table S4).
The association of DIS3 mutations with copy number Figure 1B displays the global landscape of co-occurrence of
alterations commonly found in MM disease was evaluated DIS3 mutations with other main molecular lesions.
in 847 MM cases, for which specific data were available by
whole exome sequencing (Online Supplementary Table S2). Correlation of DIS3 mutations with clinical parameters
As expected, a very significant association (P=0.0003) was We tested the clinical impact of DIS3 mutations in 930
observed between DIS3 mutations and del(13q): specifical- MM cases with available PFS and OS data. At a median fol-
ly, 62 of 86 patients harboring a DIS3 mutation for whom low-up of 889 and 868 days for PFS and OS, respectively, a
data on copy number alterations were available showed a significantly lower survival rate for both OS (log-rank
13q deletion (Online Supplementary Table S4). Next, we con- P=0.039) and PFS (log-rank P=0.021) was observed in 94
sidered the distribution of del(13q) across the different DIS3-mutated cases compared to 836 DIS3 wild-type MM
types of DIS3 mutations. Only nine of 29 MM affected by cases. Specifically, the median PFS was 800 days in
the mutational hotspots also carried del(13q); notably, the DIS3-mutated cases versus 1176 days in wild-type MM
D479 hotspot was involved in five of these nine cases. In cases (Figure 2A, B), whereas the OS evaluated at 3 years
contrast, 50 of the 54 cases harboring not-hotspot DIS3 was 65% in DIS3-mutated cases versus 79% in the wild-
mutations were associated with del(13q) (P<0.0001). type group.
Concerning the other main copy number alterations, a As described above, DIS3 mutations co-occur signifi-
very significant association (P=0.0020) was observed with cantly with del(13q) and therefore with the loss of the sec-
1q21 gain/amplification (1q gain/amp), occurring in 45 of ond allele, a finding affecting several other genes in MM.
86 DIS3-mutated patients, 38 of whom also carrying Such a result prompted us to stratify the cases in the
del(13q). In contrast, a highly significant inverse correlation CoMMpass series into four groups according to the
was found with the hyperdiploid condition (P=0.0003) and absence of both mutated DIS3 and del(13q) (381 cases); the
the occurrence of 1p22/CDKN2C loss (P=0.0205). presence of only a DIS3 mutation (24 cases); the del(13q)
Moreover, 17p13/TP53 deletions were present in only four alone (380 cases); or the occurrence of both a DIS3 muta-

A B
C D
Figure 2. Impact of DIS3 mutations on clinical outcomes. (A, B) Kaplan-Meier survival curves of 930 patients with multiple myeloma (MM) stratified according to
the occurrence of DIS3 mutations with respect to overall survival (A) and progression-free survival (B). (C, D) Kaplan-Meier survival curves of 847 patients stratified
into four molecular groups according to the presence of DIS3 mutation and del(13q) as single or bi-allelic alterations, with respect to overall survival (C) and progres-
sion-free survival (D). Log-rank test P-value measuring the global difference between survival curves and numbers of samples at risk in each group across time are
reported. Log-rank test P-values of pairwise comparisons are also reported in (C, D). Statistically significant adjusted P-values following Benjamini-Hochberg (P<0.05)
are in bold red. The median follow-up time for the overall survival analysis was 862 days (interquartile range, 594-1,092 days) while that for progression-free survival
was 828 days (interquartile range, 535-1,094 days). OS: overall survival; PFS: progression-free survival; del: deletion; mt: mutated; WT: wild-type.
tion and del(13q) (62 cases). As depicted in Figure 2C, D, with TP53 alterations. Moreover, a higher risk of having a
the presence of the bi-allelic alterations was associated shorter OS was also associated with DIS3 mutations or
with a poor prognosis in comparison to the wild-type con- del(13q) as single events (mono-allelic condition). In con-
dition, with respective 3-year OS rates of 62% versus 82%, trast, the bi-allelic condition was associated with a shorter
and a median PFS of 772 days versus 1215 days. PFS. In contrast, ISS stage I was associated with significant-
Furthermore, we tested the prognostic impact on both ly shortened time to both death and progression (Table 1).
PFS and OS of DIS3 mutations and del(13q), as single or bi- Notably, all these features retained their independent prog-
allelic lesions, along with other parameters, in 630 MM nostic power both for OS and PFS when tested in Cox
patients for whom all the information was available (Table regression multivariate analysis (Figure 3).
1). A significantly increased risk of death or progression Based on such differential effects of mono- or bi-allelic
was associated with International Staging System (ISS) DIS3 lesions, we re-analyzed the association of these
stage III and the occurrence of 1q gain/amp in association events with the known MM oncogenic lesions. Regarding

K. Todoerti et al.
A B
Figure 3. Impact of DIS3 mutations and other clinical/molecular variables on survival of patients with multiple myeloma. (A, B) Forest plots of Cox regression mul-
tivariate analyses considering all features with adjusted P-value <0.05 in univariate analysis with regards to overall survival (A) and progression-free survival (B), in
630 patients with multiple myeloma from the CoMMpass cohort for whom all considered data were available. The hazard ratio, 95% confidence interval and P-value
are reported for each variable. A global log-rank P-value is reported for each analysis. OS: overall survival; PFS: progression-free survival; ISS: International Staging
System.
the main IGH translocations, t(4;14) was exclusively relat- mutational load (RNA_ALT_FREQ), chosen on the basis of
ed to the bi-allelic condition (P=0.0170). Furthermore, a analysis of the receiving operating characteristic curve
higher prevalence of 1q gain/amp was observed in patients (area under the curve, 98%) according to DNA mutational
with bi-allelic DIS3 events (61% vs. 29%). Finally, a larger level (variant allele frequency) (Online Supplementary Table
fraction of FAM46C mutated cases was evidenced in cases S3, Online Supplementary Figure S3A). In further support of
carrying only a DIS3 mutation (5.3% vs. 3.1%) (Online this cut-off value, we found a significant global correlation
Supplementary Table S5). (Pearson correlation r=0.94) between DNA and RNA
Finally, we attempted to clarify further the prognostic mutational levels in those 56 MM cases with >20% DIS3
relevance of DIS3 mutations in relation to well-established RNA mutational load, compared to the remaining 17 MM
first-line regimens, as described in the CoMMpass dataset. cases with lower RNA somatic variant frequencies
In this respect, 930 MM patients (94 of whom harboring (r=0.30) (Online Supplementary Figure S3B, C). Furthermore,
DIS3 mutations) were grouped according to the type of significantly shorter OS (log-rank P=0.013) and PFS (log-
therapy they had received: bortezomib/ immunomodula- rank P=0.0013) were observed in the 56 MM cases with a
tory drug (IMID)-based, bortezomib-based, IMID-based, >20% DIS3 RNA mutational load compared to the 836
or carfilzomib-based (i.e., combining IMID/carfilzomib, DIS3 wild-type cases. To note, no significant difference
bortezomib/IMID/carfilzomib, or single-agent carfilzomib was appreciated between DIS3 sub-clonal mutations (i.e.,
schedules). Overall, carfilzomib-based regimens signifi- <20% DIS3 RNA mutational load) and wild-type cases
cantly improved both OS and PFS compared to all other (Online Supplementary Figure S4).
types of combination therapies (Online Supplementary For the analysis, we therefore considered a total of
Figure S1). However, no significant difference in either OS 28,346 expressed transcripts and compared the expression
or PFS was detected among DIS3-mutated cases irrespec- profiles in 56 DIS3-mutated cases versus 582 DIS3 wild-
tive of the specific therapy scheme (Online Supplementary type MM cases. A list of 7,167 DE transcripts, 6,564 of
Figure S2). These results suggest that DIS3 mutations could which annotated, was obtained at a low stringency cut-off
negatively affect carfilzomib-based therapies. However, (false discovery rate <10%). Among them, 3,464 protein-
further validation in a larger prospective cohort of patients coding genes and 2,062 lncRNA resulted mostly upregulat-
is warranted. ed (79%) in DIS3-mutated patients compared to unmutat-
ed patients (Online Supplementary Table S6, Online
Transcriptional expression changes associated with Supplementary Figure S5). Principal component analyses
DIS3 mutations and del(13q) based on the expression levels of the 6,564 DE annotated
To define the gene expression signatures and molecular transcripts stratified according to the del(13q) aberration
pathways associated with DIS3 mutations, we focused on confirmed the strong association between DIS3 mutations
cases with expression of the mutation at meaningful lev- and del(13q) (Figure 4A). Furthermore, the heatmap of the
els, thus opting for a stringent cut-off of 20% on RNA top 100 most significantly upregulated transcripts, almost

C
Figure 4. Transcriptional expression changes associated
with DIS3 mutations and del(13q). (A) Principal compo-
nent analyses of 6,564 differentially expressed tran-
scripts in 56 cases of multiple myeloma (MM) with >20%
mutated DIS3 (DIS3mt) versus 582 DIS3 wild-type (DIS3
WT) cases. (B) Heatmap of the top 100 differentially
expressed transcripts according to B statistics value, in
56 DIS3 >20% mutated versus 582 DIS3 WT MM cases.
The colored scaled bar represents standardized rows by
subtracting the mean and dividing by the standard devia-
tion. Samples in each group are further stratified accord-
ing to the occurrence of del(13q) aberration: 5 DIS3mt
and not available (nd) for del(13q) MM cases, 38 MM
cases with bi-allelic alteration, 13 MM carrying only
DIS3mt, 289 MM with del(13q) as a single lesion and
293 WT MM cases. (C) Venn diagram of differentially
expressed transcript lists resulting from 13 MM with DIS3
mutation, 289 MM with del(13q) as a single lesion, or 38
MM carrying bi-allelic alteration compared to 293 WT MM
cases.
all involving lncRNA (82%), in the stratified MM samples across patients with mono-allelic and bi-allelic lesions
revealed a stronger pattern of positive regulation in the bi- (Online Supplementary Table S7).
allelic condition with respect to DIS3 mutations or del(13q)
alone, compared to DIS3 wild-type MM cases (Figure 4B). Protein-coding genes: molecular pathways and gene
Based on these findings, we compared the global sets modulated in association with DIS3 mutations
expression profiles associated with each lesion (13 cases In order to define which molecular pathways could be
with DIS3 mutations, 289 cases with del(13q), and 38 modulated in relation to the occurrence of DIS3 mutations
cases with bi-allelic alteration) to the wild-type condition in MM, GSEA was performed on the list of DE protein-
(293 cases). We found a shared set of 430 DE transcripts coding genes that were ranked based on fold change val-
(of which 405 annotated) in all three comparisons, 305 of ues (Online Supplementary Table S6).
which were lncRNA, whereas 56 were classified as pro- The enrichment map on the top 100 GSEA gene sets
tein-coding genes (Figure 4C). Interestingly, 295 out of 405 based on gene ontology biological process terms revealed
transcripts showed a cumulative effect of deregulation a complex network of connected functional modules

K. Todoerti et al.
Table 2. Summary information on the 12 long non-coding RNA significant in multivariate analysis.
Gene stable ID Description # Chr. Start (bp) End (bp) Strand Neighbor Pearson's correlation
Gene name * Gene
Name**
ENSG00000232519 novel transcript, 1q22 155609776 155610380 -1 MSTO1 r = 0.38, P<6.08E-16
AL353807.2 AS to MSTO1
ENSG00000235919 ASH1L AS RNA 1 1q22 155562042 155563944 1 ASH1L; r= 0.34, P< 6.08E-16
ASH1L-AS1 [Source:HGNC Symbol; AL353807.4 r= 0.63, P< 6.08E-16
Acc:HGNC:44146]
ENSG00000271991 novel transcript, 2p24 19902025 19902569 1 TTC32 r = 0.40, P<6.08E-16
AC013400.1 AS to TTC32
ENSG00000271387 novel transcript, 1q25 184385753 184386704 -1 C1orf21 r= 0.69, P<6.08E-16
AL445228.2 AS to C1orf21
ENSG00000272606 novel transcript, 2p16 55617909 55618373 1 PNPT1 r=0.15, P=2.70E-05
AC015982.2 AS to PP4R3B
ENSG00000236206 novel transcript, 1q24 165598356 165624084 1 MGST3 r = 0.28, P=8.93E-15
AL356441.1 sense to MGST3
ENSG00000255647 novel transcript, 5q14 90410000 90410669 1 CETN3 r= 0.13, P=6.39E-04
AC093510.1 AS to CETN3
ENSG00000259775 novel transcript, 14q32 103331674 103332367 -1 EIF5 r= 0.30, P<6.08E-16
AL138976.2 AS to EIF5
ENSG00000260236 novel transcript, 3p21 47379089 47380999 -1 PTPN23 r= 0.11, P=4.42E-03
AC099778.1 AS to PTPN23
ENSG00000272426 novel transcript, 1p36 16904339 16904776 -1 RNU1-2 r=0.15, P=3.00E-05
BX284668.6 AS to CROCC CROCC r=0.18, P=1.36E-06
ENSG00000272716 novel transcript, 2p22 32165046 32165757 -1 SLC30A6 r= 0.07, P=8.94E-02
AL121658.1 AS to SLC30A6 SPAST r=0.15, P=2.71E-05
ENSG00000273355 novel transcript, 18p11 813274 813756 1 YES1 r=0.16, P=1.93E-05
AP000894.4 AS to YES1
*The significance in multivariate analysis is indicated for overall survival (underlined Gene stable ID), overall survival and progression-free survival (italicized Gene stable ID),
or progression-free survival (Gene stable ID in plain text). The four lncRNA in bold are those validated by quantitative reverse transcriptase polymerase chain reaction.
**Neighbor genes belonging to the list of differentially expressed genes (Online Supplementary Table S6) are marked in bold. ID: identity; Chr: chromosome; AS: antisense.
mainly concerning RNA and protein metabolism, cell- tions; notably, these transcripts corresponded to the most
cycle regulation, nucleosome organization, immune significantly deregulated ones in the global DE list (Online
response, cell proliferation and apoptosis, cell adhesion Supplementary Table S6, Online Supplementary Figure S7B).
and tissue development (Online Supplementary Figure S6). Focusing on the shared 490 coding transcripts (Online
Furthermore, GSEA revealed a significant enrichment of Supplementary Table S6, Online Supplementary Figure S7C),
transcriptional signatures some of which known to be dis- GSEA identified a number of gene sets among those pre-
tinctively associated with main IGH translocations (Online viously obtained from the global analysis; in particular, we
Supplementary Table S8), likely in agreement with the co- confirmed downregulation of the oxidative phosphoryla-
occurrence of DIS3 mutations and t(4;14) or MAF translo- tion gene set along with genes involved in RNA and
cations (Online Supplementary Table S4), or in an opposite amino acid metabolism and translation, and upregulation
manner, with the hyperdiploid condition (Online of the interferon signaling gene set (Online Supplementary
Supplementary Tables S4 and S8). Therefore, with the aim Figure S8, Online Supplementary Table S8).
of identifying DE transcripts more specifically related to Finally, the shared DE transcript list associated with
DIS3 mutations, we investigated the transcriptional pro- DIS3 mutations was particularly enriched in lncRNA
files in MM subgroups with a more homogeneous genetic (782/1542: 51% of all DE transcripts) (Online
background, each of them stratified according to the Supplementary Table S6, Online Supplementary Figure S7D),
occurrence of DIS3 mutations: i.e., patients carrying thus further supporting the notion of a distinctive and
t(4;14) and 1q gain/amp; MAF translocations and 1q stronger impact of DIS3 mutations on the ncRNA tran-
gain/amp; or hyperdiploid cases (Online Supplementary scriptome.
Figure S7A). Whereas no significant DE transcripts were
identified in cases with both MAF translocations and 1q Differential expression patterns of long non-coding
gain/amp (likely due to the limited number of cases), RNA associated with DIS3 mutations
almost 90% of the DE transcripts from the other two LncRNA transcriptional patterns specifically associated
comparisons overlapped with the original transcriptional with DIS3 mutations were further investigated. In detail,
signature (Online Supplementary Figure S7A). Overall, we based on the pronounced heterogeneity of lncRNA and
identified 1,542 shared DE transcripts that could be likely their lower expression levels as compared to coding tran-
considered as distinctively associated with DIS3 muta- scripts, we applied a more stringent analysis on the 782

shared DE lncRNA, and focused on the 50 most significant lncRNA sense to coding-genes, one long intergenic non-
ones (false discovery rate <1%). All lncRNA were upregu- protein coding RNA, two microRNA host genes and two
lated in DIS3-mutated cases compared to DIS3 wild-type divergent transcripts (Online Supplementary Table S9). Of
cases and were mainly represented by lncRNA antisense note, the novel transcript Z93930.2 is located less than 100
to coding genes (80%); the remaining cases included three bp antisense to the transcription factor XBP1, a well-estab-
Figure 5. Long non-coding RNA with clinical relevance in patients with multiple myeloma. Scheme of the genomic region of the four long non-coding (lnc)RNA val-
idated by means of quantitative reverse transcriptase polymerase chain reaction in 43 patients with newly diagnosed multiple myeloma including 13 with DIS3 wild-
type (WT), 14 with DIS3 WT and del(13q), seven with DIS3 mutation (DIS3mt), and nine with DIS3mt and del(13q). Primer positions are indicated in red below each
lncRNA. Differential expression was assessed by the Wilcoxon signed-rank test and statistically significant P-values (<0.05) are reported above each boxplot. Dunn
test. P-values for pairwise comparisons are reported in tables under each boxplot, with statistically significant P-values in bold red.

K. Todoerti et al.
lished regulator of MM, known to be altered during the in DIS3-mutated samples as compared to unmutated ones
initiation and progression of MM.27 (Online Supplementary Table S6). Finally, the expression of
Based on the recurrent evidence that the transcription of some (AC093510.1, AC015982.2, AL138976.2, and
mRNA and lncRNA appears to be closely regulated, lead- AC099778.1) of these lncRNA was validated by quantita-
ing to a cis-regulatory relationship,28-30 we investigated the tive reverse transcriptase polymerase chain reaction in 43
levels of expression of overlapping or nearby transcripts newly diagnosed MM proprietary samples previously
localized in close proximity to the 50 lncRNA (in a win- characterized for the presence of DIS3 mutations and for
dow up to 65 kb). We considered 81 mRNA-lncRNA pairs which material was available (Online Supplementary Table
and analyzed the correlation between their expression lev- S1). In detail, we found that AC093510.1, AC015982.2, and
els across the entire dataset of 767 MM cases profiled by AL138976.2 were significantly upregulated in MM with
RNA-sequencing in the CoMMpass cohort. A significant DIS3 mutations without del(13q) as compared to in DIS3
Pearson correlation (r>0.5, P<6.08x10-16) was observed for wild-type samples, whereas AC099778.1 was significantly
nine lncRNA-gene pairs; among them, AL121672.3 and upregulated only in the bi-allelic condition (Figure 5).
MIRLET7BHG, both mapping at 22q13, showed a relevant
correlation with each other (r=0.65) and with the PRR34
gene (r=0.66 and r=0.73, respectively) (Online Discussion
Supplementary Table S10).
We took advantage of the large publicly available
Clinical relevance of long non-coding RNA CoMMpass dataset to investigate the type and frequency
Next, all 50 lncRNA were tested for their relationship to of DIS3 mutations in MM and their impact on the tran-
OS and PFS using Kaplan-Meier survival analysis on the scriptional signature and clinical outcome.
767 MM cases with available RNA-sequencing and sur- In agreement with previously reported data,6,16,17 we
vival data. Groups with high versus low expression were assessed that the frequency of DIS3 mutations in newly
determined according to the mean cut-off value for each diagnosed MM is approximately 10%; notably, DIS3
lncRNA expression level across the entire dataset. mutations were associated with del(13q) as bi-allelic events
Interestingly, higher levels of expression were associated in 72% of cases. The majority of DIS3 mutations are mis-
with a poorer clinical outcome in terms of PFS for 35 out sense. Together with their clustering at particular codons,
of all the 50 tested lncRNA. Furthermore, 15 of them the lack of truncating mutations is not typical of a tumor-
showed an unfavorable prognosis in terms of OS (Online suppressor gene and may suggest an oncogenic potential
Supplementary Figure S9, Online Supplementary Table S11). for DIS3. DIS3 mutations showed a clear pattern of co-
The clinical impact of the 35 lncRNA with poorer clini- occurrence with other molecular alterations, mainly chro-
cal outcome was further investigated by Cox regression mosomal translocations. This phenomenon could be
univariate analysis. For 21 lncRNA, their higher expression explained through the interaction between the RNA exo-
level was associated with a significantly higher risk in PFS, some and the protein activation-induced cytidine deami-
and for five of them (AC015982.2, AL353807.2, nase (AID) during the process of class switching and hyper-
AC013400.1, ASH1L-AS1, and AL445228.3) also in OS mutation in B cells;31 indeed, DIS3 mutations could indi-
(Online Supplementary Table S12). Next, for all these 21 sig- rectly, through disruption of an interaction with AID,
nificant lncRNA, high and low lncRNA expression levels cause mis-targeting of the somatic hypermutation process
were tested in 630 cases, together with other clinically rel- leading to chromosomal translocations. In particular, DIS3
evant characteristics, i.e. ISS stage I and III, the occurrence mutations are associated with t(4;14), this combination
of 1q gain/amp in association with TP53 alterations, the defining a poor prognostic MM subgroup.16 However, our
presence of DIS3 mutations or del(13q) as single events for analyses pointed out that t(4;14) occurred statistically sig-
OS, or in a bi-allelic condition for PFS. Notably, all the five nificantly in patients with bi-allelic lesions, in agreement
lncRNA associated with a shorter OS retained their clini- with the very frequent association between t(4;14) and
cal impact when tested in Cox regression multivariate del(13q) alterations. Notably, the oncogenic events that
analysis. Two of them (AC015982.2 and AL445228.3) most frequently co-occurred with DIS3 mutations were
retained an independent significant prognostic power also del(13q), 1q gains, t(4;14) and MAF translocations. Overall,
for PFS; besides these two lncRNA, another seven this spectrum of molecular lesions suggests a functional
(AC099778.1, AP000894.4, AL121658.1, AL356441.1, constraint of cooperating oncogenic events in MM, with a
BX284668.6, AC093510.1, AL138976.2) were found to be selection of later lesions being restricted by the ones
independent predictors of PFS at multivariate analysis appearing first in the transformed cell. With regard to this,
(Table 2, Online Supplementary Table S13). Of note, the bi- DIS3 mutations were found to be both clonal in some
allelic condition lost its independent clinical impact in all patients and subclonal in others, meaning they might func-
the multivariate analyses of the nine lncRNA significant tion sometimes as early and sometimes as late hits.7,22
for PFS (Online Supplementary Table S13). Our study also pointed out that the clinical relevance of
Overall, from our analysis 12 lncRNA were predicted to DIS3 mutations depended strictly on the co-occurrence of
have substantial clinical relevance (Table 2). Specifically, 11 del(13q). In detail, we established that the bi-allelic lesions
of them code for novel transcripts, and four are antisense significantly affected PFS, whereas the mono-allelic condi-
to known transcripts whose expression level, when tion predicted worse OS. Notably, these alterations
detectable, was highly positively correlated (Table 2, remained valuable independent predictors even when
Online Supplementary Table S10). Interestingly, we found tested in combination with the clinical and poor prognosis
four couples of lncRNA-coding genes (antisense or nearby) molecular variables used to foresee clinical outcome. The
located on chromosome 1q with highly correlated expres- differential impact of the bi-allelic or mono-allelic lesions
sion (Table 2); these coding genes (MGST3, ASH1L, on MM outcome could be related to the fact that our data
MSTO1, and C1orf21) were also significantly upregulated highlighted two patterns of DIS3 lesions: one in which

del(13q) co-exists with non-hotspot DIS3 mutations, and prostaglandin E, which are important mediators of inflam-
a second in which hotspot DIS3 mutations rarely show mation;33 ASH1L, a methyltransferase already known to
loss of heterozygosity through bi-allelic events. Again, be involved in cancer;34 and MSTO1, important for mito-
this is a rather intriguing pattern that may suggest either chondrial fusion and intracellular distribution.35 Along
haploinsufficiency for some mutations and not others, or with these findings, the patterns observed in the context
a different function with some mutations showing loss of the DE coding transcripts associated with DIS3 muta-
and others showing gain of function. This might also tions are also interesting. Indeed, we found downregula-
explain the different clinical consequences on PFS and OS tion of gene sets related to oxidative phosphorylation,
observed with the two genetic statuses. Indeed, previous metabolism of RNA or amino acids, and translation, and
reports showed how different types of DIS3 mutations contrariwise, upregulation of interferon signaling. These
could lead to diverse biological effects either by impairing transcriptomic changes are in line with the functional role
exosome function through reduced/modified DIS3 activi- of DIS3 in RNA metabolisms36,37 and in agreement with
ty,11 or through a dominant-negative effect exerted by what has been described for yeasts in which mutations
mutated DIS3 on the other catalytic subunit, Rrp6, acting have been extensively investigated.38 In particular, the pre-
in the exosome complex.32 dicted enhanced interferon activity may represent a
DIS3 is a key component of the multisubunit RNA exo- response to the accumulation of RNA substrates in cells
some complex in eukaryotic cells involved in the process- following a deficiency of DIS3.39
ing, quality control and degradation of virtually all classes Overall, our comprehensive evaluation of the clinical
of RNA. Our study further supports and extends the and transcriptional consequences of DIS3 mutations/defi-
notion that DIS3 mutations affect the transcriptome, ciency in MM strongly indicates that they may play an
showing a stronger impact on noncoding RNA species, important role in the mechanisms of MM transformation
mainly lncRNA. Indeed, we found that approximately half and progression. Our results may provide important
of the DE transcripts predicted to be specifically related to insights for functional studies in order to better under-
the presence of DIS3 mutations are represented by novel, stand such mechanisms in MM.
largely uncharacterized lncRNA. Among them, we high-
lighted 12 lncRNA, five of which are independent predic- Disclosures
tors of poorer OS and nine of worse PFS, with two of No conflicts of interest to disclose.
them (AC015982.2 and AL445228.3) predicting both.
Moreover, the clinical impact of the nine lncRNA predict- Contributions
ing inferior PFS at higher expression levels was independ- KT designed the study, collected and analyzed data and wrote
ent of the genetic status of DIS3. The effect of these 12 the paper; DR designed the study, analyzed data and wrote the
lncRNA on the pathobiology of MM disease remains to be paper; VF, FM and FM analyzed data; NB wrote the paper; ET
fully elucidated; indeed, they are all novel transcripts, and AN designed the study, supervised the study and wrote the
none of them currently reported as being associated with manuscript
cancer. Interestingly, some of them showed a strict corre-
lation in terms of expression levels with the corresponding Funding
nearby genes, thus suggesting a cis-regulatory relationship This work was financially supported by grants from the
between the paired transcripts. Although these findings Associazione Italiana Ricerca sul Cancro (AIRC) to AN
need to be investigated further, this could be the case for (IG16722 and IG24365). NB is funded by the European
genes involved in fundamental molecular pathways, such Research Council under the European Union’s Horizon 2020
as MGST3, involved in the production of leukotrienes and research and innovation program (grant agreement n. 817997).
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Platelet Biology & its Disorders ARTICLE
The GPIba intracellular tail - role in transducing Ferrata Storti Foundation

VWF- and collagen/GPVI-mediated signaling
Adela Constantinescu-Bercu,1 Yuxiao A Wang,1 Kevin J Woollard,2
Pierre Mangin,3 Karen Vanhoorelbeke,4 James TB Crawley1 and
Isabelle I Salles-Crawley1
1
Center for Hematology, Department of Immunology and Inflammation, Imperial College
London, London, UK; 2Center for Inflammatory Disease, Department of Immunology and
Inflammation, Imperial College London, London, UK; 3Université de Strasbourg,
INSERM, EFS Grand-Est, BPPS UMR-S 1255, FMTS, Strasbourg, France and 4Laboratory
for Thrombosis Research, KU Leuven, Kortrijk, Belgium. Haematologica 2022
Volume 107(4):933-946
ABSTRACT
T
he GPIba-VWF A1 domain interaction is essential for platelet teth-
ering under high shear. Synergy between GPIba and GPVI signal-
ing machineries has been suggested previously, however its
molecular mechanism remains unclear. We generated a novel GPIba
transgenic mouse (GpIbaΔsig/Δsig) by CRISPR-Cas9 technology to delete the
last 24 residues of the GPIba intracellular tail that harbors the 14-3-3 and
phosphoinositide-3 kinase binding sites. GpIbaΔsig/Δsig platelets bound von
Willebrand factor (VWF) normally under flow. However, they formed
fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 blocker,
demonstrating that despite normal ligand binding, VWF-dependent sig-
naling is diminished. Activation of GpIbaΔsig/Δsig platelets with ADP and
thrombin was normal, but GpIbaΔsig/Δsig platelets stimulated with collagen-
related-peptide (CRP) exhibited markedly decreased P-selectin exposure
and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in
GPVI-mediated signaling. Consistent with this, while hemostasis was
normal in GpIbaΔsig/Δsig mice, diminished tyrosine-phosphorylation, (par-
ticularly pSYK) was detected in CRP-stimulated GpIbaΔsig/Δsig platelets as
well as reduced platelet spreading on CRP. Platelet responses to
rhodocytin were also affected in GpIbaΔsig/Δsig platelets but to a lesser
extent than those with CRP. GpIbaΔsig/Δsig platelets formed smaller aggre- Correspondence:
gates than wild-type platelets on collagen-coated microchannels at low,
medium and high shear. In response to both VWF and collagen binding, ISABELLE I SALLES-CRAWLEY
i.salles@imperial.ac.uk
flow assays performed with plasma-free blood or in the presence of
aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation con-
tributes to the phenotype of the GpIbaΔsig/Δsig platelets. Together, these Received: December 23, 2020.
results reveal a new role for the intracellular tail of GPIba in transducing Accepted: June 10, 2021.
both VWF-GPIba and collagen-GPVI signaling events in platelets.
Pre-published: June 17, 2021.
Introduction https://doi.org/10.3324/haematol.2020.278242
In order to fulfil their hemostatic function, platelets are recruited to sites of vessel
damage by von Willebrand factor (VWF), which interacts with exposed collagen and,
thereafter, to glycoprotein (GP) Iba on the platelet via its A1 domain. VWF-mediated Material published in Haematologica is covered by copyright.
platelet tethering facilitates platelet capture.1 Subsequent interaction of platelets with All rights are reserved to the Ferrata Storti Foundation. Use of
additional ligands (e.g., aIIbb3-fibrinogen, collagen-GPVI, collagen-a2b1) and conditions:
changes in platelet phenotype are required to stabilize the platelet plug. Although the https://creativecommons.org/licenses/by-nc/4.0/legalcode.
VWF-GPIba interaction primarily facilitates platelet recruitment, it also transduces a Copies of published material are allowed for personal or inter-
signal that causes intraplatelet Ca2+ release and activation of the platelet integrin, nal use. Sharing published material for non-commercial pur-
aIIbb3.2-5 These signaling events are highly dependent upon flow as shear forces https://creativecommons.org/licenses/by-nc/4.0/legalcode,
induce unfolding of the GPIba mechanosensitive juxtamembrane region that trans- sect. 3. Reproducing and sharing published material for com-
lates the mechanical signal into intracellular biochemical events.6,7 Signaling is mercial purposes is not allowed without permission in writing
dependent upon the binding of adaptor and signaling molecules (e.g., Src kinases, Lyn from the publisher.
and c-Src, 14-3-3 isoforms and phosphoinositide-3 kinase [PI3K]) that can associate
with the GPIba intracellular tail.8-12 Downstream activation of phospholipase Cg2

A. Constantinescu-Bercu et al.
(PLCg2), PI3K-Akt, cGMP-PKG, mitogen activated kinase and AGTATGAATGAGCGGGAGCC and subsequent Sanger
and LIM kinase 1 pathways have also been reported.13-19 By sequencing (Genewiz).
comparison to other platelet agonists (e.g., collagen, throm- Experimental procedures were performed as previously
bin, ADP, thromboxane A2), signaling through GPIba is described.29,30 Additional details are included in the Online
considered weak. VWF-GPIba signaling, which we term Supplementary Appendix.
platelet ‘priming’ rather than activation, does not induce
appreciable degranulation.5 Therefore, the contribution of
platelet ‘priming’ to normal hemostasis remains unclear as Results
the effects of the other platelet agonists have the potential
to mask those of GPIba. However, in scenarios where other Generation of GpIbaΔsig/Δsig mice
platelet agonists are either absent or in low abundance (e.g., Sequence identity between human and murine GPIba
platelet recruitment to endothelial or bacterial surfaces), the intracellular region is very high, supporting the contention
effects/importance of GPIba signaling may become more that their functions are well conserved (Figure 1A). In order
prominent.5 to evaluate the role of the GPIba intracellular tail upon both
GPVI is a collagen/fibrin receptor on the platelet surface VWF- and collagen/GPVI-mediated signaling, we generated
that non-covalently associates with Fc receptor g-chain a novel transgenic mouse (GpIbaΔsig/Δsig) using CRISPR-Cas9
(FcRg) and signals via immunoreceptor tyrosine-based acti- technology. We introduced a point mutation (Ser695Stop)
vation motifs (ITAM).20-22 Collagen binding to platelets that resulted in a premature stop codon that deletes the last
induces clustering of GPVI, which results in the phosphory- 24 a.a. of the GPIba intracellular tail (a.a. 695-718) contain-
lation of FcRg by Src family kinases, Lyn and Fyn, that asso- ing the entire 14-3-3 isoform and PI3K binding region,10,12
ciate with the intracellular domain of GPVI.23,24 This causes but maintains the upstream filamin binding site in GPIba
the recruitment and phosphorylation of Syk tyrosine (residues 668-681 in murine GPIba)31 (Figure 1A and B).
kinase, and formation of a LAT-based signaling complex Introduction of the mutation was confirmed by sequencing
that can activate PLCg2 and lead to release of intraplatelet and by western blotting using an anti-GPIba antibody that
Ca2+ stores, activation of protein kinase (PK) C, and ulti- recognizes the terminal region of the intracellular tail
mately aIIbb3 activation and both a- and dense-granule (Figure 1C to E). GpIbaΔsig/Δsig mice were viable and born with
release.21 the expected Mendelian frequencies.
Previous studies have suggested functional associations
between GPIba and GPVI and/or its co-receptor FcRg.13,25,26 GpIbaΔsig/Δsig mice platelet count, platelet size
For example, VWF-GPIba-mediated platelet responses are and hemostatic function
reportedly impaired in GPVI/FcRg deficiencies in both mice GpIbaΔsig/Δsig mice had mildly reduced (~20%) platelet
and humans.13,27 There is also evidence that VWF can poten- counts and slightly larger platelet size (Figure 2A and B), but
tiate responses after collagen mediated responses in human other hematological parameters were unaffected (Online
platelets.28 However, the molecular basis of GPIba and Supplementary Table S1). This is in contrast to the severe
GPVI receptor crosstalk has not been elucidated. Using a thrombocytopenia and giant platelets observed in complete
novel GPIba transgenic mouse in which the last 24 amino GPIba deficiency in mice or Bernard-Soulier patients.32,33
acids (a.a.) of the GPIba intracellular tail were deleted, we Expression of the major platelet receptors, GPVI, aIIbb3,
demonstrate the importance of this region not only to GPIba, and the extracellular region of GPIba was unaltered
VWF-dependent signaling in platelets, but also reveal a on GpIbaΔsig/Δsig platelet surfaces (Figure 2C).
major contribution in augmenting GPVI-mediated platelet In order to assess hemostatic function in GpIbaΔsig/Δsig mice,
signaling. we performed tail bleeding assays. Unlike Vwf-/- mice or
mice lacking the extracellular domains of GPIba,32,34,35
GpIbaΔsig/Δsig mice displayed normal blood loss following tail
Methods transection (Figure 2D), suggesting that GpIbaΔsig/Δsig platelets
can be recruited to sites of vessel damage similar to wild-
Mice type mice.
All procedures were performed with the United Kingdom There was no difference between GpIbaΔsig/Δsig mice and
Home Office approval in accordance with the Animals (Scientific wild-type littermates in a non-ablative laser-induced throm-
Procedures) Act of 1986. GpIbaΔsig/Δsig mice were generated in-house bus formation, as measured by the kinetics and extent, of
by the Medical Research Council transgenic group at Imperial both platelet accumulation and fibrin deposition (Figure 2E
College using CRISPR-Cas9 technology (Figure 1). Briefly, pronu- to G; Online Supplementary Figure S1; Online Supplementary
clear injections (CBAB6F1) were performed with Cas9 mRNA (75 Video S1).29,30,36 These results support the contention that
ng/mL), guide RNA (gRNA; 25-50 ng/mL) and single-strand oligo deletion of the GPIba does not appreciably influence either
donor DNA (25-50 ng/mL). The donor DNA (GGTAAGGCC- platelet recruitment or their ability to support thrombin
TAATGGGCGAGTGGGGCCTCTGGTAGCAGGACGGC- generation. In this model, platelet accumulation requires
GACCCTGAGCTCTGAGTCAGGGTCGTGGTCAGGACC- both VWF and thrombin but has less dependency upon col-
TATTGGGCACAGTGGGCATTA) had 50 bp homology arms at lagen exposure or GPVI signaling due to the non-ablative
the 5’ and 3’ ends (Integrated DNA Technologies). Embryos were injury.37,38
transferred to pseudo-pregnant CBAB6F1 female mice. Two
founder mice originated from the same gRNA (CGACCCT- GpIbaΔsig/Δsig platelets bind von Willebrand factor (VWF)
GACTCAGAGCTGAGGG) were bred with C57BL/6 mice. F1 normally, but exhibit decreased VWF-mediated
GpIbaΔsig/+ mice were bred to obtain GpIbaΔsig/Δsig mice, and GpIba+/+ signaling
littermates were used as controls. Genotyping was performed by In order to specifically examine the effect of the GPIba
polymerase chain reaction (PCR) amplification of a GpIba frag- intracellular tail truncation upon VWF-dependent platelet
ment (551 bp) using primers: AAGCACTCACACCACAAGCC capture, we coated microchannels with murine VWF over

The role of GPIba in GPVI-signaling
D E
Figure 1. Generation and characterization of GpIbaΔsig/Δsig mice. (A) Sequence alignment of the last 100 amino acids (a.a.) of human and mouse GPIba. Sequence
identities are highlighted in red. Filamin binding region: (a.a. 560-573) and (a.a. 668-681) for human and mouse GPIba; PI3K/14-3-3 binding region: (a.a. 580-610)
and (a.a. 688-718) for human and mouse GPIba. (B) Schematic representation of the GpIba gene with CRISPR guide target site, gRNA sequence, BbvCI restriction
enzyme site and Cas9 predicted cut site. Primers used to amplify the GpIba allele from genomic DNA are indicated in purple. Design of the 101 bp single stranded
DNA repair template with the point mutation to introduce a codon stop eliminating the BbvCI restriction enzyme site and removing the last 24 a.a. of GPIbα is also
shown. The resulting truncated a.a. sequence from GpIbaΔsig/Δsig mice is indicated in green. (C) Genomic DNA sequences from GpIba+/+ and GpIbaΔsig/Δsig mice.
Successful substitution is indicated with an arrow. (D) Diagram showing the binding of the anti-GPIbα tail Ab (Biorbyt; orb 215471). (E) Platelet lysates from GpIba+/+
and GpIbaΔsig/Δsig mice were probed with the anti-GPIba tail and b-actin antibodies. Absence of band in the GPIba western-blot confirms the successful truncation of
the GPIba intracellular tail in GpIbaΔsig/Δsig mice.

which we perfused plasma-free blood (to remove fibrino- GpIbaΔsig/Δsig platelets bound to VWF and only very few
gen and outside-in activation aIIbb3) at 1,000s-1. GpIbaΔsig/Δsig exhibited filopodia (Figure 3E and F). When these experi-
platelets were recruited normally to murine VWF-coated ments were repeated in the presence of botrocetin (a snake
surfaces with rolling velocities, surface coverage and venom that increases the affinity of VWF A1 domain for
platelet accumulation unaltered compared to GPIba+/+ GPIba)39 a large proportion (90±2.8%) of GPIba+/+ platelets
platelets (Figure 3A to D; Online Supplementary Video S2). underwent shape changes and developed filopodia (Figure
In order to investigate the impact of the deletion of the 3E and G; Online Supplementary Figure S2A and B), a well-
last 24 a.a. of GPIba on VWF signaling, we performed described consequence of VWF-GPIba signaling.9,19 This
platelet spreading assays on murine VWF, which rely upon process was significantly diminished in GpIbaΔsig/Δsig platelets
VWF-GPIba signaling. On VWF alone, very few GPIba+/+ or with only 46±2.6% platelets exhibiting filopodia (Figure 3E
A B C
D E F
Figure 2. GpIbaΔsig/Δsig mice display normal bleeding loss and platelet and fibrin accumulation in the laser-induced thrombosis model. A) Platelet counts and (B)
platelet size in GpIba+/+ (n=25) and GpIbaΔsig/Δsig mice (n=30) as determined by flow cytometry. (C) Surface expression of platelet receptors GPIba, GPIbb, aIIbb3 and
GPVI in GpIba+/+ and GpIbaΔsig/Δsig mice (n=4 for each genotype) determined by flow cytometry and expressed as % of control. (D) Bar graph analyzing blood loss after
10 minutes following tail transection in GpIba+/+ and GpIbaΔsig/Δsig mice (n=9 for each genotype). (E-G) Mice cremaster muscle arterioles were subjected to the laser-
induced thrombosis model as described in the Online Supplementary Appendix. Curves represent median integrated fluorescence intensity (IFI) from platelets (arbi-
trary units: AU) (E) or fibrin(ogen) (F) as a function of time after the injury (20 thrombi in 3 GpIba+/+ and 34 thrombi in 4 GpIbaΔsig/Δsig mice). (G) Representative com-
posite fluorescence images of platelets (green) and fibrin (red) with bright field images after laser-induced injury of the endothelium of GpIba+/+ (top panels) vs.
GpIbaΔsig/Δsig mice (bottom panels). Scale bar represents 10 mm. Each symbol represents one thrombus. Horizontal lines intersecting the data set represent the medi-
an. Data was analyzed using Mann Whitney test; ns: P>0.05. Also see the Online Supplementary Video S1 and the Online Supplementary Figure S1. FSC: foward
scatter; Hb: hemoglobin.

and G; Online Supplementary Figure S2C to D).9,19 When due to a defect in VWF-GPIba signaling manifest by a lack
experiments were performed in the presence of both botro- of activation of aIIbb3 in response to VWF-GPIba binding.
cetin and GR144053, which competitively inhibits the Taken together, these results indicate that deletion of the
interaction of aIIbb3 with VWF and/or fibrinogen, the last 24 a.a. of the intracellular tail of GPIba does not influ-
number of GPIba+/+ platelets forming filopodia was not ence platelet binding to VWF, but significantly reduces
appreciably influenced (Online Supplementary Figure S2B), VWF-GPIba downstream signaling response including
but the proportion of that formed >3 filopodia was signifi- aIIbb3 activation.
cantly reduced (37±6.7% vs. 74±6.9%) (Online
Supplementary Figure S2A), revealing the contribution of out- The intracellular tail of GPIba is important for
side-in signaling to filopodia formation. Under these condi- GPVI signaling
tions, here again although GpIbaΔsig/Δsig platelets bound VWF We next evaluated agonist-induced platelet activation in
surfaces, they had a significantly diminished ability to form GpIbaΔsig/Δsig mice. In response to ADP, washed GpIbaΔsig/Δsig
filopodia (Figure 3E and H). Moreover, GR144053 had no platelets exhibited normal aIIbb3 activation and P-selectin
effect upon filopodia formation in GpIbaΔsig/Δsig platelets exposure and normal platelet aggregation (Figure 4A to D).
(Online Supplementary Figure S2C), suggesting that the Responses to thrombin were also normal except for a slight
reduced filopodia formation in these platelets was likely significant decrease in P-selectin exposure with the lowest
A B C D
F
E
Figure 3. GpIbaΔsig/Δsig platelets exhibit normal binding to von Willebrand factor but disrupted GPIbα-mediated signaling. (A-D) Plasma-free blood from GpIba+/+ and
GpIbaΔsig/Δsig mice supplemented with anti-GPIbb-DyLight488 anitbody was perfused over murine VWF at a shear rate of 1,000 s-1. (A) Representative fluorescence
images (n≥3; scale bar 10 mm) and bar graphs analyzing the integrated fluorescence intensity (IFI) (B) and the surface coverage (C) of GpIba+/+ and GpIbaΔsig/Δsig
platelets captured by murine von Willebrand factor (VWF) after 3.5 minutes of flow. (D) Rolling velocities (median ± confidence interval [CI]) were calculated from
(approximately 10,000) platelets rolling/adhering to murine VWF within the first 30 seconds (n≥3) (E) Representative confocal images of GpIba+/+ and GpIbaΔsig/Δsig
platelets (n=3 for each genotype) spread on mVWF and stained with Phalloidin-Alexa 488, in the absence or presence of Botrocetin or Botrocetin and GR144053
(scale bar 10 mm). (F to H) Percentage of platelets from GpIba+/+ and GpIbaΔsig/Δsig mice (individual data points representing the average of 3-6 fields of view) with no
filopodia, 1-3 filopodia or >3 filopodia formed on murine VWF in the absence (F; 129 GpIba+/+ platelets and 115 GpIbaΔsig/Δsig platelets analysed) or presence of
Botrocetin (G; 511 GpIba+/+ platelets and 547 GpIbaΔsig/Δsig platelets analysed), or Botrocetin and GR144053 (H; 359 GpIba+/+ platelets and 480 GpIbaΔsig/Δsig platelets
analysed). Data represents mean ± standard error of the mean (B, C, F to H) or median ± CI (D) and was analyzed using unpaired two-tailed Student’s t-test (B and
C), unpaired Mann Whitney test (D) or using two-way ANOVA followed by Sidak’s multiple comparison test (F to H); *P<0.05, ***P<0.001, ****P<0.0001. Also see
the Online Supplementary Figure S2 and the Online Supplementary Video S2.

A B
C D
E F G
H I J
K L
Figure 4. Legend on following page.

Figure 4. GpIbaΔsig/Δsig platelets exhibit altered GPVI-mediated signaling. (A and B) Flow cytometric analysis of surface expression of activated aIIbb3 (A) and P-selectin
(B) in GpIba+/+ and GpIbaΔsig/Δsig platelets (n=8) in response to ADP (1-20 mM), a-thrombin (20-200 mU/mL), or CRP (1-10 mg/mL). MFI: geometric mean fluorescence
intensity (C) Representative aggregation traces (n=3-6) of washed platelets isolated from GpIba+/+ (blue) or GpIbaΔsig/Δsig (red) mice and stimulated with ADP (1-10 mM),
a-thrombin (20-50 mU/mL) or CRP (0.5-3 mg/mL). Aggregation was monitored using a Chronolog aggregometer over 6 minutes. (D) Bar graph analysing the maximum
aggregation (%) obtained in the conditions presented in (C). (E) Representative micrographs (n=3 for each genotype; 3 fields of view analyzed per condition; scale
bar 10 mm) of 454 GpIba+/+ and 420 GpIbaΔsig/Δsig platelets spread on CRP and stained with Phalloidin-Alexa 488. Bar graphs quantifying the surface area (F) and per-
centages (G) of platelets that remained round, formed filopodia or spread on CRP. (H) Western blot analyzing tyrosine kinase phosphorylation in platelets from
GpIba+/+ and GpIbaΔsig/Δsig mice, following stimulation with 3 mg/mL CRP for 0-180 seconds (s), using b-actin as a loading control (representative of n=3). (I) Western
blots analyzing the levels of phosphorylated and non-phosphorylated SYK, PLCg2 and Akt in platelets from GpIba+/+ and GpIbaΔsig/Δsig mice, after 0-180 s stimulation
with CRP (representative of n=3). (J-L) Bar graphs displaying the levels of phosphorylated SYK, PLCg2 and Akt in platelets from GpIba+/+ and GpIbaΔsig/Δsig mice, after
0-180 s stimulation with CRP and normalizing the intensity according to the non-phosphorylated levels of SYK, PLCg2 and Akt. For the surface area (F), the data rep-
resent the median ± confidence interval (CI) and was analyzed using the unpaired Mann Whitney test. All other data is displayed as mean ± standard error of the
mean and was analyzed using two-way ANOVA followed by Sidak’s multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Also see the Online
Supplementary Figures S2 and S3.
thrombin concentration (Figure 4A and B) but this did not reduced phosphorylation of Syk (approximately 20%)
influence thrombin-induced platelet aggregation (Figure 4C (Online Supplementary Figure S4A to C). P-selectin exposure
and D). How this reduced P-selectin exposure in response in response to rhodocytin was reduced in GpIbaΔsig/Δsig
to low thrombin concentration is manifest remains unclear, platelets while aIIbb3 activation was only diminished for
but may reflect the findings of a previous study that sug- the lowest concentration of the toxin without reaching sta-
gested the importance of 14-3-3ζ binding to GPIba specifi- tistical significance (Online Supplementary Figure S4D to E).
cally for low-dose thrombin responses.40 Despite largely These results suggest that the GPIba tail may also influence
unaffected responses to ADP and thrombin, in response to CLEC-2 ITAM-mediated signaling, but perhaps with
collagen-related peptide (CRP), GpIbaΔsig/Δsig platelets exhibit- reduced dependency.
ed markedly reduced aIIbb3 activation and P-selectin expo-
sure (Figure 4A and B). Interestingly, GpIbaΔsig/Δsig platelet The role of the GPIba intracellular tail in platelet
aggregation following CRP stimulation appeared normal recruitment and aggregation under flow
(Figure 4C and D). In order to examine the consequences of the combined
Next, we evaluated the ability of GpIbaΔsig/Δsig platelets to effects of disrupted VWF-GPIba signaling and diminished
spread on fibrinogen surfaces with and without prior stim- GPVI signaling in platelets in more physiological assays, we
ulation with thrombin. Without platelet stimulation, similar quantified platelet recruitment and aggregate formation on
to wild-type platelets, most GpIbaΔsig/Δsig platelets remained collagen-coated microchannels under flow. Experiments
round while upon stimulation with thrombin approximate- were performed at high (3,000 s-1), medium (1,000 s-1) and
ly 80% platelets spread fully with no difference observed in low (200 s-1) shear, as platelet recruitment is increasingly
the spread platelet area (Online Supplementary Figure S3A to dependent on VWF-GPIba as shear increases while subse-
E). As full spreading is highly dependent upon outside-in quent platelet aggregate formation on collagen surfaces
signaling through aIIbb3,41 this suggests that this signaling becomes more dependent upon GPVI signaling.42-44
pathway is unaffected in GpIbaΔsig/Δsig platelets. We then Perfusing whole blood at 3,000 s-1 and 1,000 s-1 over colla-
explored the ability of platelets to spread on CRP-coated gen, we observed a marked reduction in surface coverage of
surfaces. Consistent with diminished platelet activation in GpIbaΔsig/Δsig platelets when compared to GPIba+/+ platelets
response to CRP, GpIbaΔsig/Δsig platelets remained round in (Figure 5A and B; Figure 6A and B; Online Supplementary
contrast to wild-type platelets (59±3.4% vs. 19±7%; Figure Videos S3 and S4). GpIbaΔsig/Δsig platelets that bound to colla-
4E and G). This effect was also quantified by a 20% reduc- gen also formed smaller aggregates than GPIba+/+ platelets
tion in bound platelet area (Figure 4F) and in the reduced (Figures 5C and 6C), likely reflecting the subsequent effect
incidence of filopodia formation – 16±6.3% for GpIbaΔsig/Δsig of diminished collagen-GPVI signaling. Perfusing wild-type
versus 52±7.1% for GPIba+/+ (Figure 4E and G). Collectively, plasma-free blood (to remove soluble VWF and fibrinogen)
these results reveal an appreciable defect in GPVI-mediated in collagen-coated microchannels revealed a significant
signaling in GpIbaΔsig/Δsig platelets. reduction of both platelet adhesion and thrombus growth
There was an overall reduction in tyrosine phosphoryla- to similar levels observed in GpIbaΔsig/Δsig samples (Figure 6A,
tion after CRP stimulation in GpIbaΔsig/Δsig platelets compared B, D and E) showing that a small amount of VWF-indepen-
to wild-type platelets (Figure 4H). Further analysis revealed dent binding to collagen occurs at 1,000 s-1. When whole
appreciably reduced Syk kinase activation in GpIbaΔsig/Δsig blood experiments were performed in the presence of
platelets, as measured by phosphorylation of Syk on GR144053, to block aIIbb3, GPIba+/+ platelets were recruit-
Tyr525 and Tyr526 in response to CRP and lower phospho- ed to the collagen surface as a monolayer. However, addi-
rylation levels of its downstream target pPLCg2 (p-Tyr tional platelet-platelet recruitment was abolished and there-
1217), although this was less marked than for those fore there was limited thrombus growth in 3D. This was
observed with pSyk (Figure 4I to K). In addition, phospho- measured by an increase in surface coverage with a
rylation levels of Akt (p-Ser 473), a known substrate of PI3K decrease in thrombus formation (i.e., total platelet fluores-
were also appreciably diminished in GpIbaΔsig/Δsig versus cence; Figure 6A, B and D).45 Surface coverage as well as
GpIbaΔsig/Δsig (Figure 4I and L). In order to assess whether the platelet accumulation of GpIbaΔsig/Δsig platelets was similar in
effect of truncation of GPIba was specific for GPVI-mediat- both the absence and presence of GR144053 (Figure 6A, B
ed platelet responses, or whether other tyrosine-mediated and E), suggesting that lack of active aIIbb3 is part of the
signaling pathways might also be affected, we stimulated platelet phenotype. In order to more specifically examine
GpIbaΔsig/Δsig and wild-type platelets with rhodocytin (C-type the role of GPVI in this system, we performed experiments
lectin receptor 2 [CLEC-2] agonist). Tyrosine-phosphoryla- in the presence of JAQ1, an anti-murine GPVI blocking anti-
tion profile of GpIbaΔsig/Δsig platelets in response to rhodocytin body. Blocking GPVI significantly reduced surface coverage
was similar to that of GPIba+/+ platelets, with slightly and platelet accumulation in GpIbaΔsig/Δsig and GPIba+/+

platelets, revealing the important contribution of GPVI sig- GPIba with truncated intracellular tail. This circumvented
naling at 1,000 s-1 (Figure 6F to I), in stabilizing platelet the limitations associated with studying/expressing
recruitment and their subsequent aggregation. platelet receptor complexes in heterologous cellular sys-
At venous shear rates (200 s-1) where the dependencies on tems. Previously generated full knockout (GPIba-/-) and
VWF and collagen are slightly different to 1,000 s-1, surface also GPIba/IL4Ra-tg mice that lack the extracellular
coverage of GpIbaΔsig/Δsig platelets was slightly reduced com- region of GPIba do not enable analysis of VWF signaling
pared to GPIba+/+ platelets although it did not reach signifi- per se, as they lack the ability to bind VWF, meaning that
cance. However, thrombus growth was significantly dimin- one cannot dissociate the effects of loss of VWF binding
ished (Figure 7A to C; Online Supplementary Video S5). Using and/or VWF signaling upon functional effects upon the
plasma-free blood, the surface coverage was similar for platelets.32,35 Transgenic mice (hTgY605X) that express human
GpIbaΔsig/Δsig and GPIba+/+ platelets, mediated by direct (VWF- GPIba that lacks the terminal 6 a.a. of the intracellular tail
independent) interaction with collagen (Figure 7A and B). displayed reduced megakaryocyte recovery following
Similar to high-shear conditions, platelet accumulation induced thrombocytopenia,49 but more recent in vitro stud-
under plasma-free conditions of GPIba+/+ platelets was sig- ies have revealed that these mice do not lack the entire 14-
nificantly reduced compared to whole blood (Figure 7D) 3-3/PI3K binding region,9,10,12 suggesting that their VWF
similar to those observed with GpIbaΔsig/Δsig platelets (Figure signaling function may not be fully disrupted making
7E). In the presence of GR144053, we saw the same interpretation of the mouse phenotype difficult.
increase in surface coverage of GPIba+/+ platelets with GpIbaΔsig/Δsig mice had a modest reduction in platelet
reduced localized 3D-platelet thrombi (Figure 7A and B) counts compared to GPIba+/+ littermates that is likely be
although the platelet accumulation was not significantly attributable to the small increase in platelet size (Figure 2A
different to GPIba+/+ whole blood (Figure 7D) likely due to and B). Interestingly, platelet size is also moderately
the increased platelet coverage. Consistent with the results increased in the GPIba/IL4Ra-tg mice,35 but, again, this is
obtained under high-shear conditions, the effect of modest compared to the size observed in GPIba-/- or in
increased surface coverage in the presence of GR144053 Bernard-Soulier platelets.32,33 Although the major filamin
was not observed with GpIbaΔsig/Δsig platelets, nor was binding site remains intact in GpIbaΔsig/Δsig mice, our find-
platelet accumulation appreciably further diminished ings may be consistent with CHO cell studies that sug-
(Figure 7A, B and E). Finally, similar to results obtained gested the presence of additional or extended filamin
under arterial shear conditions, blocking GPVI significantly binding regions within the intracellular tail of GPIba.48 By
reduced surface coverage and platelet accumulation in both themselves, the 20% reduction in platelet count and slight
GpIbaΔsig/Δsig and GPIba+/+ platelets (Figure 7F to I). As removal increase in platelet size would not impart a hemostatic
of either VWF or blocking of GPVI had very similar effects, defect.50
this suggests that VWF-GPIba and GPVI-collagen binding GpIbaΔsig/Δsig mice exhibited normal hemostatic responses
may act synergistically to recruit platelets at low shear. to tail transection, and normal thrombus formation fol-
lowing mild laser-induced thrombosis (Figure 2D to G).
We used a non-perforating endothelial cell injury that does
Discussion not induce collagen exposure. Therefore, this non-ablasive
model is independent of collagen-mediated signaling path-
The ability of platelet GPIba binding to VWF to trans- ways.36,38 However, both the tail transection and laser-
duce intraplatelet signaling is well-known, but the hemo- induced models are sensitive to VWF function.34,37 Our
static role of the platelet ‘priming’ that follows has fre- results reveal the normal VWF-binding function of
quently been perceived as redundant due to the compara- GpIbaΔsig/Δsig platelets. Normal bleeding times were also
tively mild phenotypic changes in platelets that ensue reported in hTgY605X transgenic mice with no overt effect on
when compared to other platelet agonists (e.g., thrombin, platelet or coagulation functions.49
collagen). Using a novel GpIbaΔsig/Δsig mouse, we now Truncation of the intracellular tail of GPIba did not alter
demonstrate that the intracellular tail of GPIba is impor- expression of its extracellular domain (nor influence sur-
tant not only for transduction of VWF-GPIba signaling, face expression of GPIba, GPVI or aIIbb3) (Figure 2C).
but also collagen-GPVI-mediated responses in platelets Consequently, GpIbaΔsig/Δsig platelet capture to mouse VWF-
(Figure 8). coated surfaces was unaffected as well their rolling veloc-
The binding of GPIba to VWF, and of GPVI to collagen, ities (Figure 3A to D). Despite normal VWF binding, dele-
are critical events for platelet plug formation.42,46,47 Previous tion of the PI3K and 14-3-3 binding region in GPIba9,10,12
studies reported associations between GPIba and GPVI, significantly decreased filopodia extension upon stimula-
or its co-receptor FcRg suggesting potential interplay tion of VWF binding with botrocetin but also in the pres-
between these signaling pathways.25,26,28 Functional ence of an aIIbb3 antagonist that prevent outside-in sig-
crosstalk between these signaling pathways is supported naling induced by the VWF C4 domain binding to activat-
by the diminished VWF-GPIba-dependent responses in ed aIIbb3 (Figure 3E, G to H). Normal VWF-platelet bind-
platelets deficient in GPVI and by the ability of VWF to ing in GpIbaΔsig/Δsig mice is in line with previous studies
further potentiate platelet secretion in response to showing that deletion of the 14-3-3ζ binding site in
CRP.13,27,28 human GPIba in GPIb-IX CHO cells does not influence
In order to explore GPIba signaling function and its VWF binding, but does reduce their ability to spread.9,51
influence upon GPVI signaling, we generated GpIbaΔsig/Δsig Other studies showed that a membrane-permeable
mice by introduction of a stop codon downstream of the inhibitor of the 14-3-3ζ-GPIba interaction (MP-aC) inhib-
main filamin binding site (a.a. 668-681), but upstream of ited GPIba-dependent platelet agglutination and was pro-
the 14-3-3 isoforms and PI3K binding regions that are tective in murine thrombosis models.11,52 However,
important for VWF-GPIba signaling.8-12,48 (Figure 1) This although this peptide disrupts the interaction between 14-
resulted in uniform production of platelets that express 3-3ζ and GPIba, it may also influence 14-3-3ζ function

independent of GPIba binding. This contention is perhaps centrations.57,58 Taken together, previous studies support
supported by a recent study revealing that 14-3-3ζ defi- the contention that Bernard-Soulier patient platelets
cient mice are protected against arterial thrombosis with exhibit a partial deficit in GPVI signaling that resembles
normal VWF-GPIba-mediated platelet function.53 the deficit in GpIbaΔsig/Δsig mouse platelets.
In addition to defective VWF-mediated signaling, Platelets can interact with collagen directly through
GpIbaΔsig/Δsig platelets exhibited markedly diminished colla- GPVI and a2b1, and indirectly via GPIba binding to VWF,
gen-mediated signaling through GPVI evidenced by the latter being increasingly important as shear rates rise to
reduced surface expression of P-selectin and activation of first capture the platelets and enable the aforementioned
aIIbb3, fewer filopodia upon CRP stimulation (Figure 4A, direct interactions to take place.42,59 This is demonstrated in
B, E to G), and severely diminished platelet aggregate for- wild-type mice, similar to previous reports,43,60 by the
mation on collagen under venous and arterial shears markedly reduced binding of platelets to collagen in the
(Figures 5 to 7). Bernard-Soulier patient platelets have his- absence of plasma (and therefore VWF) at medium shear
torically been reported to respond normally to collagen in rates (Figure 6A, B and D). Although we demonstrated that
aggregation assays.54 However, the thrombocytopenia and GpIbaΔsig/Δsig platelets bind VWF normally, we saw the
giant platelets associated with full GPIba deficiency com- largest defect in platelet coverage/accumulation when
bined with the loss of VWF-dependent platelet recruit- compared to wild-type mice at 3,000 s-1 (Figure 5). Based on
ment on collagen impair full analysis of other platelet sig- these results, it seems likely that VWF-GPIba signaling is
naling pathways under physiological flow conditions. also important at these high shear rates, similar to the
Interestingly, although early studies on Bernard-Soulier importance of GPIba binding to VWF for platelet tethering.
patients reported that platelet aggregation in response to We therefore contend that under medium/high shear con-
collagen was normal, their transformation into procoagu- ditions, VWF-GPIba platelet priming induces some rapid
lant platelets was specifically impaired in response to col- activation of aIIbb3, which enable the platelets to better
lagen (but not other agonists).55 More recently, a Bernard- withstand the higher shear rates, prior to their interac-
Soulier patient with mutations in both GPIba and filamin tion/activation by collagen (Figure 8). Although most evi-
A was also reported to exhibit defects in GPVI-mediated dent at the highest shear rates, GpIbaΔsig/Δsig platelets exhib-
signaling responses.56 Although the authors contended ited reduced accumulation at venous shear rates (Figure
that this defect might be due to the filamin A mutation, 7C). Given that the surface coverage on collagen was not
this may warrant some reappraisal in light of the data pre- significantly altered at 200 s-1 in GpIbaΔsig/Δsig platelets com-
sented herein. Like Bernard-Soulier platelets, we found pared to wild-type platelets (Figure 7A and B), the deficit in
that GpIbaΔsig/Δsig platelets aggregated normally in response subsequent platelet accumulation must be due to reduced
to CRP (Figure 4C to D). The signaling deficit presumably reactivity of GpIbaΔsig/Δsig platelets. This is supported by the
allows sufficient activation of aIIbb3 for the platelets to clear importance of aIIbb3 activation to this assay, demon-
aggregate. This is perhaps unsurprising given that Gp6+/- strated by the effects of GR144053 in preventing 3D accu-
platelet aggregation is only affected at low collagen con- mulation of platelets at both 200 s-1 and 1,000 s-1 in wild-
B C
Figure 5. GpIbaΔsig/Δsig platelets have a reduced ability to

bind to collagen and form microthrombi at 3,000 s-1.
Hirudin anticoagulated whole blood from GpIba+/+ and
GpIbaΔsig/Δsig mice was labeled with anti-GPIba-DyLight488
antibody and perfused over fibrillar collagen type I (0.2
mg/mL) at a shear rate of 3,000 s-1 for 3 minutes. (A)
Representative fluorescence images (n=6) after 3 min-
utes of perfusion in whole blood (WB) from GpIba+/+ and
GpIbaΔsig/Δsig mice at 3,000 s-1. Platelet deposition (B) and
thrombus build-up measured as integrated fluorescence
intensity (IFI) (C). All data is shown as mean ± standard
error of the mean and analyzed using unpaired two-tailed
student’s t-test The maximal platelet IFI was used to com-
pare the thrombus build up data. *P<0.05, ***P<0.001.
Scale bar 100 mm. Also see the Online Supplementary
Video S3.

type platelets (Figure 6A to D; Figure 7A to D). We also GpIbaΔsig/Δsig platelets (Figure 6A, B, D and E; Figure 7A, B, D
observed an increase in the platelet coverage in wild-type and E), demonstrating a lack of aIIbb3 activation that
platelets in the presence of the aIIbb3 blocker. This is in would be consistent with a diminished GPVI-mediated sig-
line with our previous study and others showing that naling response. It is important to note that this response is
aIIbb3 blockade allows the formation of a platelet mono- diminished, rather than ablated as the addition of JAQ1 led
layer, but prevents thrombus growth in 3D and also lateral to a marked decrease in both platelet tethering and accu-
platelet-platelet aggregation (Figure 6B; Figure 7B).5,45,61,62 mulation at both 1,000 s-1 and 200 s-1 shear rates (Figure 6F
This underscores the importance in quantifying both to I; Figure 7F to I). The question remains open as to the
platelet coverage and accumulation in flow assays when precise contribution of VWF-GPIba versus collagen-GPVI
studying platelet signaling defects.45,61 Importantly, signaling deficits to the phenotype of GpIbaΔsig/Δsig platelets.
GR144053 did not alter these parameters when added to Our data suggest that both signaling pathways likely con-
A B C
D E
G H I
Figure 6. GpIbaΔsig/Δsig platelets have a reduced ability to bind to collagen and form microthrombi at 1,000 s-1. (A to E) Hirudin anticoagulated whole blood supple-
mented or not with GR144053 or plasma-free blood from GpIba+/+ and GpIbaΔsig/Δsig mice was labeled with anti-GPIba-DyLight488 antibody (Ab) and perfused over
fibrillar collagen type I (0.2 mg/mL) at a shear rate of 1,000 s-1 for 3 minutes (min). (A) Representative fluorescence images (n≥3) after 3 min of perfusion in whole
blood (WB), plasma-free blood (PFB) or WB + GR144053 from GpIba+/+ and GpIbaΔsig/Δsig mice. Platelet deposition (B) and thrombus build-up measured as integrated
fluorescence intensity (IFI) (C to E). All data is shown as mean ± standard error of the mean and analyzed using unpaired two-tailed student’s t-test (C) or one-way
ANOVA followed by Dunnett’s multiple comparison test (B, D to E). Data is compared to means from GpIba+/+ WB (B and D) or GpIbaΔsig/Δsig WB (E). The maximal platelet
integrated fluorescence intensity (IFI) was used to compare the thrombus build up data. *P<0.05. Scale bar 100 mm. Also see the Online Supplementary Video S4.
(F to I) Hirudin anticoagulated whole blood from GpIba+/+ and GpIbaΔsig/Δsig mice supplemented with JAQ1 or Rat-IgG control Ab (20 mg/mL) was labeled with anti-GPIbα-
DyLight488 Ab and perfused over fibrillar collagen type I (0.2 mg/mL) at a shear rate of 1,000 s-1 for 3 min. (F) Representative fluorescence images (n=3) after 3
min of perfusion. Platelet deposition (G) and thrombus build-up measured as IFI (H and I). All data is shown as mean ± standard error of the mean and analyzed
using unpaired two-tailed student’s t-test. The maximal platelet IFI was used to compare the thrombus build up data. *P<0.05, **P<0.01. Scale bar 100 mm.

tribute to this, as disruption of either interaction causes a GpIbaΔsig/Δsig platelets via CLEC-2, another receptor that sig-
major reduction in platelet accumulation in wild-type nals via an ITAM motif,64 was also affected, but perhaps to
platelets under both venous and arterial shear rates. a lesser extent than those mediated by GPVI (Online
GPVI belongs to the immunoglobulin superfamily and Supplementary Figure S4) suggesting that the function of the
signals via tyrosine kinase phosphorylation pathways. In GPIba intracellular tail is more important for GPVI mediat-
order to further investigate the defect in GPVI signaling in ed responses. Based on these findings, we hypothesize that
GpIbaΔsig/Δsig platelets, analysis of tyrosine phosphorylation the tail of GPIba may be important for the docking of sig-
downstream of GPVI revealed that SYK and PLCg2 phos- naling molecules such as SYK, LAT and PLCg2 that are
phorylation was reduced in GpIbaΔsig/Δsig platelets (Figure 4H downstream of GPVI and CLEC-2 on ITAM phosphorylat-
to K). Interestingly, the diminished phosphorylation was ed motif of the FcRg and CLEC-2 receptors and warrant
more pronounced for SYK than for PLCg2 perhaps high- further investigation. It would also be of interest to deter-
lighting the existence of LAT-independent mechanisms of mine if the reduction in PI3K signaling in response to CRP
PLCg2 phosphorylation.63 Interestingly, activation of stimulation (Figure 4I to L) is due to the lack of binding of
A B C
D E
G H I
Figure 7. GpIbaΔsig/Δsig platelets have a reduced ability to bind to collagen and form microthrombi at 200 s-1. (A to E) Hirudin anticoagulated whole blood supplement-
ed or not with GR144053 or plasma-free blood from GpIba+/+ and GpIbaΔsig/Δsig mice was labeled with anti-GPIba-DyLight488 Ab and perfused over fibrillar collagen
type I (0.2 mg/mL) at a shear rate of 200 s-1 for 3 minutes (min). (A) Representative fluorescence images (n≥3) after 3 min of perfusion in whole blood (WB), plas-
ma-free blood (PFB) or WB + GR144053 from GpIba+/+ and GpIbaΔsig/Δsig mice. Platelet deposition (B) and thrombus build-up measured as integrated fluorescence
intensity (IFI) (C to E). All data is shown as mean ± standard error of the mean and analyzed using unpaired two-tailed student’s t-test (C) or one-way ANOVA followed
by Dunnett’s multiple comparison test (B, D to E). Data is compared to means from GpIba+/+ WB (B and D) or GpIbaΔsig/Δsig WB (E). The maximal platelet integrated flu-
orescence intensity (IFI) was used to compare the thrombus build-up data. *P<0.05, **P<0.01. Scale bar 100 mm. Also see the Online Supplementary Video S5. (F
to I) Hirudin anticoagulated whole blood from GpIba+/+ and GpIbaΔsig/Δsig mice supplemented with JAQ1 or Rat-IgG control antobodies (20 mg/mL) was labeled with anti-
GPIba-DyLight488 Ab and perfused over fibrillar collagen type I (0.2 mg/mL) at a shear rate of 200 s-1 for 3 min. (F) Representative fluorescence images (n=3) after
3 min of perfusion. Platelet deposition (G) and thrombus build-up measured as IFI (H and I). All data is shown as mean ± standard error of the mean and analyzed
using unpaired two-tailed student’s t-test. The maximal platelet IFI was used to compare the thrombus build up data. *P<0.05. Scale bar 10 mm.

Figure 8. Proposed model for GPIba-GPVI cross talk. Under normal conditions, resting/circulating platelets (1) present aIIbb3 on their surface in its closed conforma-
tion. Plasma von Willebrand factor (VWF) (2) circulates in its globular conformation with its A1 domain hidden, preventing interaction with platelet GPIba. Upon vascular
injury, the subendothelial extracellular matrix containing collagen becomes exposed to the blood. VWF, via its A3 domain, binds to collagen and, due to shear forces,
unravels to expose its A1 domain to which platelet GPIba binds (3). Next, mechanosensitive signaling events downstream of VWF A1-GPIba that require the intracellular
tail of GPIba take place leading to some activation of surface aIIbb3 (4) while the deceleration of platelets allows for the subsequent binding of platelets to collagen
via several collagen receptors including GPVI (6). The intracellular tail of GPIbα is also crucial for optimal collagen/GPVI signaling that lead to platelet activation, shape
change and granule release (7). Ultimately, additional circulating platelets will be recruited at the site of injury to form the hemostatic plug (8).
PI3K to the intracellular tail of GPIba or it is a consequence revised the manuscript; KJW designed and performed experi-
of diminished SYK phosphorylation.65 ments and revised the manuscript; PM and KV provided critical
In summary, we generated a novel GPIba transgenic reagents and revised the manuscript; JTBC designed experi-
mouse in which their platelets bind VWF normally, but ments, prepared the figures and wrote the manuscript; IIS-C
the subsequent VWF-GPIba signaling is disrupted. designed and performed experiments, analyzed data, prepared
Intriguingly, these mice clearly reveal the molecular link the figures and wrote the manuscript.
between GPIba- and GPVI-mediated signaling in platelets
and underscore the cooperative functions of these two Acknowledgements
major platelet receptors.45 Platelets in addition to their The authors acknowledge the technical assistance of Alisha
important role in thrombosis and hemostasis contribute to Miller, Elodie Ndjetehe, Ben Moyon and Zoe Webster from
the host response to infection and inflammation.66-69 Our Central Biomedical Services and MRC transgenic group at
recent work suggests that VWF-GPIba-dependent platelet Imperial College. We thank the LMS/NIHR Imperial
priming potentiates the recruitment of neutrophils, which Biomedical Research Center Flow Cytometry Facility for support.
may represent a key early event in the targeting of We would like to thank Sooriya Soman, Dr Pavarthi Sasikumar,
pathogens, but also in the development of deep vein Dr Claire Peghaire at Imperial College for technical assistance,
thrombosis.5 The GpIbaΔsig/Δsig mice now provide an invalu- and Nilanthi Karawitage at Imperial College Healthcare NHS
able tool to probe the importance of the GPIba-mediated trust for the use of the aggregometer. We are grateful to Professor
signaling in inflammatory diseases such as atherosclerosis Johannes A. Eble (University of Münster) and Dr Craig E.
and deep vein thrombosis, as well as in the host response Hughes (University of Reading) for providing rhodocytin.
to infection but also to fully decipher the molecular
dependency of GPVI signaling upon GPIba. Funding
This work was supported by the British Heart Foundation
Disclosures grants FS/15/65/32036, PG/17/22/32868 and
No conflicts of interest to disclose. RG/18/3/33405.
Contributions Data sharing statement

AC-B designed and performed experiments, analyzed data Additional information on original data and protocols will be
and wrote the manuscript; YAW performed experiments and available upon request via email i.salles@imperial.ac.uk.
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Platelet Biology & its Disorders ARTICLE
Comparative analysis of ChAdOx1 nCoV-19 Ferrata Storti Foundation

and Ad26.COV2.S SARS-CoV-2 vector
vaccines
Stephan Michalik,1* Florian Siegerist,2* Raghavendra Palankar,3* Kati
Franzke,4* Maximilian Schindler,2 Alexander Reder,1 Ulrike Seifert,5 Clemens
Cammann,5 Jan Wesche,3 Leif Steil,1 Christian Hentschker,1 Manuela Gesell-
Salazar,1 Emil Reisinger,6 Martin Beer,7# Nicole Endlich,2# Andreas
Greinacher3# and Uwe Völker1#
1
Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Haematologica 2022
Ge-nomics, University Medicine Greifswald, Greifswald; 2Institute for Anatomy and Cell Volume 107(4):947-957
Biology, University Medicine Greifswald, Greifswald; 3Institute of Transfusion Medicine,
University Medicine Greifswald, Greifswald; 4Institute of Infectiology, Friedrich-Loeffler-
Institut, Greifswald-Insel Riems; 5Friedrich Loeffler-Institute of Medical Microbiology-
Virology, University Medicine Greifswald, Greifswald; 6Division of Tropical Medicine and
Infectious Diseases, Center of Internal Medicine II, Rostock University Medical Center,
Rostock, and 7Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel
Riems, Germany
*SM, FS, RP and KF contributed equally as co-first authors
#
MB, NE, AG and UV contributed equally as co-senior authors
ABSTRACT
V
ector-based SARS-CoV-2 vaccines have been associated with vac-
cine-induced thrombosis with thrombocytopenia syndrome
(VITT/TTS), but the causative factors are still unresolved. We
comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and
Ad26.COV2.S (Johnson & Johnson) vaccines. ChAdOx1 nCoV-19 con-
tains significant amounts of host cell protein impurities, including func-
tionally active proteasomes, and adenoviral proteins. A much smaller
amount of impurities was found in Ad26.COV2.S. Platelet factor 4
formed complexes with ChAdOx1 nCoV-19 constituents, but not with Correspondence:
purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. ANDREAS GREINACHER,
Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not andreas.greinacher@med.uni-greifswald.de
by Ad26.COV2.S. These differences in impurities together with EDTA-
UWE VÖLKER
induced capillary leakage might contribute to the higher incidence rate of voelker@uni-greifswald.de
VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.
Received: October 5, 2021.
Introduction Accepted: January 5, 2022.
Pre-published: January 20, 2022.
Vaccination is key for the control of the severe acute respiratory syndrome
coronavirus-2 (SARS-CoV-2) pandemic. Adenoviral vector-, mRNA encapsulated
in lipid nanoparticles-, and antigen-based vaccines are currently in use, all encoding https://doi.org/10.3324/haematol.2021.280154
the spike protein.1,2 Since February 2021 the rare but severe adverse reaction of vac-
cine-induced immune thrombotic thrombocytopenia (VITT; synonym thrombosis
with thrombocytopenia syndrome [TTS]) has been observed in individuals vaccinat-
ed against SARS-CoV-2. VITT/TTS occurs 5-20 days (occasionally later) after vacci- Material published in Haematologica is covered by copyright.
nation with the ChAdOx1 nCoV-19 vaccine (produced by AstraZeneca) and the All rights are reserved to the Ferrata Storti Foundation. Use of
Ad26.COV2.S vector vaccine (produced by Janssen/Johnson & Johnson). The inci- conditions:
dence rate of VITT/TTS seems to be higher for ChAdOx1 nCoV-19. The reported https://creativecommons.org/licenses/by-nc/4.0/legalcode.
rate of VITT/TTS in the USA is 0.355 cases per 100,000 people vaccinated with Copies of published material are allowed for personal or inter-
Ad26.COV2.S,3 compared to 1 per 50,000-100,000 people vaccinated with ChAdOx1 nal use. Sharing published material for non-commercial pur-
nCoV-19 in the UK.4 In Germany both vaccines were used and within this medical https://creativecommons.org/licenses/by-nc/4.0/legalcode,
system, 0.56 suspected cases per 100,000 vaccine doses for Ad26.COV2.S (3,186,297 sect. 3. Reproducing and sharing published material for com-
vaccine doses administered) and 1.49 suspected cases per 100,000 vaccine doses for mercial purposes is not allowed without permission in writing
ChAdOx1 nCoV-19 (12,692,700 vaccine doses administered) were reported.5 from the publisher.
VITT/TTS involves high-affinity, platelet-activating anti-platelet factor 4 (PF4) anti-
bodies,6–8 but the mechanisms triggering these anti-PF4 antibodies are still unre-

S. Michalik et al.
solved. VITT/TTS shows striking similarities with another precast NuPAGE™ 4 to 12% gel. Electrophoresis was performed
PF4-mediated adverse drug effect, heparin-induced throm- at 150 V. The western blots were prepared using the Trans-Blot
bocytopenia (HIT) and autoimmune HIT. In HIT, polyan- Turbo Transfer System from BioRad and the transfer of proteins to
ions form complexes with PF4, inducing a conformational the polyvinylidene fluoride membrane was verified and docu-
change, which triggers anti-PF4 antibodies. This immune mented using LICOR's Revert Total Protein Stain protocol (Doc #
response is pronounced in patients with tissue trauma and 988-19494). The specific proteins were detected with primary and
inflammation. We have shown that one or more con- secondary antibodies described in the Online Supplementary
stituents of the ChAdOx1 nCoV-19 vaccine interact with Methods.
PF4, forming complexes which contain PF4 and the aden-
ovirus hexon protein. This might trigger conformational Proteasome activity assays
changes in the positively charged chemokine PF4 leading to Chymotrypsin-like activity was assessed in vaccine or
the formation of a neo-antigen and then subsequent activa- HEK293T cell lysate using 0.2 mM fluorescently tagged Suc-LLVY-
tion of B cells in a pro-inflammatory environment.9 These AMC (Bachem, Bubendorf, Switzerland) quantified with a fluo-
activated B cells then produce high avidity anti-PF4 anti- rometer using a 380/460 nm filterset. To confirm proteasomal
bodies that bind PF4 and trigger an activation cascade of activity 100 nM bortezomib14 or 200 nM carfilzomib
platelets and granulocytes, leading to NETosis and massive (Selleckchem, Houston, TX, USA)15, was added.
thrombin production. However, it is not known which vac-
cine components, beside the hexon protein, interact with Dynamic light scattering and zeta potential
PF4 and which additional factors influence this interaction. All dynamic light scattering measurements were performed in a
Both vaccines (ChAdOx1 nCoV-19 and Ad26.COV2.S) fixed scattering angle Zetasizer Nano-S system (Malvern
are produced in human cell lines, T-REx-293 cells (human Instruments Ltd., Malvern, UK). The hydrodynamic diameter
embryonic kidney cells, a HEK293 derivate) for ChAdOx1 (nm) was measured at 25°C, and light scattering was detected at
nCoV-19 and PER.C6 TetR cells (human embryonic retinal 173°. Surface 𝜁 potential was performed in folded capillary 𝜁 cells
cells) for Ad26.COV2.S. We and others have previously (DTS1070, Malvern Instruments Ltd., Malvern, UK). Data were
shown that the ChAdOx1 nCoV-19 vaccine contains a large analyzed using Zetasizer software, version 7.13 (Malvern
number of host cell proteins (HCP).9,10 Here we report the Instruments Ltd., Malvern, UK).
results of a comprehensive, comparative analysis of the
ChAdOx1 nCoV-19 and Ad26.COV2.S vaccines, using pro- Immunoelectron and transmission electron microscopy
teomics, transmission electron microscopy, dynamic light- Vaccines or the purified adenovirus particles were incubated
scattering, single-molecule light microscopy, and an in vivo with biotinylated PF4 and transferred to formvar-coated transmis-
capillary leakage assay. sion electron microscopy grids. After washing, samples were
Our data reveal substantial differences in composition labeled with an anti-adenovirus monoclonal antibody detected by
and functional properties between the two vaccines, which a gold conjugate. The same samples were labeled with a strepta-
may contribute to the different incidences of VITT/TTS. vidin-gold conjugate to detect PF4-biotin. Grids were stained with
1% phosphotungstic acid and analyzed with a Tecnai-Spirit trans-
mission electron microscope (FEI, Eindhoven, Germany).
Methods
Super resolution single-molecule light microscopy
Comprehensive details of the Methods are described in the Diluted vaccine or purified virions were incubated with human
Online Supplementary Material. PF4 and immobilized on cleaned coverslips. After fixation and
All experiments were performed in accordance with local and blocking, PF4 and adenoviral hexon were visualized using second-
national ethics standards and German animal protection legisla- ary (PF4) and primary (hexon) immunofluorescence detected by
tion, overseen by the “Landesamt für Landwirtschaft, Alexa Fluor 488 and Cy5. Coverslips were mounted in Everspark
Lebensmittelsicherheit und Fischerei, Rostock” of the federal state dSTORM buffer (Idylle Labs, France)16 and blinking sequences
of Mecklenburg - Western Pomerania. imaged on a Zeiss Elyra PS.1 super resolution system. Single-mol-
ecule localization microscopy data were processed in FIJI using
Sample preparation and liquid chromatography tandem NanoJ core17 and Thunderstorm18 and analyzed using custom FIJI19
mass spectrometry. scripts.
Vaccines were precipitated using salt-acetone precipitation.11
Adenovirus particles were purified using subsequent sucrose- Zebrafish vascular permeability assay
cushion and sucrose-gradient ultracentrifugation. Protein was A novel zebrafish-based in vivo assay was developed to deter-
digested with trypsin as described by Blankenburg et al.12 Liquid mine local changes of vascular permeability following intramuscu-
chromatography tandem mass spectrometry (LC-MS/MS) exper- lar injections: Transgenic zebrafish at 5 days post-fertilization
iments were performed on an Orbitrap ExplorisTM 480 mass spec- expressing a 78 kDa GFP-tagged plasma protein20,21 were injected
trometer (Thermo Scientific, Bremen, Germany) coupled to an intramuscularly with 1 nL of native vaccine, purified virions, 100
UltimateTM 3000 RSLCnano HPLC (Dionex/ Thermo Scientific, µM EDTA or 0.9% NaCl. Fluorescence intensity ratios (intravas-
Waltham, MA, USA). The mass spectrometry proteomics data cular vs. intramuscular) were measured in the direct vicinity of the
have been deposited with the ProteomeXchange Consortium via injection site at t=0 and t=10 min.
the PRIDE18 partner repository13 with the dataset identifier
PXD027344.
Results
Sodium dodecylsulfate gel electrophoresis and western
blot analysis Comparative profiling of ChAdOx1 nCoV-19 and
For protein separation, one-fiftieth of one vaccine dose, as well Ad26.COV2.S vaccines
as dilutions of HEK293 total protein lysate, were loaded onto a Comparative profiling of ChAdOx1 nCoV-19 and

Comparative analysis of SARS-CoV-2 vector vaccines
Ad26.COV2.S (three different lots each) consistently host cell-derived human proteins. A dilution series of a lab-
revealed significant differences: (i) the total protein concen- oratory HEK293 cell line lysate, which was used instead of
tration of the ChAdOx1 nCoV-19 vaccine was approxi- the cell line used for vaccine production, confirmed the
mately 3.4-times higher than that of the Ad26.COV2.S vac- quantities of host-cell proteins (54% for ChAdOx1 nCoV-
cine (mean: 102 ng/µL vs. 29.8 ng/µL) (Figure 1A); (ii) silver- 19 and 1.5% for Ad26.COV2.S) (Online Supplementary Figure
nitrate staining of vaccines separated by sodium dodecyl- S3). None of the top ten most abundant human proteins in
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ChAdOx1 nCoV-19 was detected in Ad26.COV2.S (Online
displayed a markedly more complex protein pattern than Supplementary Table S2). Adenoviral proteins accounted for
expected for pure virions for ChAdOx1 nCoV-19 compared 40.8-55.5% (ChAdOx1 nCoV-19) and 99.04-99.74%
to Ad26.COV2.S (Figure 1B). (iii) mass spectrometric analy- (Ad26.COV2.S) of total ion intensity, while the SARS-CoV-
sis (Online Supplementary Table S1) identified a much higher 2 spike protein was detected only in the ChAdOx1 nCoV-
proportion (44.5% to 59.2% vs. only 0.26% to 0.96%) 19 vaccine (3 different lots) (Online Supplementary Figure S1).
(Figure 1D, Online Supplementary Figures S1 and S2) and Western blot analysis confirmed the significant abundance
number (N = 1571±31 vs. N
ChAdOx1 nCoV-19 = 59±14; two-
Ad26.COV2.S of 11 selected host-cell proteins in the ChAdOx1 nCoV-19
sided t-test P=8.709x10-6) (Online Supplementary Figure S2) of vaccine, which even exceeded the abundances detected in
A B C
Figure 1. Analysis of the protein composition of ChAdOx1 nCoV-19 and Ad26.COV2.S vaccines. (A) Determination of the protein concentration of the two vaccines
(3 different lots each). Protein concentration was determined with a quantitative bicinchoninic acid (BCA) assay. Protein concentration per 500 µL (vaccination dose)
and 1 µL vaccine (secondary axis) of three lots of ChAdOx1 nCoV-19 or Ad26.COV2.S vaccine, respectively, are shown. Statistical testing was performed using a two-
sided t-test. (B) Protein patterns of silver nitrate-stained sodium dodecylsulfate polyacrylamide gel electrophoresis of ChAdOx1 nCoV-19 or Ad26.COV2.S (3 lots each)
vaccines along with a dilution series of a laboratory HEK293 cell line extract for comparison. The HEK293 cell extract was loaded onto the gel at 1.5, 1.0, 0.5, or
0.25 µg per lane, and 10 µL (1/50th of a vaccine dose) were used for each vaccine. (C) Western blot analysis of HSP90-a protein, using the same gel loading scheme
as for the silver nitrate-stained gel. (D) intensity-based absolute quantification (iBAQ) protein intensities and theoretical molecular mass of identified proteins. Protein
intensities of ChAdOx1 nCoV-19 or Ad26.COV2.S (exemplarily shown for lot 3) were calculated using the iBAQ algorithm (minimum of 3 unique peptides per protein)
and plotted against the theoretical molecular mass. Proteins are color-coded according to their respective class. Blue dots indicate vector proteins; gray dots repre-
sent human proteins; the red dot indicates the SARS-CoV-2 spike protein. Points highlighted with a cross indicate proteins additionally analyzed by western blotting
in Online Supplementary Figures S4-S7.

S. Michalik et al.
the HEK293 cell line. None of these proteins was detected varied in the different lots according to the proteasome sub-
in the Ad26.COV2.S vaccine (Figure 1C, Online unit expression level (Figure 2A, C). Inhibition of the pro-
Supplementary Figure S4). teasome activity by 100 nM bortezomib14 or 200 nM carfil-
In summary, per vaccine dose (500 µL) of ChAdOx1 zomib15 confirmed assay specificity (Online Supplementary
nCoV-19 we detected 19.1-33.8 µg host-cell proteins and Figure S6).
23.3-26.3 µg chimpanzee adenovirus proteins and for the
Ad26.COV2.S vaccine 0.04-0.19 µg host-cell proteins and Platelet factor 4-vaccine cluster formation
10.2-19.2 µg adenoviral proteins. Since the approximately PF4 is the key protein involved in the immune response
5x1010 virions per vaccine dose weigh about 12.5 µg, both causing VITT. We assessed the interaction of PF4 with the
vaccines contain unassembled virus proteins, mostly hexon native vaccines and the purified adenoviral particles of
proteins. However, the amount of approximately 10-14 µg ChAdOx1 nCoV-19, which were obtained by sucrose cush-
unassembled virus proteins in ChAdOx1 nCoV-19 was ion and gradient ultracentrifugation. The purity of isolated
again much larger compared to the 0-6.5 µg in ChAdOx1 nCoV-19 virions was confirmed by transmission
Ad26.COV2.S. electron microscopy and silver-staining of one-dimensional
SDS-PAGE (Online Supplementary Figures S7 and S8).
Proteasome activity in the different vaccines Dynamic light scattering confirmed PF4-induced cluster-
Proteasome subunits were identified by mass spectrome- ing of the non-purified ChAdOx1 nCoV-19 vaccine (Figure
try and verified by western blot analysis (Figure 2A, Online 3A, left panel). The hydrodynamic diameter increased from
Supplementary Figure S5). Chymotrypsin-like activity associ- 88±2.4 nm up to 151±12 nm and 320±45 nm with 10
ated with the proteasomal b-5 subunit showed lot-depen- µg/mL and 50 µg/mL PF4, respectively (Figure 3A; the PF4
dent high levels in ChAdOx1 nCoV-19, while in dose-dependent size change is shown in Online
Ad26.COV2.S only minimal proteasome activity was Supplementary Figure S9). This complex formation was
found in one of three lots (Figure 2B, C). Substrate turnover reversible upon the addition of unfractionated heparin and
A B
Figure 2: Analysis of proteasome proteins and activity in the vaccines. (A)

Western blot analysis of proteasome 20S subunit beta 5 (the original western
blot image is provided in Online Supplementary Figure S4). (B) Fifty microliters
(1/10th of the vaccination dose) of different ChAdOx1 nCoV-19 (n=5) and
Ad26.COV2.S (n=3) lots were analyzed for the chymotrypsin-like activity of the
proteasome and compared with the activity of 0.25 µg of HEK293 cell lysate.
The mean ± standard deviation of two technical replicates are shown. (C)
Proteasomal activity was confirmed by inhibition with 100 nM bortezomib. The
mean of two technical replicates is shown.

can be attributed to weakened electropositive surface virions (𝜁 potential -1.7±4.7) and the untreated
potential of PF4 in the presence of highly negatively Ad26.COV2.S vaccine (𝜁 potential -4.5±5.7) showed only a
charged unfractionated heparin thus decreasing its ability to minimal negative charge (Figure 3B).
interact electrostatically with vaccine components. In con- Consistent with dynamic light scattering findings and as
trast, the addition of PF4 (50 µg/mL) only marginally described before,6 PF4 induced the formation of electron-
increased the particle size when incubated with purified dense aggregates with ChAdOx1 nCoV-19 (Figure 4A),
virions from ChAdOx1 nCoV-19 (from 79.6±10.3 nm to which contained unassembled hexon proteins (Online
86.7±6.22 nm; P=0.2404) (Figure 3A, middle panel) or the Supplementary Figure S11). In contrast, no comparable aggre-
Ad26.COV2.S vaccine (from 85.7±2.2 nm to 91.3±2.83 nm; gates were detected after incubation of PF4 with purified
P=0.0620) (see particle size-frequency distribution plots in virions from ChAdOx1 nCoV-19 (Figure 4B) or the
Online Supplementary Figure S10). Ad26.COV2.S vaccine (Figure 4C).
Complex formation of PF4 with the ChAdOx1 nCoV-19 PF4 single-molecule density analysis using single-mole-
vaccine was charge-dependent, as the negative charge of cule localization microscopy (Online Supplementary Figures
ChAdOx1 nCoV-19 (𝜁 potential -27.5±4.7) was neutralized S12 and S13) revealed that PF4 clusters formed on or in
by PF4. In comparison, both, purified ChAdOx1 nCoV-19 close vicinity to ChAdOx1 nCoV-19 adenoviral hexon pro-
B
Figure 3. Dynamic light scattering analysis of vaccine-
induced platelet factor 4 clustering. Analysis of
ChAdOx1 nCoV-19 vaccine, purified ChAdOx1 nCoV-19
virions and Ad26.COV2.S vaccine by dynamic light
scattering. (A) The hydrodynamic diameter (mean ±
standard deviation [SD], n=9) of ChAdOx1 nCoV-19 or
Ad26.COV2.S particles before and after addition of 10
µg/mL or 50 µg/mL platelet factor 4 (PF4) was deter-
mined. A dose-dependent increase in the size of
ChAdOx1 nCoV-19 aggregates in the presence of PF4
was detected. This effect was markedly reduced for
both purified ChAdOx1 nCoV-19 virions and
Ad26.COV2.S. Addition of unfractionated heparin
(UFH; 1 IU/mL) dissociated complexes between PF4
and vaccine components. (B) The ζ-potential (mean ±
SD, n=9) of ChAdOx1 nCoV-19 was lower than the puri-
fied ChAdOx1 nCoV-19 virions or of Ad26.COV2.S; and
largely neutralized by PF4. In the presence of UFH, a
charge reversal to net negative charge was observed
in both vaccines and purified virions from ChAdOx1
nCoV-19 vaccine. Statistical analysis was performed
by one-way analysis of variance on ranks/Kruskal-
Wallis followed by correction for multiple comparison
by two-stage Benjamini, Krieger, & Yekutieli controlling
the false discovery rate procedure (n=9) and a <0.05
was considered significant.

S. Michalik et al.
teins (PF4 single-molecule density ratio 7.09±1.38, Figure microscopy analysis revealed absence of intact virions in
4D, G; arrowheads in Online Supplementary Figure S12), but the supernatant fraction of both ChAdOx1 nCoV-19 and
not on Ad26.COV2.S (1.13±0.14, P<0.0001) or purified Ad26.COV2.S, while the pellet was enriched in virions
ChAdOx1 nCoV-19 virions (2.33±0.44, P=0.0115, mean ± (Figure 5A, B). Intriguingly, we observed amorphous elec-
standard error of mean; P-values refer to comparison with tron-dense irregularly shaped particulate material in
ChAdOx1 nCoV-19) (Figure 4D-G). ChAdOx1 nCoV-19 supernatant that was absent in
Dynamic light scattering experiments with ultracen- Ad26.COV2.S. No significant PF4-dependent complex
trifugation-separated virions from the vaccines and the formation was detected by dynamic light scattering with
resulting supernatant confirmed the single-molecule den- the pellet fractions of ChAdOx1 nCoV-19 and
sity analysis of PF4 binding (Figure 5). Electron Ad26.COV2.S, but the supernatant fraction of ChAdOx1
A B C
D E F
Figure 4. Ultrastructural analysis of vaccine-induced platelet factor 4 clustering.

(A-C) Representative micrographs of streptavidin-gold immunoelectron
microscopy (arrow); biotinylated, human platelet factor 4 (PF4) was incubated
with either ChAdOx1 nCoV-19 (A), purified virions from ChAdOx1 nCoV-19 (B) or
Ad26.COV2.S (C). Virions are exemplarily labeled by asterisks, and immunogold-
labeled aggregates by arrowheads; scale bars represent 200 nm. (D-F) Single-
molecule light microscopy show representative dual PF4 and hexon polypeptide
reconstructions after incubation with either ChAdOx1 nCoV-19 (D), purified viri-
ons from ChAdOx1 nCoV-19 (E) or Ad26.COV2.S (F). As indicated by the green
signal in close proximity to adenoviral particles in (D), PF4 was found in dense
clusters after incubation with ChAdOx1 nCoV-19 whereas PF4 formed a more
homogeneous layer on glass after incubation with purified ChAdOx1 nCoV-19 (E)
or Ad26.COV2.S (F). For quantification, single-molecule particle analysis was
performed and particle density ratios (on viral particles/on glass) analyzed
(Online Supplementary Figure S10). (G) Relative PF4 particle densities, showing
a statistically significant affinity of PF4 to adenoviral particles predominantly on
ChAdOx1 nCoV-19 but not on purified ChAdOx1 nCoV-19 virions or Ad26.COV2.S.
Statistical analysis of n=92 particles was performed with the Kruskal-Wallis test
with the Dunn correction for multiple comparisons. Respective P-values are indi-
cated in the plot, red lines and whiskers indicate mean ± standard deviation.
The dashed line is at y=1 (equal affinity of PF4 for adenoviral hexon and glass).
Full-field of view images and single-channel reconstructions are provided in
Online Supplementary Figure S9. Scale bars represent 100 nm.

nCoV-19 showed clear PF4 complex formation potential size. This was reversed by addition of heparin, indicating
(Figure 5B). However, it is important to note that under in charge-related binding of PF4 to the virions. This is again
vitro conditions, PF4 binds to both chimpanzee aden- consistent with the data obtained by cryo-electron
ovirus Y25 (ChAdOx1) and human adenoviruses (HAdV- microscopy and in silico modeling of ChAdOx1 nCoV-19,
D26 and HAdV-C5) through weak electrostatic interac- which established a possible electrostatic interaction of
tions that were abrogated in the presence of fondaparin- positively charged PF4 and negatively charged adenovirus
ux, a heparin pentasaccharide22. Proteomic analysis hexon polypeptide.22 However, the interaction Kd of
showed that the host-cell protein content in the pellet about 300 nmol was rather weak. This is likely the reason
fraction was reduced compared to the non-fractionated that we could not demonstrate distinct complexes of PF4
vaccine and a large proportion of the ChAdOx1 nCoV-19 with Ad26.COV2.S or purified ChAdOx1 nCoV-19 virion
host-cell proteins was located in the supernatant fraction. preparations. This further supports a role of impurities in
As expected, the viral proteins were enriched in the pellet ChAdOx1 nCoV-19 for the observed formation of large
fraction (Figure 5E, Online Supplementary Figures S14-S16). complexes in ChAdOx1 nCoV-19 after addition of PF4.9
We have shown that unassembled adenoviral hexon pro-
ChAdOx1 nCoV-19 induced vascular hyperpermeability teins are part of the PF4-complexes, but we still cannot
To study the effect of the two vaccines and EDTA (100 exclude a contribution of additional constituents of the
µM present in the ChAdOx1 nCoV19 vaccine) on vascu- vaccine supernatant.
lar permeability, we used in vivo microscopy of transgenic Antibody formation against PF4 is enhanced by antigen
zebrafish larvae expressing an eGFP-tagged plasma pro- presentation in an inflammatory environment. Recently,
tein (gc-eGFP, 78kDa20). Intramuscular injections (Online we have shown (in collaboration with the laboratory of
Supplementary Video S1) of 1 nL of 100 µM EDTA or Prof. Thomas Renne, Universitätsmedizin Hamburg
ChAdOx1 nCoV-19 locally increased vascular permeabil- Eppendorf, Germany) that intradermal injection of
ity, indicated by leakage of eGFP from the intravascular to ChAdOx1 nCoV-19 leads to EDTA-induced capillary
the intramuscular compartment (P=0.0001), but this was leakage in the Mile skin edema assay, thus increasing the
not observed after injection of purified ChAdOx1 nCoV- vaccine’s intravascular distribution.9 Our zebrafish model
19 virions, Ad26.COV2.S or physiological saline (Figure allows intramuscular injection, which recapitulates the
6). actual mode of vaccination. EDTA and ChAdOx1 nCoV-
19 but not Ad26.COV2.S rapidly induced local vascular
hyperpermeability. Such an increase of local capillary
Discussion leakage might facilitate direct contact of the immune sys-
tem with vaccine components, as does accidental
Our comprehensive analyses revealed major differ- intravascular administration of the vaccine.24
ences between ChAdOx1 nCoV-19 and Ad26.COV2.S Inflammation early after vaccination may also be
vaccines. Confirming our previous observation, a high enhanced when host-cell proteins are recognized by
proportion of host-cell proteins (54%) was found in the endogenous natural IgG.25 These natural antibodies bind
ChAdOx1 nCoV-19 vaccine, but only a very low level proteins of degrading cells and can form immune com-
was found in Ad26.COV2.S (1.5%). This observation plexes.
suggests very different purification approaches and purifi- Furthermore, proteasome activity was detectable in
cation efficiencies for the two vaccines, with a more thor- both vaccines, again with a remarkable difference
ough purification of adenoviruses in Ad26.COV2.S. Such between Ad26.COV2.S (only low proteasome activity
differences might be caused by the use of detergent treat- and protein abundance in one lot) compared to substan-
ment of the infected production cell culture for ChAdOx1 tially higher proteasome activities in almost all lots of
nCoV-19, which will make subsequent purification ChAdOx1 nCoV-19. This is of particular interest since
strategies more complicated.23 Hauler et al. showed that adenoviral capsid proteins are
The SARS-CoV-2 spike protein was detected only in intracellularly processed by proteasomal degradation
the ChAdOx1 nCoV-19 vaccine. There may be several which is mediated by the chaperone p97/VCP.26 In our
reasons for this, since different cell lines and procedures proteomic analysis, we identified VCP as one of the top
were used for production and purification of the two vac- five most abundant proteins of the ChAdOx1 nCoV-19
cines. Both suppliers use systems in which the TetR vaccine (Online Supplementary Tables S1 and S2).
repressor suppresses the expression of the SARS-CoV-2 Proteasomal degradation of adenoviral components such
transgene during the production of the recombinant ade- as the hexon polypeptide and/or host-cell proteins might
noviral vectors. A different degree of leakage of repres- lead to a reduction in vaccine efficiency. Whether protea-
sion might occur in the cell lines thus allowing different somal activity may also create potentially immunogenic
residual expression of the spike protein. Another reason or immunoreactive neo-antigens remains unresolved.
could be depletion of the spike protein during purification VITT/TTS occurs clinically after vaccination with
of Ad26.COV2.S. This can only be differentiated by in- ChAdOx1 nCoV-19 or Ad26.COV2.S and is mediated by
process sampling at different production steps of the vac- platelet-activating anti-PF4 antibodies. Recently, we and
cines. others have shown that anti-spike protein antibodies and
The two vaccines display differences in their ability to anti-PF4 antibodies react independently of each other,
interact with PF4. We confirm the previously observed therefore making it unlikely that VITT is caused by cross-
complex formation of PF4 with ChAdOx1 nCoV-19. reacting anti-spike protein antibodies.27,28 The current
Consistent with a recent study using cryo-electron data indicate that adenovirus particles and free hexon
microscopy of ChAdOx1 nCoV-19,22 in our study addi- proteins are the common features of both vaccines. We
tion of PF4 to purified ChAdOx1 nCoV-19 virions or to only observed formation of larger complexes of PF4 with
Ad26.COV2.S also resulted in a slight increase in particle ChAdOx1 nCoV-19. This indicates that an additional

S. Michalik et al.
Figure 5. Analysis of vaccine components prepared by ultracentrifugation. (A, C) Representative transmission electron micrographs of (A) ChAdOx1 nCoV-19 and (C)
Ad26.COV2.S vaccine and their respective supernatants and pellets obtained after ultracentrifugation. Asterisks and arrows indicate virions and electron dense amor-
phous vaccine components, respectively. Scale bar represents 200 nm. (B, D) Changes in the hydrodynamic diameter (in nm) of (B) ChAdOx1 nCoV-19 and (D)
Ad26.COV2.S vaccine and their respective supernatants and pellets obtained after ultracentrifugation of vaccines before and after addition of 10 µg/mL or 50 µg/mL
platelet factor 4 (PF4) assessed by dynamic light scattering. Dissociation of complexes between PF4 and the vaccine component was achieved by the addition of
unfractionated heparin (UFH; 10 IU/mL) (E) Western blot analysis of vaccine and pellet or supernatant fraction is shown. Primary antibodies for viral vector (anti-
hexon antibody) or human protein contaminants (anti-HSP90 antibody, anti-Epsilon 14-3-3 antibody, anti-PSMB5 antibody) were used. Statistical analysis was per-
formed by one-way analysis of variance on ranks/Kruskal-Wallis followed by correction for multiple comparisons by two-stage Benjamini, Krieger, & Yekutieli control-
ling the false discovery rate procedure (n=9) and a<0.05 was considered significant.

A F
Figure 6. Vascular hyperpermeability assay. Five days post-fertilization Tg(fabp10a:gc-eGFP) zebrafish larvae were microinjected with either physiological saline, 100
µM EDTA, ChAdOx1 nCoV-19, purified ChAdOx1 nCoV-19 or Ad26.COV2.S in four adjacent myotomes. Local fluorescence intensities of the myotomes were measured
at 0 min and 10 min post-injection (p.i.) (red asterisks) and normalized to the respective intravascular fluorescence (red arrows). Injection of 100 µM EDTA, as well
as ChAdOx1 nCoV-19, resulted in a significantly elevated extravascular leakage of the 78 kDa gc-eGFP compared to the saline control (A, B, C, F). However, injection
of Ad26.COV2.S, purified ChAdOx1 nCoV-19 or physiological saline did not cause an increase of local vascular permeability (A, D, E, F). The scale bar in (E) represents
200 µm without zoom and 100 µm with zoom.
cofactor is needed. This cofactor is present in the super- of pregnancy or organ transplantation. Systematic screen-
natant of the ChAdOx1 nCoV-19 vaccine. However, this ing of vaccinated individuals excluding or confirming
does not exclude complex formation of PF4 and such alloantibodies should be performed to clarify
Ad26.COV2.S in vivo. Several studies have shown the whether this theoretical concern requires further meas-
interaction of different adenoviruses with platelets and ures.
on the platelet surface they may also interact with PF4. In Limitations of our study include its in vitro design.
addition, hexon proteins or the virions may form com- Furthermore, our findings do not exclude the contribution
plexes with PF4 when they come into contact with addi- of certain cofactors (e.g., within the interstitial fluid, lym-
tional lymphatic or plasma proteins. phatic system, plasma, or cell surfaces) to the induction of
Beyond VITT/TTS, the potential for alloantibody for- the anti-PF4 immune response. We also did not investi-
mation by protein contaminants in vaccines might be a gate involvement of B-cell and T-cell populations.
matter of concern since the PregSure® vaccine, used in Moreover, since both adenoviral vector-based vaccines
cattle, induced alloantibody-driven bovine neonatal pan- have been discontinued in Germany, we cannot compare
cytopenia, a vaccine-induced alloimmune disease that the immune responses among vaccinated individuals.
was observed in young calves of PregSure®-vaccinated Finally, the lack of a suitable in vivo model for induction of
cows and is characterized by hemorrhage, pancytopenia, VITT/TTS limits the ability to reach definite conclusions
and severe destruction of hematopoietic tissue. The plas- regarding in vivo mechanisms.
ma of cows that gave birth to affected calves contained In summary, we show that process-related impurities
alloantibodies, which were likely induced by alloantigen- in the form of host-cell derived proteins, active proteases
expressing protein contaminants of the vaccine from the and unassembled hexon proteins differ in quality and
bovine kidney cell line used for vaccine production.29 quantity between ChAdOx1 nCoV-19 and Ad26.COV2.S
Alloantibodies will only cause clinical effects in the case SARS-CoV-2 vaccines. EDTA-induced capillary leakage

S. Michalik et al.
and host-cell protein impurities might further facilitate to visualize the data. SM, FS, RP, KF, MB, NE, AG, and UV
induction of an anti-PF4 immune response by intravascu- wrote the first draft of the manuscript. All authors contributed to
lar translocation of vaccine constituents and induction of the interpretation of results and manuscript editing. All authors
an early inflammatory response after vaccination. These approved the final version of the manuscript.
factors might explain the higher incidence rate of
VITT/TTS for ChAdOx1 nCoV-19 compared to Acknowledgments
Ad26.COV2.S vaccines. However, the authors would like We thank Katrin Schoknecht for support in the proteomics
to point out again that only comprehensive vaccination of analyses and Mandy Jörn for graphical design of electron
the human population can effectively contain the SARS- microscopy micrographs.
CoV-2 pandemic.
Funding
Disclosures This study was funded by Deutsche Forschungsgemeinschaft
AG reports personal fees and non-financial support from (DFG, German Research Foundation) grants: 374031971 - A06
Aspen, Boehringer Ingelheim, Instrumentation Laboratory, and and A11-TRR240, 398967434 - SFB/TR261, A11 - SFB877,
Roche; grants from Ergomed, Rovi, Sagent, Portola, Fa. Blau P6 - KFO306, B8 - SFB841, and INST 2026/13-1 FUGG, the
Farmaceutics, Prosensa/Biomarin, DRK-BSD Baden- Ministerium für Wirtschaft, Arbeit und Gesundheit Mecklenburg-
Würtemberg/Hessen, and Biokit; personal fees from Bayer Vital, Vorpommern (project COVIDPROTECT), “Structure and
Chromatec, Sanofi-Aventis, and GTH e.V; grants and personal Function of the Proteasome System in Platelets“ GR2232/8_1 and
fees from Macopharma; as well as grants and other from DRK- SE 885/2-1 (DFG), Leibniz WissenschaftsCampus –
BSD NSTOB, In addition, AG reports having a patent, appli- ComBioCat – W10/2018, by the Federal Ministry of Education
cation n. 2021032220550000DE, pending. and Research (BMBF, grant 01GM1518B, STOP- FSGS), the
Südmeyer fund for kidney and vascular research (“Südmeyer-
Contibutions Stiftung für Nieren- und Gefäßforschung”), the Dr. Gerhard
SM, FS, RP, KF, AR, US, CC, JW, LS, CH, MG-S, MB, Büchtemann fund, Hamburg, Germany and the PeNe_C19
NE, AG, and UV conceived the research. SM, FS, RP, KF, AR, study by the Ministerium für Wirtschaft, Arbeit und Gesundheit
US, CC, JW, LS, CH, MG-S, MB, NE, AG, and UV defined Mecklenburg-Vorpommern.
the methodology. SM, FS, RP, KF, AR, MS, US, CC, JW, LS,
CH, and MG-S. conducted the analysis. SM, FS, RP, KF, AR, Data-sharing statement
US, CC., JW, LS, CH, MG-S, MB, NE, AG, and U.V. inter- The mass spectrometry proteomics data have been deposited to
preted the data. SM, FS, RP, KF, AR, and MS generated ways ProteomeXchange (dataset identifier PXD027344).
Weisser K, Kyrle PA, Eichinger S. Activity probe for in vivo profiling of the
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ARTICLE Red Cell Biology & its Disorders
Ferrata Storti Foundation Brain injury pathophysiology study by a

multimodal approach in children with sickle
cell anemia with no intra or extra cranial
arteriopathy
Valentine Brousse,1,2,3,4 Corinne Pondarre,5,6 Manoelle Kossorotoff,7
Cecile Arnaud,5 Annie Kamdem,5 Mariane de Montalembert,1,2
Benedicte Boutonnat-Faucher,1 Slimane Allali,1,2,8 Hélène Bourdeau,9
Haematologica 2022 Keyne Charlot,10 Sebastien Bertil,11 Lydie da Costa,2,9,12,13
Volume 107(4):958-965 Philippe Connes,2,14,15# David Grévent16# and Suzanne Verlhac17,18#
1
AP-HP, Hôpital Necker Enfants Malades, Service de Pédiatrie Générale et Maladies
infectieuses, Paris; 2LABEX GR-Ex, Paris; 3Institut National de la Transfusion Sanguine,
UMR_S1134, INSERM, Paris; 4AP-HP, Hôpital Universitaire Robert Debré, Immuno-
Hématologie, Paris; 5Centre Intercommunal de Créteil, Service de Pédiatrie, Créteil;
6
Paris XII University, INSERM U955, Créteil; 7AP-HP, Hôpital Necker Enfants Malades,
Service de Neurologie Pédiatrique, Paris; 8Laboratory of Cellular and Molecular
Mechanisms of Hematological Disorders and Therapeutical Implications, Paris
Descartes – Sorbonne Paris Cité University, Imagine Institute, INSERM U1163, Paris;
9
AP-HP, Hôpital Robert Debré, Service d’Hématologie Biologique, Paris; 10Unité de
Physiologie des Exercices et Activités en Conditions Extrêmes, Département
Environnements Opérationnels Institut de Recherche Biomédicale des Armées,
Brétigny-sur-Orge; 11AP-HP, Service d’Hématologie Biologique, Hôpital Européen Georges
Pompidou, Paris; 12Service d'Hématologie Biologique, Université de Paris, Paris;
13
Hematim UR 4666, Amiens; 14Laboratoire LIBM EA7424, Equipe « Biologie Vasculaire
et du Globule Rouge », Université Claude Bernard Lyon 1, Villeurbanne; 15Institut
Universitaire de France, Paris; 16AP-HP, Hôpital Necker Enfants Malades, Service
d’Imagerie Pédiatrique, Paris; 17Centre Hospitalier Intercommunal de Créteil, Service
d’Imagerie Médicale, Créteil and 18AP-HP, Hôpital Universitaire Robert Debré, Service
d’Imagerie Médicale, Paris, France
#
PH, DG and SV contributed equally as co-senior authors
ABSTRACT
D
espite its high prevalence in children with sickle cell anemia
Correspondence: (SCA), the pathophysiology of silent cerebral infarcts (SCI)
VALENTINE BROUSSE remains elusive. The main objective of this study was to explore
valentine.brousse@gmail.com the respective roles of major determinants of brain perfusion in SCA chil-
dren with no past or current history of intracranial or extracranial vascu-
lopathy. We used a multimodal approach based notably on perfusion
Received: December 21, 2020. imaging arterial spin labeling (ASL) magnetic resonance imaging (MRI)
Accepted: April 14, 2021. and near infra-red spectroscopy (NIRS), as well as biomarkers reflecting
Pre-published: April 22, 2021. blood rheology and endothelial activation. Out of 59 SCA patients (mean
age 11.4±3.9 yrs), eight (13%) had a total of 12 SCI. Children with SCI
had a distinctive profile characterized by decreased blood pressure,
https://doi.org/10.3324/haematol.2020.278226 impaired blood rheology, increased P-selectin levels, and marked anemia.
Although ASL perfusion and oximetry values did not differ between
©2022 Ferrata Storti Foundation groups, comparison of biological and clinical parameters according to the
Material published in Haematologica is covered by copyright. level of perfusion categorized in terciles showed an independent associ-
All rights are reserved to the Ferrata Storti Foundation. Use of ation between high perfusion and increased sP-selectin, decreased red
published material is allowed under the following terms and blood cell deformability, low hemoglobin F level, increased blood viscos-
conditions:
https://creativecommons.org/licenses/by-nc/4.0/legalcode. ity and no a-thalassemia deletion. NIRS measurements did not yield
Copies of published material are allowed for personal or inter- additional novel results. Altogether, these findings argue for early MRI
nal use. Sharing published material for non-commercial pur- detection of SCI in children with no identified vasculopathy and suggest
https://creativecommons.org/licenses/by-nc/4.0/legalcode, a potential role for ASL as an additional screening tool. Early treatment
sect. 3. Reproducing and sharing published material for com- targeting hemolysis, anemia and endothelial dysfunction should reduce
mercial purposes is not allowed without permission in writing the risk of this under diagnosed and serious complication.
from the publisher.

Multimodal brain study in children with SCA
Introduction compared several biomarkers (markers of endothelial

injury and hemorheology), level of brain oxygenation (near
Brain injury is a major complication in sickle cell anemia infra-red spectroscopy [NIRS]) and perfusion imaging
(SCA) patients because of its high prevalence and devastat- (TCD and arterial spin labeling [ASL] MRI) between chil-
ing consequences. Historically, overt stroke used to be the dren with newly discovered and without SCI and ii) tested
most distressing clinical complication in childhood but its the association between these parameters.
prevalence has dropped from 10% to 1-3% in high-income
countries since the implementation of a preventive screen-
ing strategy by transcranial Doppler (TCD) coupled with Methods
chronic transfusion therapy in case of abnormal results.1,2,3,4
In sharp contrast, the contribution of silent cerebral infarcts Consecutive patients from two French referral centers for sickle
(SCI) to global neurovascular burden has emerged, following cell disease (University Hospital Necker-Enfants Malades and
the improved ability to detect subclinical lesions by magnet- Centre Hospitalier Intercommunal de Creteil) were screened dur-
ic resonance imaging (MRI). SCI are found in up to 37% of ing routine visits according to the following inclusion criteria: i) SS
SCA children before the age of 14 years, can be detected as or S-b°thalassemia genotype; ii) no past history of abnormal or
early as 1 year old and their prevalence increases with conditional transcranial and extracranial TCD; iii) at steady state (>
age.3,5,6 In addition, SCI are frequently associated with neu- 3 months from any vaso-occlusive event, transfusion or infection);
rocognitive impairment.7,8 While overt ischemic stroke iv) age between 5 and 17 years. The study was offered to all chil-
mainly occurs in the arterial territory of internal carotid or dren meeting the inclusion criteria and regularly followed up since
middle cerebral artery (usually with an associated stenosis or neonatal screening in the participating centers, between March
occlusion), silent infarcts tend to occur in the border zone 2015 and July 2016, with a target of 60 children (based on the
areas of the brain, mainly in the deep white matter, includ- expected number of SCA patients meeting the inclusion criteria in
ing in patients without large vessel arteriopathy. This obser- these centers and the pilot design of the study). Treatment by
vation highly suggests that SCI may be related to alterations hydroxyurea was not an exclusion criterion, but chronic transfu-
of perfusion and oxygenation.9 However, no clear patho- sion was. After written informed consent was obtained, a visit dur-
physiological process in SCI genesis has been demonstrated ing which all investigations were performed was scheduled.
thus far, although anemia, both chronic and acute, seems to The protocol was approved by the ethics committee “Comité pour
be a significant contributor.6 Conventional MRI shows brain la Protection des Personnes Ile de France III” (014-A01575-42) and
injury in a pattern suggestive of hemodynamic compromise registered in clinicaltrials gov. Identifier: NCT 02909283.
but it does not directly assess cerebral hemodynamics. By Clinical parameters as well as relevant past medical events were
contrast, perfusion analysis by arterial spin labeling (ASL) collected and are available in the Online Supplementary Appendix.
allows to better evaluate the cerebral hemodynamic risk at a Biological parameters were measured for each patient: routine
regional level, in a non-invasive manner, and may serve as a hematological and blood rheological parameters, and markers of
screening tool to identify children at risk of SCI. endothelial activation (see the Online Supplementary Appendix).
Blood rheology is a key determinant of blood flow, vas- TCD imaging (TCDI) was performed using a LOGIQ E9 XDclear
cular resistance and tissue perfusion.10 Increased blood vis- 2.0 ultrasound system (GE Healthcare, Milwaukee, WI, USA) at
cosity in both adults and children with SCA has been asso- Necker – Enfants Malades and an Acuson S 2000 ultrasound sys-
ciated with a risk of frequent vaso-occlusive crises. tem (Siemens Healthineers, Erlangen, Germany) at Centre
Moreover, SCA individuals with the greatest reduction in Hospitalier Intercommunal de Creteil.
red blood cell (RBC) deformability would be at higher risk MR imaging was performed with a GE Signa HDxt 1.5-T system
for developing leg ulcers, glomerulopathy and pri- (GE Medical Systems, Milwaukee, WI, USA) and a 12-channel
apism.11,12,13 These studies also reported increased RBC head-neck-spine coil in non-sedated children.
aggregates strength in SCA patients with glomerulopathy SCI were defined on MRI as hypersignals of cerebral parenchyma
or frequent priapism. Although a contribution of blood of at least 3 mm on T2 FLAIR sequence. Details on MRI sequences
rheology has been speculated in overt stroke in SCA,14 and image analysis including calculation of hypersignal volume are
there is no study focusing on SCI and blood rheology in available in the Online Supplement Appendix.
this disease in the literature. Surprisingly, a study by Brown Cerebral blood flow was measured using a three-dimensional
et al.15 performed in patients with paraproteinemia or pseudo-continuous arterial spin labeling (pCASL) sequence (repeti-
leukemia observed no association between cerebral blood tion time msec/echo time msec, 4,428/10.5; postlabeling
flow and blood viscosity. Instead, the authors reported an delay =1,025 msec; 80 axial partitions; field of view, 240x240x4
inverse relationship between cerebral blood flow and arte- mm; acquisition matrix, eight spiral arms in each three-dimension-
rial oxygen content, showing that regulatory mechanisms al partition with 512 points per arm; flip angle, 155°; acquisition
can maintain cerebral oxygen transport despite increased time, 4 minutes 17 seconds).
blood viscosity. The same negative association has been Transcranial NIRS was performed using a dual-channel absolute
reported in children with SCA.16 However, the associations cerebral oximeter FORE-SIGHT® (Branford, CT, USA).
between brain oxygenation, cerebral blood flow in the dif-
ferent brain areas and blood rheology have never been Statistics
investigated, and more particularly in the context of SCI. In We first compared the different measured parameters in patients
order to better decipher the mechanisms involved in sub- with and without SCI using an unpaired student t test.
clinical cerebral injury in children with SCA, we sought to The cohort was divided into terciles of ASL values: hypo-perfu-
explore the respective roles of hemolysis, abnormal blood sion (Low), normal perfusion (Middle) and hyper-perfusion (High)
rheology, cerebral oxygen supply and brain perfusion char- groups. the three groups were then compared using a one-way
acteristics in children with no past or current history of ANOVA with Tukey post hoc test. A c2 test was used for the qual-
intracranial or extracranial vasculopathy. We used a multi- itative analyses. The significance level was defined as P<0.05
modal approach in a multi-centric pilot study where we i) (SPSS, v. 20, IBM SPSS Statistics, Chicago, IL).

V. Brousse et al.
Results rial abnormalities on MRA analysis. Twelve SCI (3-5 mm:

n= 8; 5-15 mm: n=4, >15 mm: n=0) were observed in eight
General characteristics of the population of 59 (13%) children. The median volume of SCI was
Fifty-nine SCA patients (mean age 11.4±3.9 yrs) at 41,57 mm (range, 15,8-362 mm). SCI were located in the
steady state were enrolled, of which 34 (57.6%) were right anterior border zone in six patients. Five patients had
treated with hydroxyurea (HU). Patients main clinical and a single lesion, while three patients had >1 lesion, includ-
biological characteristics are summarized in Table 1. ing two with bilateral lesions.
Transcranial Doppler and magnetic resonance imaging Analysis of patients with silent cerebral infarcts
analysis Children with SCI had a lower past vaso-occlusive crisis
At the time of assessment, all TCD velocities were (VOC) rate compared to children with no SCI (Table 1).
within normal ranges (<170 cm/s) and there were no arte- Despite a comparable frequency of HU treatment in both
Table 1. General characteristics of the population.

All (n=59) no-SCI (n = 51) SCI (n = 8) P
Sex (M/F) 20/39 16/35 4/4 0.301
Age (yrs) 11.4 ± 3.9 11.4 ± 3.7 11.6 ± 4.9 0.865
Ongoing HU ttt at inclusion (N; %) 33; 56 30; 59 3; 38 0.259
Time since HU initiation (yrs) 3.1 ± 3.1 3.2 ± 3.2 1.6 ± 1.3 0.170
Daily dose (mg/kg/day) mean (SD) 23 ± 4.6 24.1 ± 3.3 12,8 ± 0.5 0.001
Age at beginning of HU initiation (yrs) 8.3 ± 4.3 8.2 ± 3.9 9.0 ± 5.4 0.170
Alpha thalassemia (N; %) 24; 41 23; 46 1; 13 0.074
G6PD deficiency (N; %) 4; 7 4; 8 0; 0 0.412
VOC rate (events/yr) 0.58 ± 0.58 0.65 ± 0.60 0.17 ± 0.20 0.001
ACS rate (events/yr) 0.08 ± 0.10 0.09 ± 0.10 0.03 ± 0.05 0.157
Transfusion history (%) 23.7 % 23% 25% 0.96
Mean number of transfusions (n, SD) 8 (11) 8 (10) 12 (17) 0.16
SpO2 (%) 99.0 ± 1.1 99.1 ± 1.0 98.6 ± 2.0 0.599
DBP (mmHg) 68 ± 11 69 ± 11 61 ± 7 0.047
SBP (mmHg) 110 ± 11 111 ± 11 106 ± 10 0.283
MAP (mmHg) 82 ± 9 83 ± 9 76 ± 8 0.047
Pulse Pressure (mmHg) 42 ± 12 42 ± 12 45 ± 6 0.437
Hb (g/dL) 8.8 ± 1.2 8.9 ± 1.1 8.2 ± 1.1 0.047
Hct (%) 25.0 ± 3.3 25.3 ± 3.2 23.0 ± 3.0 0.025
MCV (fl) 82 ± 12 83 ± 12 77 ± 10 0.186
MCHC (g/dL) 35.0 ± 1.5 34.9 ± 1.5 35.8 ± 0.9 0.037
Reticulocyte count (109/L) 225 ± 102 224 ± 105 229 ± 88 0.91
Platelets (109/L) 343 ± 154 331 ± 141 418 ± 220 0.141
WBC count (109/L) 8.98 ± 3.61 8.78 ± 3.64 10.29 ± 3.36 0.277
HbF (%) 16.0 ± 10.1 16.0 ± 10.4 16.1 ± 8.2 0.986
ASAT UI/L 49 ± 16 48 ± 16 52 ± 13 0.548
LDH UI/L 484 ± 164 485 ± 173 479 ± 108 0.938
Soluble E-selectin (ng/mL) 73.4 ± 29.6 73.2 ± 21.7 74.5 ± 39.7 0.911
Soluble P-selectin (ng/mL) 59.3 ± 21.3 57.1 ± 20.7 73.5 ± 21.7 0.047
CD34* cell (/mL) 9185 ± 6738 9803 ± 6773 5788 ± 6229 0.301
RBC aggregation (%) 52 ± 6 52 ± 7 51 ± 4 0.811
RBC disaggregation threshold (s-1) 378 ± 201 359 ± 191 491 ± 236 0.037
RBC deformability (a.u.) 0.49 ± 0.09 0.50 ± 0.09 0.44 ± 0.07 0.025
Blood viscosity (cP) 5.35 ± 0.93 5.40 ± 0.94 5.04 ± 0.83 0.312
Hematocrit/blood viscosity (a.u.) 4.72 ± 0.59 4.73 ± 0.58 4.62 ± 0.69 0.627
Right ctSO2 (%) 63 ± 10 63 ± 10 61 ± 9 0.662
Left ctSO2 (%) 63 ± 10 63 ± 10 64 ± 9 0.828
M: male; F: female; SD: standard deviation; SCI: silent cerebral infarct; HU: hydroxyurea; VOC: vaso-occlusive crisis; ACS: acute chest syndrome; DBP: diastolic blood pressure; SBP:
systolic blood pressure; MAP: mean arterial pressure; MCHC: mean corpuscular hemoglobin concentration; MCV: mean corpuscular volume; Hct:hemtocrit; HbF: hemglobin F;
ASAT: aspartate aminotransferase; LDH: lactate dehydrogenase; RBC: red blood cell; a.u.: artbitrary units; CP: centipoise; ctSO2: cerebral tissue hemoglobin oxygen saturation;
P-values are given for the comparison between the non-SCI and SCI groups.

Table 2. Cerebral blood flow values in patients with or without silent cerebral infarct.
no-SCI (n = 51) SCI (n = 8) P
Right hemisphere perfusion (mL/100 g/min)
ACA 116.5 ± 17.8 (112.0-121.0) 126.1 ± 17.4 (114.0-138.0) 0.162
Anterior Junctional 122.9 ± 21.1 (117.0-129.0) 123.8 ± 16.2 (113.0-135.0) 0.909
Superficial MCA 111.9 ± 17.9 (107.0-117.0) 116.5 ± 13.8 (107.0-126.0) 0.489
Caudate nucleus 93.3 ± 16.2 (88.8-97.8) 98.5 ± 16.4 (87.1-110.0) 0.406
Putamen 82.5 ± 14.2 (78.6-86.4) 90.0 ± 13.5 (80.7-99.3) 0.168
posterior Junctional 120.6 ± 23.3 (114.0-127.0) 120.5 ± 16.9 (109.0-132.0) 0.988
Left hemisphere perfusion (mL/100 g/min)
ACA 116.5 ± 19.4 (111.0-122.0) 132.0 ± 23.9 (115.0-149.0) 0.117
Anterior Junctional 121.6 ± 22.1 (116.0-128.0) 122.8 ± 15.5 (112.0-134.0) 0.887
Superficial MCA 111.3 ± 18.9 (106.0-116.0) 121.0 ± 16.1 (110.0-132.0) 0.174
Caudate nucleus 94.3 ± 16.0 (89.9-98.7) 102.8 ± 14.2 (93.0-113.0) 0.165
Putamen 83.0 ± 14.4 (79.0-87.0) 93.4 ± 11.0 (85.8-101.0) 0.058
Posterior Junctional 119.9 ± 26.4 (113.0-127.0) 122.5 ± 24.3 (106.0-139.0) 0.794
Posterior fossa perfusion
Right Cerebellar hemisphere 88.1 ± 8.2 (85.8-90.3) 84.1 ± 8.2 (78.4-89.8) 0.295
Left Cerebellar hemisphere 89.6 ± 16.9 (85.0-94.2) 85.4 ± 8.1 (79.8-91.0) 0.277
No significant difference in cerebral blood flow values was evidenced between the two groups. Values are given as means ± standard deviation (95% Confidence Interval):
SCI: silent cerebral infarcts; MCA: middle cerebral artery; ACA: anterior cerebral artery.
Table 3. Characteristics of the population according to the level of perfusion assessed by arterial spin labeling in the right anterior cerebral artery
and categorized in terciles.
Lower CBF (N=20) Middle CBF (N = 19) Higher CBF (N = 20)
Age (yrs) 12.4 ± 4.2 10.5 ± 4.2 11.2 ± 3.2
HU (%) 71 56 43
a thalassemia (%) 46 29 25*
G6PD deficiency (%) 31 35 35
VOC rate (events/yr) 0.68 ± 0.53 0.47 ± 0.43 0.63 ± 0.73
ACS rate (events/yr) 0.09 ± 0.11 0.06 ± 0.08 0.09 ± 0.11
SpO2 (%) 99.3 ± 1.0 99.1 ± 1.2 98.8 ± 1.1
DBP (mmHg) 70 ± 12 67 ± 11 66 ± 10
SBP (mmHg) 114 ± 12 108 ± 12 108 ± 10
MAP (mmHg) 85 ± 10 81 ± 9 80 ± 9
Hb (g/dL) 9.2 ± 1.2 8.9 ± 1.1 8.3 ± 1.0*
Hct (%) 26.3 ± 3.4 25.2 ± 3.3 23.6 ± 2.6*
MCV (fL) 82 ± 13 81 ± 11 84 ± 11
MCHC (g/dL) 35.0 ± 1.3 35.2 ± 1.0 34.9 ± 2.0
Reticulocyte count (109/L) 193 ± 84 229 ± 112 255 ± 105*
Platelets (109/L) 315 ± 123 314 ± 141 388 ± 159
White blood cells (109/L) 7.99 ± 3.37 9.23 ± 3.89 9.91 ± 3.51
HbF (%) 19.4 ± 11.3 18.4 ± 10.2 11.9 ± 6.5*#
ASAT UI/L 42 ± 8 50 ± 15 55 ± 18*
LDH UI/L 407 ± 126 515 ± 154 545 ± 186
CRP mg/L 5.8 ± 8.4 6.0 ± 5.0 5.4 ± 9.2
E-selectin 67.0 ± 22.3 68.5 ± 35.0 85.0 ± 28.4
P-selectin 49.3 ± 14.7 58.2 ± 21.3 68.9 ± 23.1**
RBC aggregation (%) 52 ± 6 53 ± 7 50 ± 5
RBC disaggregation threshold (s-1) 390 ± 222 399 ± 225 356 ± 174
RBC deformability (a.u.) 0.53 ± 0.06 0.51 ± 0.09 0.46 ± 0.08*#
Blood viscosity (cP) 5.75 ± 1.03 5.23 ± 0.80 5.18 ± 0.93*
Hematocrit/blood viscosity (a.u.) 4.66 ± 0.70 4.80 ± 0.40 4.67 ± 0.67
Right ctSO2 (%) 64 ± 10 62 ± 9 63 ± 10
Left ctSO2 (%) 64 ± 13 64 ± 8 64 ± 10
a.u.: arbitray unit; G6PD: glucose-6-phosphate dehydrogenase; HU: hydryurea; VOC: vaso-occlusive crisis; ACS: acute chest syndrome; DBP: diastolic blood pressure; SBP: systolic
blood pressure; MAP: mean arterial pressure; Hb: hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; ASAT:
aspartate aminotransferase; LDH: lactate dehydrogenase; RBC: red blood cell; ctSO2: cerebral tissue hemoglobin oxygen saturation. Significantly different from the lower cerebral
blood flow (CBF) group: *P<0.05; **P<0.01. Significantly different from the Middle CBF group: #P<0.05.

V. Brousse et al.
groups, significant differences in biological profiles were and without SCI. Of note, there was no association
evidenced: children with SCI had increased plasma between cerebral blood flow (CBF) values and the pres-
P-selectin level and RBC disaggregation threshold, and ence of SCI (Table 2).
lower hemoglobin (Hb) level and RBC deformability. Of
note, daily dose of HU was significantly lower in patients Arterial spin labeling perfusion imaging
with SCI compared to those with no SCI at the time of In the whole group of patients, CBF values showed no
data collection but the sample size was too small (n=3 of major asymmetry and correlated across all territories
8) to further interpret this finding. (r ranging from 0.46 to 0.91; P ranging from <0.01 to
In addition, diastolic blood pressure (DBP) and mean <0.001). Interestingly, comparison of patient subgroups
arterial pressure (MAP) were lower in patients with SCI. according to CBF terciles in the arterial territories showed
Oximetry data (right and left cerebral oxygenation levels) marked differences (see Table 3 for the right anterior cere-
showed no significant difference between patients with bral artery territory, as an illustration). The group with
Figure 1. Regions of interest on the arterial spin

labeling sequence. Left column: T1 weighted imag-
ing. Right column: arterial spin labeling (ASL) imag-
ing. First row: axial section tangential to the upper
wall of the lateral ventricles, second row: axial sec-
tion crossing the anterior and posterior white com-
missures (AC-PC), third row: axial section at the level
of the internal auditory canals. All axial images are
parallel to CA-CP. ACA: anterior cerebral artery area;
AW: anterior watershed area; MCA: middle cerebral
artery area; PW: posterior watershed area; C: cau-
date nuclei; LN: lenticulostriate nuclei; PF: posterior
fossa.

higher CBF had increased markers of hemolytic anemia Table 4. Correlations between cerebral oxygenation (ctSO2) and bio-
(lower Hb, increased aspartate aminotransferase (ASAT), logical parameters
lactate dehydrogenase [LDH] and reticulocyte count), Left ctSO2 (%) Right ctSO2 (%)
lower HbF level, increased marker of endothelial activa-
tion (sP-selectin value), lower RBC deformability and Age (yrs) r = -0.12 r = -0.22
blood viscosity compared to the group with lower CBF. In SpO2 (%) r = 0.35* r = 0.43**
addition, the frequency of patients with co inherited α- DBP (mmHg) r = -0.08 r = 0.14
thalassemia was lower. An ordinal multivariate analyses SBP (mmHg) r = -0.09 r = -0.08
was performed to test the independent associations
MAP (mmHg) r = -0.09 r = 0.08
between the perfusion level (tercile groups) and the main
parameters influencing blood flow. Blood viscosity was Hb (g/dL) r = 0.48*** r = 0.53***
retained for the model because it is a key determinant of Hct (%) r = 0.53*** r = 0.56***
blood flow.10 Since blood viscosity is highly dependent on MCV (fL) r = 0.07 r = 0.17
hematocrit/Hb and colinearity was indeed very strong MCHC (g/dL) r = -0.26 r = -0.30*
between blood viscosity and hematocrit and Hb (variance
Reticulocyte count (109/L) r = -0.06 r = -0.12
inflation factor [VIF] = 36.6 and 37.4, respectively), hema-
tocrit and Hb were not included in the multivariate Platelets (109/L) r = -0.17 r = -0.14
model. Likewise, ASAT and reticulocyte count were not White blood cells (109/L) r = -0.26 r = -0.24
considered for the model as, like LDH, they reflect HbF (%) r = 0.44** r = 0.45**
hemolysis. In the multivariate model, which was highly ASAT UI/L r = -0.37* r = -0.24
s i g n i f i c a n t
(P<0.001), all the parameters except LDH (P=0.058), were LDH UI/L r = -0.51*** r = -0.43**
independently associated with the level of perfusion CRP mg/L r = -0.07 r = -0.24
(sP-selectin: P<0.01; RBC deformability: P<0.01; HbF E-selectin r = -0.16 r = -0.20
level: P<0.05; blood viscosity: P<0.05; a-thalassemia: P-selectin r = 0.12 r = 0.07
P<0.05).
RBC aggregation (%) r = 0.03 r = -0.10
Oximetry measurements RBC disaggregation threshold (s-1) r = -0.30* r = -0.27*
In the whole population, cerebral oximetry results RBC deformability (a.u.) r = 0.54*** r = 0.51***
showed a significant positive correlation with Hb, SpO2, Blood viscosity (cP) r = 0.30* r = 0.30*
RBC deformability, blood viscosity and HbF and a nega- Hematocrit/blood viscosity (a.u.) R = 0.21 R = 0.17
tive correlation with markers of hemolysis (total bilirubin DBP: diastolic blood pressure; SBP: systolic blood pressure; Hb: hemoglobin; Hct: hem-
and LDH) as well as the RBC disaggregation threshold tocrit; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin con-
(Table 4). Of note, blood pressure, age, reticulocyte count, centration; ASAT: aspartate aminotransferase; LDH: lactate dehydrogenase; CRP:
C-reactive protein; a.u.: arbitrary units; RBC: red blood cell; ctSO2: cerebral tissue
level of selectins and TCD velocities did not correlate hemoglobin oxygen saturation Significant correlations: *P< 0.05; **P< 0.01; ***P<
with oximetry results. A multivariate analysis was per- 0.001.
formed for the right and left cerebral tissue hemoglobin
oxygen saturation including SpO2, Hb, RBC deformabili- Discussion
ty, HbF, LDH and the RBC disaggregation threshold. Hb
was preferred to blood viscosity as tissue oxygenation is In this study of 59 young children highly selected for no
thought to be highly dependent on the number of RBC present or past history of cerebral and extra cranial vascu-
and Hb concentration. None of the parameters was sig- lopathy, a prevalence of eight of 59 (13%) children with SCI
nificant (P=0.38 and P=0.35 for the right and left ctSO2, was found, illustrating the residual burden of neurovascular
respectively), which may be explained by the fact that all injury in patients having received recommended follow-up.
are interrelated to affect brain oxygenation. SCI predominated in the fronto-parietal white matter at the
junction between the anterior and the middle cerebral
Effect of hydroxyurea treatment artery territories, as previously described.17 Expectedly, on
HU treatment did not significantly impact the results of T2 FLAIR sequence, no SCI >15 mm were found which
neurovascular explorations in this cohort, except for sE- would have presumably translated in clinically detectable
and sP-selectin levels that were significantly lower in chil- symptoms. Likewise, volumes of SCI were within previ-
dren receiving treatment. Treated children received a ously described ranges.18 Consistent with the inclusion cri-
daily dose of HU that was not a maximum tolerated dose teria of no past conditional or abnormal TCD or transient
but was a fairly high dose (23+/-4.6 mg/kg/day) and both vasculopathy, there was no arterial abnormality on MRA
groups had indeed comparable levels of Hb or HbF. analysis. SCI and more specifically their size, volume and
Although compliance was not specifically addressed, the localization may impact cognitive function. In this study
decreased level of selectins in treated children argues for cognitive testing was not performed and subsequently cog-
an effect of HU on alleviating endothelial injury, in addi- nitive consequences related to SCI cannot be inferred.
tion to its known effect on hemolytic anemia. The cross- Nevertheless, the frequency of SCI in these highly selected
sectional design of the study, however, does not allow to children plead for early identification of silent lesions in
actually compare groups at baseline, i.e., before treatment order to further explore cognitive functions for early imple-
initiation and precludes further interpretation. In line, the mentation of supportive learning skills. In line with recent
specific effect of HU in children with SCI (or the lack of, recommendations, MRI screening, is therefore highly rec-
given the significantly low dose) is limited by the small ommended in all children with SCA, including in those
sample size (n=3). with no identified vasculopathy.19

V. Brousse et al.
Given the absence of large vessel vasculopathy in these would have increased cerebral perfusion and oxygenation.
children, SCI are presumably unrelated to ischemia occur- However, because NIRS is unable to measure cerebral
ring downstream of a large vessel stenosis. SCI may nev- oxygenation at a depth of interest and is limited to the
ertheless share common risk factors with large vessel vas- anterior territories, its additional predictive value remains
culopathy and result from impaired perfusion. Indeed, we to be demonstrated, particularly in patients at risk of
found distinctive clinical and biological features in chil- white matter SCI.
dren with SCI. In line with previous reports, a markedly This study has a number of limitations. Its cross-sec-
increased hemolytic and anemic profile favoring SCI was tional design does not allow associations to be interpreted
evidenced, consistent with a lower pain rate.20,21 HU treat- as causalities and its sample size was small, particularly
ment did not significantly impact these data, but interpre- regarding the number of patients with SCI treated by HU.
tation is very limited given the small sample size of treat- It is possible that the lack of association between elevated
ed children with SCI (n=3), and the low daily dose at the CBF values and the presence of SCI was due to the low
time of data collection. Interestingly, despite significantly power of the study, for instance. Regarding imaging tech-
greater anemia in children with SCI, blood viscosity did niques, ASL MRI has also inherent limitations. In particu-
not significantly differ across groups because RBC lar the transit time is reduced in SCA patients relative to
deformability was reduced in the former group, which non-anemic children. Thus, the labeling efficiency and
exerts opposite effects on blood viscosity.22 Intravascular postlabeling delay may not be adequate in every patient
hemolysis is a major determinant of vascular and endothe- and may modify the perfusion signal measured by ASL.28
lial dysfunction.23 Consistent with this, an increased level Another parameter, the fixed value of the T1-longitudinal
of sP-selectin, a marker of endothelial activation, was evi- relaxation time of blood used for CBF quantification may
denced and was associated with SCI. sP-selectin could not be adequate in every individual patient, given its
potentially serve as a biomarker of cerebral injury in chil- dependence on hematocrit and blood composition.
dren.6 In contrast with previous studies,24 a lower diastolic Another limitation is that CBF measurements were made
and mean arterial pressure was found in children with in grey matter, while silent infarcts are located preferen-
SCI. Altogether, this data strengthens the hemodynamic tially in white matter. At a magnetic field strength of 1.5
pathophysiology of SCI, whereby a further drop in pres- Tesla, the signal-to-noise ratio of the resulting perfusion
sure and/or blood flow in children with severe anemia map is too low in the cerebral white matter to allow accu-
results in ischemia in the border zone region, character- rate measurements. Despite these limitations, however,
ized by terminal arterial supply. Furthermore, hemorheo- our ASL results adequately reflected the level of perfusion
logical exploration of these patients suggests that in different territories of the brain. In fact, our results were
increased RBC aggregate strength and decreased red cell consistent with previous reports regarding the influence of
deformability may also influence the risk of SCI. both anemia and age and further allowed novel coherent
Deformable RBC mostly flow in single file in capillaries results regarding the influence of endothelial activation
and RBC aggregates need to be fully dispersed before and blood rheology for instance, and more generally the
entering into the microcirculation.22,10 Consequently, possible pathophysiology of SCI genesis.
decreased RBC deformability and increased RBC aggre- Altogether the findings of this study suggest that SCI
gates strength may both impair blood flow at the entry of may, like overt stroke, preferably occur in otherwise pauci-
small capillaries and affect tissue perfusion. symptomatic children with marked hemolytic anemia and
Cerebral blood flow begins at a low level in the perina- endothelial dysfunction. Cerebral blood flow measure-
tal period, increases to a peak value at 3-8 years of age and ments by ASL MRI may help assess the quality of perfu-
then gradually decreases to adult levels with a negative sion at a microvascular level in children with no vasculopa-
correlation with age.25 Several reports have documented thy but nevertheless at risk of subclinical injury. An early
elevated CBF in the grey matter of children with SCA, disease-modifying treatment like HU, which improves all
compared to normal controls,26-28 a finding attributed to afore-mentioned factors associated with SCI may there-
the compensatory increase in blood velocity and flow sec- fore decrease SCI risk as well, in addition to its known ben-
ondary to baseline anemia. This study allowed further eficial effect on TCD velocities and stroke risk.27,32,33
insight by showing that, regardless of concomitant HU Decreasing hemolytic anemia, improving RBC rheological
treatment, the level of perfusion was independently asso- characteristics and limiting endothelial injury will help
ciated with blood viscosity (which is negatively correlated avoid cerebral injury, particularly in case of further aggres-
to anemia), HbF level, endothelial activation, a-tha- sion such as arterial stenosis, acute hypoxia, drop in Hb or
lassemia status, and marginally associated with the level increased metabolic demand. Large prospective trials eval-
of hemolysis. ASL perfusion may therefore serve as an uating the protective effect of HU on SCI are ongoing and
additional screening tool that integrates all these parame- will hopefully confirm such beneficial effect. It is possible
ters to identify children at risk of subclinical injury, that new therapeutic approaches such as P-selectin block-
beyond the known risk factor, and regardless of cerebral ade by monoclonal or pan antibodies, may have further
vasculopathy. Thresholds for risk assessment will need to protective effects in this particular complication.
be determined by further prospective studies. Disclosures
Oximetry analysis yielded expected and coherent VB, CP and MdM report honoraria and expert/consultancy
results. NIRS is a non-invasive measurement of cerebral testimony for Addmedica.
oxygenation, that varies with SpO2 and Hb. In line with
previous reports, we show that cerebral oxygenation is Contributions
low in SCA patients.29,30,31 Our results also confirmed an VB, MK, DG and SV designed the study; VB, CP, CA, AK,
association between RBC rheological parameters and MdM, BBF and SA enrolled patients; HB and LdC were
cerebral oxygenation, suggesting that patients with the responsible for biological data, except for E- and P-selectins that
most deformable RBC and less robust RBC aggregates were measured by SB; SV and DG were responsible for imaging

data; KC and PC were responsible for oximetry and hemorheo- Hullier-Amar and Isabelle Buffet from the Fondation Institut
logical data interpretation; VB, PC, CP, MK, DG, and SV ana- Imagine.
lyzed the data; PC was responsible for statistical analysis; VB
and PC wrote the manuscript. All authors reviewed, edited and Funding
approved the manuscript. This research was funded by LABEX-GR-Ex, grant number
GR-EX14/ParisDiderot/ and an additional grant was obtained
Acknowledgments from AddMeddica.
We wish to thank Wiam BHIA, Nicholas Renaud, Elisabeth
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LETTERS TO THE EDITOR
TET2-deficiency in hematopoietic stem cells predisposes
Loss of 5-hydroxymethylcytosine expression is to myeloid leukemias.5 Mutations in epigenetic modify-
near-universal in B-cell lymphomas with variable ing genes, such as DNMT3A, TET2, or IDH2, are associ-
mutations in epigenetic regulators ated with lack of 5hmC expression and recurrent in lym-
phomas of follicular helper T-cell origin.6 Mutations in
Epigenetic alterations are increasingly recognized in epigenetic modifying genes (viz., EZH2) are also com-
human malignancies, with loss of 5-hydroxymethylcyto- mon in BCL, including follicular lymphoma (FL) and ger-
sine (5hmC) expression by immunohistochemistry as a minal center-derived DLBCL, supporting the rationale
potential correlational proxy for malignant epigenetic for 5hmC evaluation in BCL. An earlier study of TET1-
change. In this study, nearly 100 cases of a broad range deficient mice associated loss of 5hmC immunostaining
of B-cell lymphomas (BCL) studied via 5hmC immuno- with development of BCL, suggesting a relationship
histochemistry showed pervasive loss of 5hmC expres- between TET1 expression, 5hmC content, and lym-
sion. Whole exome sequencing (WES) performed on a phomagenesis.7 With this background, we evaluated a
subset of diffuse large B-cell lymphoma (DLBCL) spectrum of BCL for 5hmC expression and the associa-
showed a significant fraction of cases (58.8%) with mis- tion of expression with mutations in epigenetic path-
sense mutations in such genes CREBBP, IDH1, IDH2, way-related genes.
TET1, TET2, and WT1. After review of histology, 92 cases of BCL (5 whole
Epigenetic pathways related to gene expression, section and 87 on tissue microarray [TMA]) were stained
including DNA methylation, are often dysregulated in for 5hmC (polyclonal 1:1,000, active motif, see Figure 1).
human cancers. 5hmC is increasingly gaining recogni- Five normal lymph nodes were also examined in tan-
tion as an important epigenetic mediator, indicative of dem. A subset of the whole section cases were subject to
active demethylation and corresponding gene double stain performed for CD3 (red)/5hmC (brown) to
activation.1,2 While patterns of gene expression resulting assess 5hmC in the T-cell compartment. All staining was
from epigenetic modifications are often variable, 5hmC performed on the Leica Bond IIITM autostainer. Staining
loss (as a surrogate for TET loss-of-function and 5mC was scored dichotomously as absent or present.
persistence) is associated with some human cancers, WES 17 DLBCL cases from whole sections was also
including melanoma, mesothelioma, acute myeloid performed. Tumor DNA from 17 large-cell lymphoma
leukemias, and myelodysplastic syndrome.3 samples within the above cohort was isolated using the
TET2 mutations in lymphoid cells exert pleiotropic AllPrep DNA/RNA FFPE kit (Qiagen) and matched
effects depending on the cellular compartment. germline DNA was obtained using peripheral blood with
Conditional knockout of TET2 in immature B cells leads the DNeasy Blood/Tissue kit (Qiagen). One case show-
to development of lymphoblastic leukemias,4 whereas ing slightly increased large cells, morphologically closer
A Figure 1. 5-hydroxymethylcytosine
expression by lymphoma subtypes. (A)
Lymphomas were organized into three
groups: high-grade B-cell lymphoma
(HGBCL) /diffuse large B-cell lymphoma
(DLDCL) low-grade (including mantle cell
lymphoma [MCL]), and Hodgkin lym-
phoma [HL]; with constituent subtypes
specified, and average percent 5-hydrox-
ymethylcytosine (5hmC) loss per group
tabulated by total numbers of cases.
Overall, 94% of large B-cell lymphomas,
94% low-grade B-cell lymphomas
(including MCL) and 88.5% of HL
showed loss of 5hmC expression. One
very weakly stained MCL case was con-
sidered within the negative group. The
HGBCL cases included 1 double-hit lym-
phoma and one HGBCL-not otherwise
specified (NOS) as per World Health
B Organization 2017 schema. Graphs
were generated within Plotly and Visual
Studio code.14 (B) Three sample cases of
lymphomas demonstrating loss of
expression in the neoplastic cells (black
arrowhead) depicting Hodgkin/Reed-
Sternberg cell, diffuse DLBCL as well as
neoplastic follicles of follicular lym-
phoma (FL). Background endothelial
cells and reactive lymphoid cells demon-
strate strong retained expression of
5hmC (blue arrows). NLPHL: nodular
lymphocyte predominant Hodgkin lym-
phoma; cHL: classical Hodgkin lym-
phoma; B-NHL: B-cell non-Hodgkin lym-
phoma; LPL: lymphoplasmacytic lym-
phoma; MZL: marginal zone lymphoma;
SLL: small lymphocytic lymphoma;
THRBCL: T-cell/histiocyte-rich B-cell lym-
phoma; PMBL: primary mediastinal
(thymic) large B-cell lymphoma; RT:
Richter transformation.

to nodular lymphocyte predominant Hodgkin lym- significant majority of intrafollicular (likely CD4 follicu-
phoma (NLPHL), was excluded from WES analysis to lar helper T cells) and extrafollicular T cells showed loss
maintain homogeneity. of 5hmC on the CD3/5hmC double stain (Figure 2A).
The Agilent SureSelect XT Human All Exon v6 Kit Among all lymphoma cases (n=92), the majority of
captured whole-exome and untranslated regions (UTR), high-grade, low-grade B-cell and Hodgkin lymphomas
with reads generated using Illumina HiSeq2500 and (HL) showed loss of 5hmC in neoplastic cells (94%,
NovaSeq6000 at Theragen Bio Co., Ltd, due to perform- 94%, and 88.5% of cases, respectively, Figure 1B, Figure
ance of sequencing over two separate experiments. See 2B and D). 5hmC loss occurred in over 90% of lym-
the Online Supplementary Table S1 for additional methods phoma cells in any given case. Partial/variable loss only
on read alignment. Somatic mutations were then called occurred in four classical HL (cHL) (2 were considered
using Mutect2 through GATK4 either paired germline 5hmC retained, while 2 with extensive loss in over 90%
DNA or best practices provided panel of normals. of cells were considered 5hmC lost), demonstrating
Identified mutations were then post-processed by filter- occasional weak staining in a subset of Hodgkin cells.
ing according to best practices and annotated using the When the DLBC-not otherwise specified (NOS) with
vcf2maf tool,8 which integrates the annotation tool available cell of origin (COO) data were stratified by
Variant Effect Predictor (VEP) from Ensembl and a for- COO, there were 11 GCB cell, 11 non-GCB cell, and one
mat conversion step. The final annotated mutations undetermined COO, with all cases showing 5hmC loss.
from WES were then analyzed in R (version 4.0.3) using Cases with weak staining and partial loss was not
tidyverse (version 1.3.0) best practices and the package observed, except in one mantle cell lymphoma (MCL).
maftools (version 2.4.12).9 See the Online Supplementary Rare cases of high-grade BCL (HGBCL) with retained
Table S1 and Online Supplementary Figure S1 for results 5hmC expression consisted of one primary mediastinal
pertaining to cell of origin status and predicted signifi- BCL and one DLBCL-Richter transformation (RT) from
cance of observed mutations and their locations. The chronic lymphocytic leukemia and small lymphocytic
study was approved by the UOC Institutional Review lymphoma (CLL/SLL). The only two low-grade BCL
Board (IRB13-1297). with retained 5hmC expression were both CLL/SLL. HL
Normal lymph nodes retained 5hmC in the mantle, with retained 5hmC expression were split between two
marginal, and paracortical areas with isolated loss only cHL (9% of cHL) and one NLPHL (25% of NLPHL).
in the germinal center B (GCB) cells with scattered follic- Only two DLBCL-RT from CLL/SLL were included
ular dendritic cells showing retained 5hmC. Notably, a within the DLBCL-NOS set. One case showed retained
B C D
Figure 2. T-cell panel: CD3 (red)/5-hydroxymethylcytosine (brown) double stain on reactive node and B-cell lymphoma. (A) Low power magnification of germinal
center-mantle interface. Noted normal mantle cells positive for 5-hydroxymethylcytosine (5hmC) while perifollicular T cells are in red. High power magnification
of the paracortical areas (panel on right top) show several T cells with only CD3 (red) while 5hMC (brown) is negative in these cells. Germinal center areas at
high power with scattered T cells (likely CD4+ follicular helper T cells) with loss of 5hmC. Normal follicular dendritic cells (large doublets) express strong 5hmC
while germinal center B cells are also negative. (B, C and D) Correspond to 1 case each of classical Hodgkin Lymphoma (cHL), diffuse large B-cell lymphoma
(DLBCL), and T-cell/histiocyte-rich B-cell lymphoma (THRBCL) with neoplastic cells negative for 5hMC (black arrowheads) with a significant component of
microenvironment T cells also negative for nuclear 5hmC.

5hmC expression and the other showed concordant and both showed uniform loss of 5hmC expression.
5hmC loss in both the high-grade DLBCL and residual Their study also found that all CLL and most MCL cases
low-grade CLL/SLL components. In most lymphomas, retained 5hmC expression, with only two of 11 MCL
reactive background small lymphoid cells with strong demonstrating loss. In contrast, our study noted loss of
retained 5hmC expression served as internal controls. expression in the majority of CLL cases (6/8) and all four
However, in some Hodgkin and DLBCL cases (including MCL cases (Figure 1A). Their study included one
transformed FL), a significant fraction of non-neoplastic DLBCL-RT, but staining pattern details in the trans-
background milieu also demonstrated loss of 5hmC formed component were not reported. We expected an
expression (Figures 2B and C; Online Supplementary increased likelihood of 5hmC loss in the transformed
Figure S1). component, but noted an inverse pattern with retained
WES analysis interrogating for mutations in epigenetic 5hmC in one DLBCL-RT and loss in one CLL compo-
regulators (IDH1, IDH2, TET1, TET2, KMT2D, EZH2 nent, suggesting that loss is not correlated with progres-
and CREBBP) showed missense mutations in ten of 17 sion in BCL. Our data in HL cases is aligned with the
DLBCL tested (58.8% of cases) with CREBBP seen most observations of Siref et al. which reported near-universal
frequently (Figure 3). Details on the nature of these loss of expression in cHL.11
mutations and effect on protein are detailed in the Online Additionally, we assessed 17 DLBCL cases via WES for
Supplementary Figure S2. missense mutations explanative of 5hmC expression loss
This study demonstrated near-universal loss of 5hmC in the aforementioned epigenome-related genes
expression by immunohistochemistry across a wide (DNMT3A, TET1, TET2, IDH1, IDH2 and WT1). While
range of HGBCL and LGBCL (>90%) and HL (88%). The about half of these cases demonstrated mutations in one
observations support results from prior studies in BCL or more of these genes, most without alterations still
by Matsuda et al. and Siref et al., and extends the obser- showed 5hmC loss. This aligns with observations by
vation to additional BCL including MCL (typical and Lemonnier et al. in T-cell lymphoma where a significant
blastoid variants) as well as DLBCL-NOS.10,11 proportion of cases with 5hmC loss did not show muta-
The Matsuda study evaluated four subtypes of BCL tions in TET2 or DNMT3A.6 From a mechanistic perspec-
(follicular lymphoma [FL], chronic lymphocytic leukemia tive, BCL carry mutations in these epigenetic regulator
[CLL], MCL, and Burkitt lymphoma [BL]) and noted uni- genes less frequently. Rather, a subset of FL and DLBCL
form loss of 5hmC expression in FL and BL.10 Lack of (enriched for germinal center COO) harbor mutations in
staining in FL is congruent with our findings. We includ- the epigenetic modulator EZH2 that promotes increased
ed two cases of HGBCL (1 double hit, another without), suppressive trimethylation via H3K27me, affecting 5mC
Figure 3. Whole exome sequencing data looking at epigenetic regulators (IDH1, IDH2, TET1, TET2) mutations in 17 diffuse large B-cell lymphomas. (A)
Distribution of cases stratified by mutations and cell of origin (COO) status showing that most cases with mutations were enriched in the non-germinal center
COO. For more information, see the Online Supplementary Figure S2.

hydroxylation.12,13 The minimal number of mutated conducted the research and edited significant portions of the manu-
cases impedes speculation since we focused on just script.
DLBCL to ensure homogeneity. However, non-GCB pre- Acknowledgements: we thank Veronique Saada and Cyril
dominance in mutation-positive DLBCL in our study
Quivoron, Department of Translational Hematology and Pathology,
suggests that 5hmC loss is likely independent of EZH2
Gustave Roussy Cancer Campus, Villejuif, France for help with
mutation status, although the exact mechanism remains
poorly understood. From a translational perspective, it proofing the manuscriptand Jessica Robertson of the lymphoma pro-
was recently demonstrated that 5hmC in circulating cell- gram for help with logistics of the Hoogland Lymphoma Biobank
free DNA assessed by chemical labeling-based sequenc- tissue array data.
ing technology correlated with prognosis in newly-diag- Funding: the study was funded in part by the Hoogland
nosed DLBCL and hence examining TET1, TET2 in con- Lymphoma Biobank at the University of Chicago Medicine.
junction with 5hmC and 5mC may have prognostic util-
ity in the setting of BCL.2 References
In summary, we corroborate previously published data
and extend current insights by demonstrating loss of 1. Branco MR, Ficz G, Reik W. Uncovering the role of 5-hydrox-
ymethylcytosine in the epigenome. Nat Rev Genet. 2011;13(1):7-13.
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nostically useful in establishing a malignant B-cell phe- ymethylcytosines from circulating cell-free DNA in diffuse large B-
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ecular data. However, the loss of 5hmC in reactive back- 3. Chapel DB, Husain AN, Krausz T. Immunohistochemical evaluation
ground T cells (in normal and malignant nodes) indicates of nuclear 5-hydroxymethylcytosine (5-hmC) accurately distinguish-
that 5hmC loss in T cells is not a surrogate of es malignant pleural mesothelioma from benign mesothelial prolifer-
ations. Mod Pathol. 2019;32(3):376-386.
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Kevin S. Tanager,1* Jovian Yu,2* Brian C-H Chiu,3 Timothy 5. Moran-Crusio K, Reavie L, Shih A, et al. Tet2 loss leads to increased
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1 6. Lemonnier F, Poullot E, Dupuy A, et al. Loss of 5-hydroxymethylcy-
The University of Chicago Medicine, Department of Pathology, tosine is a frequent event in peripheral T-cell lymphomas.
Chicago, IL; 2The University of Chicago Medicine, Department of Haematologica. 2018;103(3):e115-e118.
Hematology/Oncology, Chicago, IL; 3The University of Chicago 7. Cimmino L, Dawlaty MM, Ndiaye-Lobry D, et al. TET1 is a tumor
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JK and GV contributed equally as co-senior authors Media; 2020.
9. Mayakonda A, Lin DC, Assenov Y, Plass C, Koeffler HP. Maftools:
Correspondence: efficient and comprehensive analysis of somatic variants in cancer.
GIRISH VENKATARAMAN - girish.venkataraman@uchospi- Genome Res. 2018;28(11):1747-1756.
10. Matsuda I, Imai Y, Hirota S. Distinct global DNA methylation status
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doi:10.3324/haematol.2021.279648 sine and 5-hydroxymethylcytosine. J Clin Exp Hematop.
2014;54(1):67-73.
Received: July 19, 2021.
11. Siref A, McCormack C, Huang Q, Lim W, Alkan S. Diminished
Accepted: November 30, 2021. expression of 5hmc in Reed-Sternberg cells in classical Hodgkin lym-
phoma is a common epigenetic marker. Leuk Res. 2020;96:106408.
Pre-published: December 16, 2021.
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Disclosures: no conflicts of interest to disclose phoma. J Clin Exp Hematop. 2016;56(2):71-78.
13. Schmitz R, Wright GW, Huang DW, et al. Genetics and pathogenesis
Contributions:: KST and JY conducted the research and wrote
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uated only after one CAR-T cell therapy course.

Immunophenotypic changes in leukemic blasts in All patients underwent routine diagnostic
children with relapsed/refractory B-cell precursor immunophenotyping and MRD detection by 8-10-color
acute lymphoblastic leukemia after treatment with flow cytometry according to the standard protocols of
CD19-directed chimeric antigen receptor (CAR)- the Moscow-Berlin group.7,8 During the study period,
expressing T cells MFC was performed on FACS Canto II, FACS Celesta
(both from Becton Dickinson, BD, US), CytoFlex and
Implementation of CD19 targeting with the bispecific Navios (both from Beckman Coulter, BC, US) flow
engager blinatumomab and T cells harboring chimeric cytometers. EuroFlow guidelines for machine perform-
antigen receptor (CAR-T) has resulted in significant ance monitoring were used.9 Cytometer Setup and
improvement in therapy results in children with Tracking beads (BD), Flow-Check Pro Fluorospheres
relapsed/refractory (R/R) B-cell precursor acute lym- (BC) and CytoFLEX Daily QC Fluorospheres (BC) were
phoblastic leukemia (BCP-ALL).1,2 However, some used for daily cytometer optimization. Normal lympho-
patients do not respond to therapy or experience cytes were used as the control for positivity/negativity
relapse.1,3 Minimal residual disease (MRD) persistence or definition. The tumor cell immunophenotype was ana-
reappearance during CD19-directed therapy is a key sign lyzed with a focus on markers applicable for MFC-MRD
predicting failure of pan-B-cell antigen targeting. Thus, investigation. The Online Supplementary Table S2 pro-
accurate detection of residual tumor cells has emerged as vides a list of monoclonal antibodies used for MFC-MRD
a key tool in evaluating the efficacy of such immunother- monitoring. CD22 and CD24 were additionally studied
apy. Loss of targeted antigen, which is the main escape as suggested by S. Cherian et al.10 MFC-MRD results
mechanism for BCP-ALL under selective pressures of were controlled with IG/TR gene rearrangements and
CD19-directed treatment, is a significant obstacle for fusion genes monitoring by molecular techniques with
MRD monitoring by multicolor flow cytometry (MFC). high concordance obtained (85% of qualitatively concor-
As MFC-MRD assessment is ordinarily based on CD19- dant MRD results for CD19-negative relapses and 79%
positive compartment analysis,4 possible CD19 negativ- for those who retained CD19 positivity). Expression of
ity of tumor cells increases the significance of other surface antigens was deemed positive if the antigen was
immunophenotypic markers for MFC data analysis. expressed on more than 20% of tumor cells.7 An
Nevertheless, antigens applicable for immunophenotyp- increase/decrease in the expression of each single anti-
ic aberrations evaluation are also known to undergo gen was defined as a change in the percentage of positive
expression modulation during therapy.5 Recently, we cells of more than 25%. Proportions of patients with sta-
demonstrated that each antigen useful for MFC-MRD ble and changed expression of each single antigen
detection can be either increased or decreased in chil- between CD19-negative and CD19-positive entities
dren treated with blinatumomab.6 As longer CAR-T cell were compared using Fisher’s exact test.
therapy persistence leads to prolonged pressure on The CD19 expression status on leukemic cells after
leukemic cells compared to blinatumomab,3 this phe- CAR-T cell therapy is depicted in Figure 1. The blasts of
nomenon could probably also affect the immunopheno- all patients except one were totally CD19-positive prior
typic stability of the leukemia, making MFC-MRD to CAR-T administration. In the remaining patient, only
assessment in such patients very challenging. The cur- 30% of BM cells expressed the target antigen, while in
rent report briefly summarizes our data on MFC-MRD cerebrospinal fluid (CSF), all leukemic BCP were CD19-
and relapse detection in patients treated with CAR-T cell positive. In five patients, the leukemic cells at relapse
therapy with a main focus on changes in the expression were CD19-positive, and in 11 relapsed patients, CD19
of markers that are relevant for MFC-MRD investiga- negativity was found. Two of 11 CD19-negative relapses
tion. displayed a small CD19-positive subpopulation (5% and
We carried out a retrospective review of 39 pediatric 15% respectively). Patients who were resistant to CAR-
patients with R/R ВCP-ALL who received CAR-T cell T cell therapy had preserved CD19 expression. The
therapy between February 2018 and September 2020 blasts of children who had leukemic cells in the BM on
and in whom tumor blasts were detectable in the bone MFC-MRD level only (n=19) were either CD19-positive
marrow (BM) at least once after CAR-T cell infusion. (n=10) or CD19 negative (n=9). CD19-negative leukemia
Among them, there were four patients resistant to CAR- appeared a little less frequent in children who have
T cell therapy, 16 who experienced relapse (>5% blast already received CD19 targeting (8 of 16, 50%) compar-
cells by MFC) and 19 children with blasts in the BM on ing to those who were not pretreated with blinatu-
MFC-MRD level. The characteristics of the patients, momab (12 of 19, 63%, P=0.506).
including cytogenetic data, are presented in the Online The immunophenotypic changes of leukemic cells in
Supplementary Table S1. The study was approved by the the 16 relapsed patients are presented in Figure 2A. For
Ethics Committee of the Dmitry Rogachev National all antigens applicable for MFC-MRD assessment except
Medical Research Center of Pediatric Hematology, CD58, expression changes were demonstrated, either
Oncology and Immunology, and informed consent for increased or decreased expression (Figure 3). On the
the collection and investigation of samples and for par- other hand, the proportion of patients with noted
ticipation in current study was obtained from patients’ changes was relatively small: CD34 was found to be the
parents or legal guardians. most unstable antigen (changes in 4 of 16 patients,
Subjects reported here were either included in a 25%). We did not find differences in the frequencies of
prospective trial of CD19 CAR-T cell therapy with the 4- changes in marker expression among CD19-negative
1BB costimulatory domain (clinicaltrials gov. Identifier: and CD19-positive relapses (Figure 2A). The expression
NCT03467256) or treated on a compassionate use basis. of CD22 and CD24, which are suggested to be candi-
Morphological examination and MFC-MRD detection in dates for CD19 substitution,10 displayed remarkable sta-
BM aspirates were performed before CAR-T cell infu- bility, although CD24 was tested only in part of the
sion, on days 14 and 28, and 2, 3, 4, 6, 8, 10, 12, and 24 study group. We analyzed 19 patients who did not
months after the beginning of CAR-T cell therapy. The relapse but had leukemic cells at the MRD level in the
immunophenotypic stability of leukemic cells was eval- BM by MFC at least once during the follow-up period. In

Figure 1. CD19 expression in the studied patients

(n=39). The study group included resistant patients
(n=4), patients with relapse (5 CD19-positive and 11
CD19-negative) and patients with blasts detected by
multicolor flow cytometry (MFC) at the minimal resid-
ual disease (MRD) level in the bone marrow at least
once (n=19).
addition to explicable downmodulation of CD19 in near- Supplementary Figure S1). Among the 11 CD19-negative
ly half of the patients (9 of 19), as in the relapsed relapses, six were preceded by CD19-negative MFC-
patients, CD34 displayed the highest instability (expres- MRD, and one was preceded by a progressive decrease
sion changes in 10 of 19 patients), while other antigens in CD19 expression on MFC-MRD, while in others,
again were rather stable, with no changes noted in the relapse developed directly after MFC-MRD negativity
expression of CD38, CD58 and CD22 (Figure 2B). The (Online Supplementary Figure S1).
leukemic cells in four resistant patients had a rather sta- As CD19 downregulation was noted in more than half
ble immunophenotypic profile with very rare cases of of the patients, for antigens useful for primary B-lineage
antigen expression changes (Figure 2C). gating (CD10, CD22, CD24)10,12 not only the stability
As CD19 is the best known target for immunotherapy but also homogeneous positivity on tumor cells is vital.
in BCP-ALL,1,2 CD19-directed CAR-T cells are widely In the current study, neither of these antigens displayed
used as the salvage approach for R/R patients.3 Thus, significant changes, although their expression prior to
lack or preservation of CD19 expression is crucial for fur- CAR-T cell infusion was not always satisfactory. Indeed,
ther treatment strategy choice. D. Libert et al. have in six of 33 and eight of 25 patients, less than 90% of the
shown that CD19 negativity occurs in nearly 65% of leukemic population before the CAR-T cell course was
relapses after CAR-T cell application11 with a massive CD22-positive (median 64%, range, 4-73%) and CD24-
disproportion in frequency between 4-1BB- and CD28- positive (median 0%, range, 0-79%), respectively. As
containing platforms (85% vs. 22% of relapsed patients, expected,13 in four patients with low CD22 and in five
respectively).11 In addition, as discussed previously,6 loss patients with low CD24, rearrangements involving the
of CD19 could break the well-established conventional KMT2A gene (KMT2A-r) were found. CD10
algorithm of MFC-MRD gating, as cytometric residual negativity/low expression (median 9%, range, 0-52%)
leukemia detection is based on B-cell compartment was also not restricted to KMT2A-r cases: it was found
investigation.4 In our study, 4-1BB CAR-T cells were in 13 patients (7 with KMT2A-r) and remained stable in
used, and the frequency of CD19-negative relapses was nine children. Moreover, in two of three cases of CD10
68.8%. Taking these patients together with the resistant upmodulation, this marker expression after CAR-T cell
patients (all remained CD19-positive) and the patients therapy was still not total.
with only MFC-detected MRD tumor cells, the total inci- Previously, we showed the results of a similar study
dence of CD19 negativity was 51.3% in all children in that included patients with R/R BCP-ALL treated with
whom tumor cells were detectable in the BM during fol- blinatumomab only.6 Compared to that after blinatu-
low-up after the first CAR-T cell therapy course. In all momab treatment, the proportion of cells with CD19
three patients in whom CD19-positive relapse was pre- loss after CAR-T cell treatment was significantly higher
ceded by MFC-MRD reappearance, the residual (51.3% vs. 27.1%), although no lineage switches were
leukemia had preserved expression of CD19 (Online noted in the current work (comparing 3 of 30 relapses

Figure 2. Frequency of changes in the immunophenotype of leukemic blasts. (A) Relapsed patients. (B) Patients who had detectable blasts in the bone marrow
only at the minimal residual disease (MRD) level. (C) Resistant patients.

after blinatumomab). Generally, the antigen profile of tered in this study (probably due to relatively few
leukemic cells after CAR-T treatment displayed relative- patients investigated), although they were rather fre-
ly higher stability, although CD58 was the most stable quent in case of blinatumomab application.6 Described
and CD34 was the most unstable marker irrespective of immunophenotypic features make this genetic subgroup
the type of immunotherapy given. Moreover, no differ- a very hard target for MFC-MRD technique. Being
ences in the immunophenotype of CD19-negative and notably rare in the general BCP-ALL population,
CD19-positive relapses and MFC-MRD persistence were KMT2A-r are present in a significant part of R/R
noted after CAR-T cell therapy, while after BiTE treat- patients14,15 (17.9% in the current study). Considering
ment, CD45 and CD38 changed more frequently in the this, addition of at least one early B-lineage antigen
case of CD19 loss.6 (iCD79a preferably) and careful analysis of the whole
With its peculiar immunophenotype and tendency immunophenotype with searching both for initial anti-
towards lineage switch, KMT2A-r BCP-ALL cases genic patterns and for cells different from normal coun-
require special solutions for MFC-MRD monitoring after terparts (with respect to specific regeneration patterns)16
CD19 targeting. In our cohort, there were seven patients could lead to reliable results, highly comparable with
with various types of KMT2A-r. Before CAR-T cell ther- molecular techniques data (86% with IG/TR rearrange-
apy, they were either CD10-negative (n=6) or borderline ments monitoring by next-generation sequencing and
CD10-positive (n=1, CD10 decreased at MFC-MRD 98% with polymerase chain reaction-based fusion genes
reappearance). Among six children with available CD22 transcript detection). Moreover, additional MRD confir-
and CD24 expression, only two were totally CD22-pos- mation tools, such as fluorescence in situ hybridisation or
itive and one was CD24-positive. Moreover, in three chimerism studies17 from sorted suspicious cells, can
patients of this subgroup, ALL recurrence was CD19- help in MFC-MRD monitoring of such a complicated
negative. Fortunately, no lineage switches were regis- BCP-ALL subgroup.
A B
Figure 3. Representative examples of immunophenotypic plasticity in

patients after T cells harboring chimeric antigen receptor. (A) Case of
CD19-negative relapse. (B) Case of reappearance of CD19-negative mini-
mal residual disease (MRD). (C) Case of phenotypically stable resistance to
the immunotherapy. CAR-T: CD19-directed chimeric antigen receptor (CAR)-
expressing T cells.

Despite the more stable antigen profile of leukemic for sustained remissions in leukemia. N Engl J Med.
cells after CAR-T cell therapy than after blinatumomab 2014;371(16):1507-1517.
treatment, more frequent loss of CD19, which is the cor- 3. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in chil-
dren and young adults with B-cell lymphoblastic leukemia. N Engl J
nerstone of B-lineage gating during conventional MFC- Med. 2018;378(5):439-448.
MRD detection, necessitates usage of additional B-lin- 4. Dworzak MN, Gaipa G, Ratei R, et al. Standardization of flow cyto-
eage antigens such as CD22, CD24, CD10 and iCD79a metric minimal residual disease evaluation in acute lymphoblastic
for primary B-cell compartment gating. Therefore, the leukemia: multicentric assessment is feasible. Cytometry B Clin
final search for MFC-MRD becomes more sophisticat- Cytom. 2008;74(6):331-340.
ed6,18 than conventional CD19-based methods, especial- 5. Dworzak MN, Gaipa G, Schumich A, et al. Modulation of antigen
ly taking into account specific BM regeneration patterns expression in B-cell precursor acute lymphoblastic leukemia during
that are more visible after CD19 targeting.16 induction therapy is partly transient: evidence for a drug-induced
regulatory phenomenon. Results of the AIEOP-BFM-ALL-FLOW-
Thus, considering the frequent loss of CD19 as well as MRD-Study Group. Cytometry B Clin Cytom. 2010;78(3):147-153.
the modulation of the expression of all other antigens 6. Mikhailova E, Gluhanyuk E, Illarionova O, et al.
relevant to MFC-MRD monitoring, a large panel of anti- Immunophenotypic changes of leukemic blasts in children with
bodies including additional B-lineage markers (CD22, relapsed/refractory B-cell precursor acute lymphoblastic leukemia,
CD24, iCD79a, etc.) combined with modified gating who have been treated with Blinatumomab. Haematologica.
strategy and consideration of specific background varia- 2020;106(7):2009-2012.
tions, should be applied to increase the effectiveness of 7. Novikova I, Verzhbitskaya T, Movchan L, et al. Russian-Belarusian
multicenter group standard guidelines for childhood acute lym-
MFC-MRD detection in BCP-ALL patients after CD19
phoblastic leukemia flow cytometric diagnostics. Oncohematology.
targeting by CAR-T cell therapy. 2018;13(1):73-82.
8. Popov A, Belevtsev M, Boyakova E, et al. Standardization of flow
Ekaterina Mikhailova, Olga Illarionova, Larisa Shelikhova, cytometric minimal residual disease monitoring in children with B-
Elena Zerkalenkova, Olga Molostova, Yulia Olshanskaya, cell precursor acute lymphoblastic leukemia. Russia–Belarus multi-
Galina Novichkova, Alexey Maschan, Michael Maschan and center group experience. Oncohematology. 2016;11(4):64-73.
Alexander Popov 9. Kalina T, Flores-Montero J, Lecrevisse Q, et al. Quality assessment
program for EuroFlow protocols: summary results of four-year
Dmitry Rogachev National Medical Research Center of Pediatric (2010-2013) quality assurance rounds. Cytometry A. 2015;87(2):
Hematology, Oncology and Immunology, Moscow, Russian Federation 145-156.
Correspondence: 10. Cherian S, Miller V, McCullouch V, et al. A novel flow cytometric
assay for detection of residual disease in patients with B-lym-
ALEXANDER M POPOV - uralcytometry@gmail.com phoblastic leukemia/lymphoma post anti-CD19 therapy.
doi:10.3324/haematol.2021.279677 Cytometry B Clin Cytom. 2018;94(1):112-120.
11. Libert D, Yuan CM, Masih KE, et al. Serial evaluation of CD19 sur-
Received: July 21, 2021. face expression in pediatric B-cell malignancies following CD19-tar-
Accepted: December 2, 2021 geted therapy. Leukemia. 2020;34(11):3064-3069.
12. Mejstrikova E, Hrusak O, Borowitz MJ, et al. CD19-negative relapse
Pre-published: December 16, 2021.
of pediatric B-cell precursor acute lymphoblastic leukemia following
Disclosures: no conflicts of interest to disclose. blinatumomab treatment. Blood Cancer J. 2017;7(12):659.
Contributions: EM and AP designed the study, acquired and ana- 13. De Zen L, Bicciato S, te Kronnie G, Basso G. Computational analysis
of flow-cytometry antigen expression profiles in childhood acute
lyzed cytometric data and wrote the paper; OI acquired and analyzed
lymphoblastic leukemia: an MLL/AF4 identification. Leukemia.
cytometric data; LSh collected clinical data and wrote the paper; EZ 2003;17(8):1557-1565.
acquired and analyzed cytogenetic and molecular genetic data, and 14. Queudeville M, Schlegel P, Heinz AT, et al. Blinatumomab in pedi-
wrote the paper; OM collected clinical data; YuO acquired and ana- atric patients with relapsed/refractory B-cell precursor acute lym-
lyzed cytogenetic and molecular genetic data; GN and MM designed phoblastic leukemia. Eur J Haematol. 2021;106(4):473-483.
the study and acted as the general supervisor; AM designed the study, 15. Winters AC, Bernt KM. MLL-rearranged leukemias-an update on
acted as the general supervisor and wrote the paper. science and clinical approaches. Front Pediatr. 2017;5:4.
16. Mikhailova E, Semchenkova A, Illarionova O, et al. Relative expan-
Funding: the KMT2A rearrangement assessment study was sup- sion of CD19-negative very-early normal B-cell precursors in chil-
ported by RFBR grant number 7-29-06052 and Presidential grant dren with acute lymphoblastic leukaemia after CD19 targeting by
number MK-1645.2020.7 and 075-15-2020-338. Flow cytometric blinatumomab and CAR-T cell therapy: implications for flow cyto-
minimal residual disease evaluation was supported by RFBR grant metric detection of minimal residual disease. Br J Haematol.
number 18-29-09132. 2021;193(3):602-612.
17. Semchenkova A, Brilliantova V, Shelikhova L, et al. Chimerism eval-
uation in measurable residual disease-suspected cells isolated by
References flow cell sorting as a reliable tool for measurable residual disease
verification in acute leukemia patients after allogeneic hematopoiet-
1. Topp MS, Gokbuget N, Zugmaier G, et al. Phase II trial of the anti-
CD19 bispecific T cell-engager blinatumomab shows hematologic ic stem cell transplantation. Cytometry B Clin Cytom.
and molecular remissions in patients with relapsed or refractory B- 2020;100(5):568-573.
precursor acute lymphoblastic leukemia. J Clin Oncol. 18. Cherian S, Stetler-Stevenson M. Flow cytometric monitoring for
2014;32(36):4134-4140. residual disease in B lymphoblastic leukemia post T cell engaging
2. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells targeted therapies. Curr Protoc Cytom. 2018;86(1):e44.

sperm are under development.6 In fertility preservation

Early testicular maturation is sensitive to deple- programs, patients with SCD constitute up to 35% of all
tion of spermatogonial pool in sickle cell disease patients with non-malignant diseases.4 Based on limited
case series, even before HU therapy, most young men
Cryopreservation of testicular tissues before with SCD have semen quality below the World Health
hematopoietic stem cell transplant (HSCT) is offered by Organization standard minimum criteria and spermato-
specialized centers worldwide to boys with sickle cell gonial numbers in prepubertal boys have been shown to
disease (SCD). In order to elucidate whether hydroxyurea be reduced.5,7-8 A recent study with a small cohort size
(HU) therapy or disease-related factors affect the sper- concluded that spermatogonial numbers in HU exposed
matogonial quantity of SCD patients, we collected clini- patients (n=17) were not significantly different from
cal data of 29 boys (age range, 2.8-15.1 years) and calcu- those without HU exposure (n=13).9
lated a Z-score. Our results show that most spermatogo- The current study was designed to evaluate if dose,
nial numbers (n=17) were below the reference values of exposure time, age at HU initiation, iron load or disease
healthy boys. There was a correlation between the num- severity affects spermatogonial quantity in SCD patients.
ber of spermatogonia and the age at HU initiation Insights enable clinicians to prospectively counsel
(P=0.029, r=0.476). This study suggests that besides fac- patients and parents about the risks for reduced sper-
tors intrinsic to SCD, an HU exposure in early life may matogonial numbers prior to HSCT.
lead to further depletion of spermatogonia reducing the Testicular tissues from 29 boys with SCD (age range,
potential for successful fertility preservation. 2.8-15.1 years) participating in the fertility preservation
SCD is the most common corpuscular anemia, with programs Androprotect (German network, n=19) and
approximately 300,000 affected babies born anually.1 Nordfertil (Nordic network, n=10) between 2013-2020
SCD manifests in hemolytic anemia and episodes of were included. Also, testicular tissue from two adult SCD
microvascular vaso-occlusion leading to end-organ patients (age range, 21.5-48.5 years, University Hospital
ischemia-reperfusion injury and organ damage. Münster) were available. All age-appropriate patients and
Treatment to alleviate complications involves HU.14 In the guardians gave written informed consent for the use
high-resource countries, allogeneic HSCT is proposed as of testicular tissues for research. All procedures were in
a cure. However, there are significant risks, including accordance with the Declaration of Helsinki and Ethical
acute toxicity and late effects, e.g., infertility caused by approval was obtained from the responsible Ethics
previous gonadotoxic conditioning.3 Boards.
As fertility preservation, a growing number of centers Hormone levels, testicular volume, and markers of
worldwide cryopreserve either sperm or testicular tissues chronic hemolysis were determined, information detail-
of prepubertal boys prior to HSCT.4,5 Cryopreservation of ing transfusions, HU therapy and SCD complications was
testicular tissue containing undifferentiated germ cells, collected, and severity scores were calculated (Table 1;
termed spermatogonia, remains an experimental Online Supplementary Table S1).10,11
approach as protocols to differentiate spermatogonia into Patients underwent open testicular biopsy during
Table 1. Clinical characteristics and Spearman correlations for mean spermatogonial numbers per round tubular cross section and fertility
index Z-scores of the 24 patients with testicular samples containing a minimum of 25 evaluated tubules.
Clinical characteristics S/T Z-score FI Z-score
N Median (range) P r P r
Age 24 7.1 (2.8-15.1) 0.015* 0.492* 0.073 0.373
Hematological parameters
HbS level, % of total Hb 16 54.2 (9.4-85.3) 0.778. - 0.077 0.571 - 0.153
HbF level, % of total Hb 13 12.0 (3.0-34.0) 0.553 0.181 0.734 0.105
MCV, fL 15 86.0 (76.0-123.0) 0.512 0.184 0.838 - 0.058
Platelets, G/L 22 294.0 (104.0-1,103.0) 0.497 0.153 0.990 0.003
Neutrophils, G/L 20 4.2 (0.5-7.9) 0.925 0.023 0.853 0.044
Biomarkers of chronic hemolysis
Hb, mg/L 23 100.0 (74.0-134.0) 0.667 - 0.095 0.645 - 0.101
LDH, U/L 20 158.0 (86.2-262.3) 0.405 0.197 0.046* 0.451*
Biomarker of iron status
Ferritin, mg/L 19 305.0 (51.0-3,244.0) 0.304 0.249 0.209 0.302
HU therapy†
Dose, mg/kg 24 22.5 (0.0-45.0) 0.766 - 0.064 0.537 - 0.132
Exposure time, d 24 750.5 (0.0-2,839.0) 0.856. - 0.039 0.565 - 0.124
Age at HU start, y 24 5.0 (1.0-9.9) 0.029* 0.476* 0.024* 0.490*
Dose multiplied by
exposure time, mg x d 24 17,434.0 (0.0-1,01636.0) 0.984 0.004 0.720 - 0.077
Complications
Pain crisis per year, n 19 0.4 (0.0-4.0) 0.486 0.170 0.158 0.337
Number of ATS, n 24 0.0 (0.0-3.0) 0.703 - 0.082 0.802 0.054
Score (Sebastiani et al.)10 24 0.4 (0.3-0.6) 0.439 - 0.166 0.748 - 0.069
Score C (Van den Tweel et al.)11 24 50.0 (5.0-80.0) 0.168 0.291 0.149 0.304
ATS indicates acute thorax syndrome; FI: feritlity index; S/T: spermatogonia per round tubular cross-section; Hb: hemoglobin; HbF: fetal hemoglobin; HbS: sickle hemoglobin;
HU: hydroxyurea; y: year; x d: per day; LDH: lactate dehydrogenase; MCV: mean corpuscular volume; P: P-value and r, Spearman correlation coefficient. *P<0.05. † Washout
period of 2 weeks - 4 months was used prior to biopsy for three HU-exposed patients. All 3 patients received transfusions.

which less than 20% of the testicular volume of one containing somatic Sertoli cells. Mean spermatogonial
testis was sampled. Two-thirds of the tissues were trans- numbers per round tubular cross section (S/T) were
ported to the research center, partly overnight, and were assessed to obtain comparable spermatogonial numbers
cryopreserved. The remaining third was fixed in formalin across samples (Online Supplementary Table S1).
in the Nordfertil program. Tissues from the Androprotect Moreover, the fertility index (FI) as the percentage of
program and adult patients were fixed in Bouin's solu- tubular cross-sections containing spermatogonia was
tion. After embedding in paraffin, all tissues were sec- determined.
tioned (3-5 µm), and two independent sections (distance In order to control physiological variation in spermato-
>15 µm) were immunostained with MAGEA4, following gonial numbers during development, Z-scores were cal-
published protocols.12,13 At Karolinska Institutet, a fluo- culated for S/T and FI using reference means.14 For statis-
rescence microscope (Eclipse E800, Nikon; Japan) was tical analysis, only samples with >25 round tubules were
employed for analysis. In Münster, images were captured considered, resulting in the inclusion of n=24 prepuber-
using the PreciPoint M8 microscope/scanner and subse- tal/pubertal patient samples (Online Supplementary Table
quently analysed using the ViewPoint light software S1). Spearman correlation coefficient (r) and ROC analy-
(1.0.0.9628, PreciPoint, Freising, Germany). sis were performed to determine the relationships
Spermatogonia were identified based on their morpholo- between spermatogonial quantity, age and treatment
gy (size, shape), location and MAGEA4 expression characteristics using IBM SPSS Statistics V26.0 software
(Online Supplementary Figure S1). Using a blinded (IBM Corporation, Armonk, NY; US) and GraphPad
approach, all round tubular cross-sections within the tis- Prism Version 8.4.3(471) (GraphPad Software, San Diego,
sue sections were quantified (mean: 94, range, 0-344) and CA, US).
classified as tubules with spermatogonia and tubules only Out of the 29 prepubertal/pubertal samples, the major-
B C
D E
Figure 1. Number of spermatogonia per round tubular cross-section and fertility index in patients with sickle cell disease. (A) Representative images showing
MAGEA4-positive spermatogonia (immunohistochemical staining) in patients with sickle cell disease (SCD) and controls (n=3, each) of different ages. Scale
bars: 20 μm (4-10 years), 50 μm (42-48 years), arrows indicate spermatogonia and arrowheads Sertoli cells. Scale bars were added to the images using Adobe
Photoshop CS2 (Adobe Systems, California, US). Objective: Olympus PlanC N 60x/0.80 (PreciPoint, Freising, Germany). (B) Spermatogonia per round tubular
cross-section (S/T) and (C) S/T Z-score as well as the fertilty index (FI) (D) and FI Z-scores (E) by age in 29 patients with SCD. In (B) and (D) data are plotted on
lines corresponding Z-scores for the mean reference values (Adapted from Funke et al.).14 A significant correlation exists between younger age and lower S/T Z-
scores (P=0.015, r=0.492). SD: standard deviation.

ity (n=17) scored below previously published reference 0.84, 95% confidence interval [CI]: 0.67–1.00) with 75%
values of S/T (Z-score <-3), but only four were devoid of sensitivity and 82% specificity to identify testicular sam-
spermatogonia (Figure 1A to C). Both adult patients ples containing very low spermatogonial numbers (S/T
showed spermatogonial numbers below the reference Z-score <-7). Therefore, testicular maturation during the
values (Online Supplementary Table S1). Our observations first 3 years of life may be especially sensitive to deple-
confirm previous reports of severely decreased spermato- tion of the spermatogonial pool in SCD patients.
gonial numbers.5,7,9 Additionally, most patients (n=21/29) Significant correlation however does not prove that early
had FI Z-scores below the reference values (Figure 1D HU initiation determines the low S/T Z-score at very
and E), illustrating that spermatogonia were only focally young age.
present. Older age correlated significantly with higher S/T Z-
In line with Gille et al.,9 no difference was observed scores (P=0.015, r=0.492) and a cut-off age of 8.8 years
between the numbers of spermatogonia in HU-exposed identified with 100% sensitivity and 80% specificity
(n=21) and non-exposed groups (n=3) although the small (AUC 0.84, 95% CI: 0.64–1.00) S/T Z-scores within the
sample size results in limited informative value normal range (>-3). Our data confirm that the pubertal
(P=0.9999). Three patients with a wash out period of 2 increase in spermatogonial numbers does occur in SCD
weeks to 4 months without HU prior to biopsy were patients in line with a previous report.8 Also in cases of
included in the HU-exposed group. There was no corre- early HU exposure we cannot exclude that spermatogo-
lation between spermatogonial numbers and HU dose or nia maintain their capacity of expansion at puberty.
exposure time (Table 1; Figure 2A and B). Besides the potentially harmful effects of early HU
Importantly, younger age correlated significantly with exposure, other factors intrinsic to SCD need to be con-
lower S/T (P=0.015, r=0.492) Z-score (Table 1; Figure sidered. There is wide variability in the phenotypic sever-
1C). Since 2014, an international consensus has recom- ity of SCD. This variation can be explained partly by dif-
mended HU therapy for all infants with SCD aged 9 ferences in the total hemoglobin concentration, the mean
months or older to reduce complications.2 Our observa- corpuscular hemoglobin concentration, iron load, coin-
tions reflect this recommendation; younger patients had heritance of a-thalassemia and fetal hemoglobin persist-
an earlier HU initiation (P<0.001, r=0.713), leading to HU ence.15 In line with this, low hemoglobin values observed
exposure at an earlier time of testicular development in the present study correlated with a high frequency of
compared to patients diagnosed before 2014. The young pain crises (P=0.026, r=-0.524). However, we could not
age at the HU initiation further correlated with lower S/T show any correlation between spermatogonial numbers
(P=0.029, r=0.476) and FI Z-scores (P=0.024, r=0.490) and the severity of SCD indicated by the total number of
(Figure 2C and D). By using ROC analysis, we were able pain crises per year, the number of acute thorax syn-
to unveil that an age of 2.4 years at HU initiation showed dromes or SCD severity scores (Table 1; Figure 2E and F).
a good diagnostic value (area under the curve [AUC]: This absence of correlation may be due to the limited
A B C
D E F
Figure 2. The impact of hydroxyurea dose, age at hydroxyurea therapy initiation and the number of pain crises as disease severity marker on the numbers of
spermatogonia per round tubular cross-section and fertility index in patients with sickle cell disease. Graphical display of the numbers of spermatogonia per
round tubular cross section (S/T) and fertility index (FI) Z-score by hydroxyurea (HU) dose (A and B), age at HU therapy initiation (C and D) and the numbers of
pain crises (E and F). In linear regression analysis, the age at HU therapy initiation reached statistical significance. P indicates P-value and r Spearman correla-
tion coefficient.

number of patients enrolled. Altogether seven patients Received: July 5, 2021.

were under regular transfusion regimes and six patients Accepted: December 7, 2021.
received pre-operative transfusions prior to testicular
biopsy (Table 1). With limited informative value, the Pre-published: December 9, 2021.
serum ferritin showed no correlation with spermatogo- Disclosures: no conflicts of interest to disclose.
nial numbers suggesting that iron load plays no signifi- Contributions: KMBF collected and analyzed data, prepared fig-
cant role in the testicular toxicity of this patient popula- ures, drafted the manuscript; NN and JBS prepared samples, set up
tion. the experimental design, collected, analyzed and interpreted data, and
In summary, most boys with SCD in this study had drafted the manuscript; AJ set up the experimental design and collected
spermatogonia within their testicular tissue although at data; AKL, CL, HAMBO, JMDP, MS and VN collected data; CK
reduced numbers. Importantly, an association between and AS collected data and were responsible for clinical patient care, SK
reduced spermatogonial numbers and the young age was supervised clinical patient care, clinical and laboratory testing and docu-
found while boys older than 8.8 years showed spermato- mentation in the University Hospital of Mun̈ ster; KJ set up the experi-
gonial numbers within the normal range. Reduced sper- mental design, collected and interpreted data, and drafted the manu-
matogonial numbers were further shown to correlate script. All authors had significant intellectual contribution in reviewing
with the age at HU initiation. An age of 2.4 years or less the manuscript and approved the final article.
at initiation associated with severely decreased sper- Acknowledgments: the authors would like to thank Dr. E. Oliver
matogonial numbers suggesting that spermatogonia (Karolinska Institutet, and Karolinska University Hospital, Stockholm,
and/or testicular somatic cells may be especially sensitive Sweden) for language editing. Moreover, the authors would like to
to disturbances such as HU exposure or disease compli- thank all clinical units involved in the study, especially Prof. Dr. U.
cations. The potential risk of further compromising the Kontny, Dr. L. Lassay, Prof. Dr. N. Wagner (RWTH Aachen,
already limited fertility by early HU initiation, must be University Hospital, Aachen, Germany), Dr. B. Bernbeck, Prof. Dr.
weighed against the overwhelming evidence that HU sig- D. T. Schneider (Klinikum Dortmund, Dortmund, Germany), Prof.
nificantly decreases morbidity and increases the lifespan Dr. A. Borkhardt, Prof. Dr. R. Meisel, Dr. F. Schuster (Department of
among SCD patients.2 Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-
For all boys that are mature enough, it is most impor- Heine-University, Duesseldorf, Germany), Dr. M. Höfs, Prof. Dr. D.
tant that they are offered sperm cryopreservation. Reinhardt, Dr. S. Schönberger, B. Schwert (Essen University Hospital,
Moreover, testicular function should be studied if HU Essen, Germany), Prof. Dr. P. Bader, Dr. A. Barnbrock (University
therapy must be discontinued to increase our knowledge Hospital Frankfurt, Frankfurt, Germany), Dr. M. Bleeke (Center for
of possible testicular recovery. For prepubertal boys, it is Obstetrics and Pediatrics, Sektion für Pädiatrische
essential that they are given the opportunity to cryopre- Stammzelltransplantation und Immunologie University Medical Center
serve immature testicular tissue prior to HSCT after Hamburg-Eppendorf, Hamburg, Germany), Prof. Dr. M. Fisch
appropriate counseling regarding potentially decreased (Department of Urology, University Medical Center Hamburg-
spermatogonial numbers. Eppendorf, Hamburg, Germany), Dr. M. Bleeke and Prof. Dr. I.
Müller (Center for Obstetrics and Pediatrics, Sektion für Pädiatrische
Klara M. Benninghoven-Frey,1* Nina Neuhaus,1* Atte K. Stammzelltransplantation und Immunologie, University Medical
Lahtinen,2,3 Claudia Krallmann,1 Joana M.D. Portela,4 Andrea Center Hamburg-Eppendorf, Hamburg, Germany), Prof. Dr. S.
Jarisch,5 Verena Nordhoff,1 Armin Soave,6 Hajar A.M. Ba Rutkowski (Center for Obstetrics and Pediatrics, University Medical
Omar,7 Mikael Sundin,8,9 Cecilia Langenskiöld,10 Sabine Center Hamburg-Eppendorf, Hamburg, Germany), PD Dr. A.
Kliesch,1 Jan-Bernd Stukenborg7 and Kirsi Jahnukainen7,11 Claviez (University Medical Center Schleswig-Holstein, Kiel,
1
Center of Reproductive Medicine and Andrology, University of Germany), and PD Dr. T. Nüßlein (Gemeinschaftsklinikum
Münster and University Clinic Münster, Münster, Germany; 2Applied Mittelrhein, Koblenz, Germany). This project was supported by J.
Tumor Genomics Research Program, Faculty of Medicine, University of Salzig and J. Dabel (Center of Reproductive Medicine and Andrology,
Helsinki, Helsinki, Finland; 3Department of Medical and Clinical University of Münster and University Clinic Münster, Münster,
Genetics / Medicum, Faculty of Medicine, University of Helsinki, Germany), who provided excellent technical support.
Helsinki, Finland; 4Center for Reproductive Medicine, Research Funding:this study was supported by grants from The Swedish
Institute Reproduction and Development, Amsterdam Universitair Childhood Cancer Foundation (PR2019-0123; TJ2020-0023;
Medische Centra, University of Amsterdam, Amsterdam, the PR2015-0073, TJ2015-0046) (JBS, KJ), the Jane and Dan Olssons
Netherlands; 5Division of Stem Cell Transplantation and Immunology, Foundation (2016-33) (JBS), the Finnish Cancer Society (KJ), the
Department of Children and Adolescent Medicine, University Hospital Finnish Foundation for Pediatric Research (KJ), the Magnus Bervalls
Frankfurt, Johann Wolfgang Goethe University, Frankfurt am Main, Foundation (JBS), the Swedish Research Council (2018-03094; 2016-
Germany; 6Department of Urology, University Medical Center 01296) (JBS, KJ), the Birgitta and Carl-Axel Rydbeck’s Research
Hamburg-Eppendorf, Hamburg, Germany; 7NORDFERTIL Grant for Pediatric Research (2020-00348; 2020-00335) (JBS, KJ),
Research Lab Stockholm, Department of Women’s and Children s and the Väre Foundation for Pediatric Cancer Research (AKL, KJ).
Health, Karolinska Institutet, and Karolinska University Hospital, This work was also supported by the German Research Foundation
Stockholm, Sweden; 8Division of Pediatrics, Department of Clinical Grand CRU326 (NN). Moreover, we thank the Faculty of Medicine
Science, Intervention and Technology, Karolinska Institutet, Stockholm, of the University of Münster for the financial support of KMBF.
Sweden; 9Section of Hematology, Immunology and HCT, Astrid
Lindgren Children’s Hospital, Karolinska University Hospital, References
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Supplementary Table S1) were cultured in PMX supple-

Targeting B-cell maturation antigen increases sensi- mented with the prosurvival cytokines interleukin 6 (IL-
tivity of multiple myeloma cells to MCL-1 inhibition 6) and APRIL, which are found at high concentrations in
the bone marrow and plasma of MM patients.5 The com-
Interfering with the mechanisms by which malignant bination of both cytokines preserved MM cell viability
plasma cells develop drug resistance is critical for pre- significantly better than in unstimulated controls (Online
venting relapse in multiple myeloma (MM). Supplementary Figure S1A). Primary MM cell survival in
Downregulation of target antigen is one of the escape PMX was significantly higher than in two-dimensional
mechanisms limiting the success of promising therapeu- (2D) cultures, as determined by cell viability percentages
tic approaches such as B-cell maturation antigen and by absolute cell number (Figure 1A to C).
(BCMA)-targeted immunotherapy.1,2 Despite high Compound diffusion in PMX was evaluated by exposing
response rates and depth of responses after treatment PMX-cultured MM cells to molecules added to the
with BCMA-targeting agents, patients eventually supernatant. In antibody-dependent cellular cytotoxicity
relapse.1–3 In most cases, the residual MM cells persisting (ADCC) and complement-dependent cytotoxicity (CDC)
after BCMA-based immunotherapy express reduced assays with the anti-CD38 antibody Daratumumab, sur-
BCMA levels,1,2 suggesting that eliminating this face-bound anti-CD38 was detected in all cells (Online
BCMAlow MM cell pool could significantly delay or pre- Supplementary Figure S1B), and MM cell line sensitivity to
vent relapse. An optimal therapeutic approach may CD38 targeting in PMX was comparable to that in 2D
therefore involve sequential treatment strategies target- culture (Online Supplementary Figure S1C), showing that
ing the critical anti-apoptotic pathways in refractory both small and large molecules diffuse efficiently in this
MM cells selected right after BCMA-based immunother- hydrogel. Importantly, MM cells retained expression of
apy. However, ex vivo identification of potentially rele- plasma cell-associated markers when cultured in PMX.
vant sequential treatment strategies involving culture of BCMA expression significantly increased over time,
primary MM cells for several days, has been limited due while CD38 levels remained mostly stable (Figure 1D
to the low viability of malignant plasma cells outside the and data not shown). Due to limited plasma cell viability
bone marrow niche. Here, we describe a reproducible in 2D culture, previous reports testing ex vivo chemosen-
culture system for primary MM cells based on a synthet- sitivity of primary MM cells are based on their co-culture
ic hydrogel where viability of primary myeloma cells is with either MSC or MS-5 cells. 8,10,11 In order to relate to
preserved even in the absence of additional stromal sub- these previous studies, we compared MM drug respons-
sets. Drug screening of MM samples in this three-dimen- es in PMX supplemented with either cytokines or MSC.
sional (3D) platform revealed a dynamic interplay Specific apoptosis induced by MCL-1i in PMX + IL-
between BCMA and MCL-1, showing that BCMAlow 6/APRIL was similar to that measured in PMX + MSC
MM cells are highly sensitive to MCL-1 inhibitors (MCL- co-cultures (Figure 1E). Furthermore, it has been report-
1i), and that pretreatment with BCMA-blocking antibod- ed that primary MM cells with a t(11;14) translocation
ies significantly increases MCL-1i efficacy in MM cells. are highly sensitive to BCL-2 inhibition,12 and we con-
The BCMA (TNFRSF17) surface receptor is selectively firmed this observation in PMX-based drug screenings
present on plasma cells, and its expression is significant- (Online Supplementary Figure S1D).
ly higher in high- versus low-risk MM and in Over culture in the presence of IL-6 and APRIL, sensi-
relapsed/refractory versus newly diagnosed patients.4 tivity of MM cells to MCL-1i decreased significantly
This progressive BCMA increase is due to loss of cells (Figure 1F), evocative of drug sensitivity loss in the phys-
expressing a relatively lower level of this receptor,4 sug- iological niche. Both IL-6 and APRIL have been linked to
gesting that malignant cells with highest BCMA expres- acquisition of drug resistance in MM, while previous
sion may have a selective advantage over the course of reports rely mostly on the study of cell lines.5,13,14
the disease. Multiple BCMA-targeting approaches are Next, we assessed BCMA levels on MM cells that per-
being tested in the clinic for the treatment of MM, sisted after each treatment. Remarkably, BCMA expres-
including antibody-drug conjugates, bispecific sion on malignant cells that survived MCL-1 inhibition
BCMAxCD3 antibodies, and anti-BCMA chimeric anti- was significantly higher than on cells left untreated or
gen receptor (CAR) T cells. 1–3 Signaling through the exposed to other agents (Figure 2A). Distribution of
APRIL-BCMA axis promotes MM cell proliferation and BCMA expression in viable MM cells indicated that
survival, and induces the expression of the anti-apoptot- MCL-1 inhibition preferentially spares BCMAhi cells
ic proteins BCL-2 and MCL-1.5 MCL-1 is a key pro-sur- (Figure 2B). Taken together, these data reveal a connec-
vival factor for healthy and malignant plasma cells, and tion between BCMA expression and dependence on
elevated MCL-1 expression in MM is associated with MCL-1, and indicates that MCL-1i treatment eliminates
chemoresistance and shorter event-free survival.6 Several BCMAlow MM cells. This observation is especially rele-
compounds targeting MCL-1 are currently tested in clin- vant considering that residual MM cells remaining after
ical trials, including the potent MCL-1 inhibitor (MCL- BCMA CAR-T cell therapy show significantly reduced
1i) S63845.7 MM cell sensitivity to MCL-1i treatment is BCMA levels, suggesting immune selection for BCMA-
highly variable between patients,8 as is observed with dim/negative clonal variants.1,2 Our data indicates that
BCMA-targeting immunotherapy,2,9 stressing the need combining MCL-1 inhibitors with BCMA-targeting
for primary MM cell-based ex vivo studies on the factors immunotherapies may increase their efficacy by deplet-
conditioning the response to these treatment modalities. ing residual BCMAlow cells.
Our culture approach for patient-derived MM cells is We next addressed the relation between BCMA block-
based on Puramatrix (PMX) hydrogel, which has previ- ade and MCL-1i-induced apoptosis by treating primary
ously been validated for MM cell co-culture with mes- MM samples with a BCMA-targeting antibody (anti-
enchymal stromal cells (MSC).10 A major advantage of BCMA). MM cell sensitivity to single-agent anti-BCMA
PMX over other gels such as Matrigel or fibrin scaffolds was heterogeneous and did not correlate with BCMA
is its chemically-defined composition (R-A-D-A repeats), expression in MM cells before treatment (Online
which eliminates batch-to-batch variability issues. MM Supplementary Figure S2A and B). Importantly, there was
cells (patient information is listed in the Online a clear inverse correlation between MCL-1i and anti-

BCMA sensitivity in MM samples: MCL-1i-resistant ment, neither by fluorescence-activated cell sorting

MM cells were highly sensitive to anti-BCMA, and vice (FACS) nor by western blot analysis (Figure 3B; Online
versa (Figure 3A). We did not observe changes in MCL-1, Supplementary Figure S2C and D). Considering that MCL-
BCL-2 or BCL-XL protein levels after anti-BCMA treat- 1i spares MM cells with highest BCMA expression,
A B
Figure 1. Primary multiple myeloma cell viability,

phenotype, and drug sensitivity testing after cul-
ture in Puramatrix hydrogel. (A) Representative
flow cytometry plots of multiple myeloma (MM)
cells after culture. MM bone marrow mononuclear
cells were seeded in Puramatrix (PMX) in the pres-
C D ence of IL-6 and APRIL (100 ng/mL each), for 7
days. All experiments involving primary MM cell
culture were performed in this setting, unless oth-
erwise stated. MM cells were gated as CD38+
CD138+ (left), and viable cells were identified as
DiOC6+ TOPRO3- cells (right). (B) Frequency and
(C) absolute cell number (expressed as fold-
increase relative to two-dimensional [2D] controls)
of MM cells after 7 days in culture in 2D or PMX
(n=18). Bars indicate mean + standard error of the
mean. (D) Mean fluorescence intensity (MFI) of B-
cell maturation antigen (BCMA) in MM cells was
measured by flow cytometry after 2 or 7 days in
PMX culture (n=12). (E) Simple linear regression
analysis comparing specific apoptosis (%) induced
E F by the MCL-1 inhibitor (MCL-1i) S63845, in n=12
MM samples cultured in PMX supplemented with
either IL-6 + APRIL or mesenchymal stromal cells
(MSC, 80,000/well 10). MCL-1i (1,000 nM) was
added on day 1, and MM viability was measured
by flow cytometry 24 hours later. Specific apopto-
sis (%) was calculated by applying the following for-
mula: [(%viable Nil - %viable treated)/ %viable Nil]
x 100. (F) Specific apoptosis induced by MCL-1i in
PMX-cultured MM cells (n=12). MCL-1i (100 nM)
was added on day 1 or 6, and viability was meas-
ured 24 hours later (day 2 or 7, respectively). Each
dot represents an individual sample. Statistical dif-
ferences between 2 groups were analyzed using
paired t-tests. *P<0.05; **P<0.01.
Figure 2. BCMAlow multiple myeloma cells are sen-

sitive to MCL-1 inhibition. (A) Mean fluorescence
intensity (MFI) of B-cell maturation antigen (BCMA)
(expressed as x-fold of MFI in untreated controls)
as measured by flow cytometry in alive multiple
myeloma (MM) cells after treatment with either 4
nM Bortezomib (Bor), 1,000 nM dexamethasone
(Dex), 100 nM BCL-2 inhibitor (BCL-2i; ABT-199), or
100 nM MCL-1i (S63845) for 24 hours (n=4-9).
B Each dot represents an individual sample.
Statistical differences between groups were ana-
lyzed using a one-way ANOVA with Bonferronis’
multiple comparison test. **P<0.01. (B)
Representative histograms showing BCMA expres-
sion in MM cells from 4 different primary samples
(MM1-MM4) cultured in Puramatrix (PMX).
Histograms show BCMA in alive MM cells untreat-
ed (black) or after a 24-hour treatment with 100
nM MCL-1i (red).

which may largely rely on BCMA signaling for survival, cultured in the presence of anti-BCMA for 6 days before
we next evaluated if co-treatment with MCL-1i and anti- a 24-h culture with MCL-1i. Strikingly, apoptosis
BCMA has synergistic effects on MM cell apoptosis. In induced by MCL-1 inhibition was significantly higher
five of seven primary MM samples tested, MCL-1i + after a 6-day pretreatment with anti-BCMA, as com-
anti-BCMA combination had a more than additive effect pared to treatment with an isotype control or a shorter
on MM cell killing (Figure 3C and D), but we did not (24 hours) BCMA blockade period (Figure 3E and F).
observe consistent synergy when combining these drugs Increased MM cell susceptibility towards sequential
with primary MM samples. Next, we addressed whether BCMA and MCL-1 targeting was also observed follow-
increased MCL-1i efficacy in a context of BCMA blocking co-culture with MSC in PMX (Online Supplementary
ade could be further enhanced by following a sequential Figure S2E to H). Pretreatment with either Daratumumab
treatment strategy. To this end, primary MM cells were (anti-CD38) or tocilizumab (anti-IL6R) did not sensitize
A B
C D
E F
Figure 3. Pretreatment with anti-BCMA maturation antigen increases MCL-1 inhibitor efficacy in multiple myeloma cells. (A) Simple linear regression analysis
comparing specific apoptosis after 24-hour treatment with either 100 nM MCL-1i or 5 ug/mL anti-B-cell maturation antigen (anti-BCMA) (a-BCMA; Vicky-1)
(n=12). (B) Flow cytometry analysis of MCL-1 (left) and BCL-2 (right) expression in primary multiple myeloma (MM) cells cultured for 2 or 7 days with IL-6 + APRIL
(100 ng/mL each) in the presence of 5 ug/mL isotype control antibody (black) or anti-BCMA (grey). Mean + standard error of the mean (n=3). (C) Specific apop-
tosis induced by 100 nM MCL-1i (black), 5 ug/mL anti-BCMA (grey), or their combination (blue) in 7 primary MM samples cultured in Puramatrix (PMX). Drugs
were added on day 6, and MM cell viability was measured 24 hours later. (D) Plots comparing expected (EXP) to observed (OBS) specific apoptosis induced by
combining anti-BCMA 5 ug/mL and MCL-1i (100 nM) for 24h (n=7). Hypothetical expected (EXP) specific apoptosis assumes an additive effect of the two com-
bined drugs, and was calculated by using the formula: [(apoptosis drug A + apoptosis drug B) – (apoptosis drug A x apoptosis drug B)/100]. In 5 of 7 samples,
this combination showed a more than additive pro-apoptotic effect. (A and D) Each dot represents an individual sample. (E) Representative flow cytometry plots
showing the proportion of alive MM cells (CD38+ TOPRO3-) after the indicated treatments. MM samples were cultured for 6 days in the presence of either 5
ug/mL isotype control antibody (black), anti-BCMA (red), or no antibody (blue). After this time, cells were treated with 100 nM MCL-1i (black, red) or co-treated
with MCL-1i + anti-BCMA (blue). MM viability was analyzed 24 hours later (i.e., on day 7) by fluorescence-activated cell sorting. (F) Cumulative plots showing
apoptosis induced by MCL-1i in the conditions specified in (E). Mean + standard error of the mean (n=5). Statistical differences between 2 groups (D) were ana-
lyzed using paired t-tests. Statistical differences between 3 or more groups (F) were analyzed using a one-way ANOVA with Bonferronis’ multiple comparison test.
*P<0.05; ** P<0.01.

MM cells to MCL-1 inhibition, suggesting that increased Dutch Parelsnoer Institute for providing MM bone marrow samples.
sensitivity to MCL-1i is specifically related to blockade We are grateful to D. van den Blink, C. Steenhuis, N.J.G. Wissing-
of the APRIL-BCMA axis and not a general consequence Blokland, and M.J.M. Dijkstra-Boerkamp from the Central
of ADCC (Online Supplementary Figure S2I and J). Diagnostic Laboratory (CDL) of the UMCU.We thank S.J. Vastert for
Limiting the potential toxicities associated with MCL1i providing Tocilizumab. We thank L. Abbink for her help with ADCC
therapy is important: MCL-1 is expressed on different and CDC assays. We thank Servier for providing the MCL-1-specific
healthy tissues, including cardiomyocytes, hematopoiet- inhibitor S63845. We thank R. Raijmakers, M. Jak, T. Kimman and
ic stem cells, oocytes, and lymphocytes.15 It is, therefore all VP laboratory members for helpful discussions.
relevant to find strategies to enhance the efficacy of Funding: this investigation was supported by a Bas Mulder Award
MCL-1 inhibitors specifically in MM cells, which may to VP from the Dutch Cancer Foundation (KWF)/Alped’HuZes foun-
lower the required doses for a persistent therapeutic dation (award no. UU 2015-7663) and a project grant to VP from the
effect. Our results suggest that blocking the APRIL- Dutch Cancer Foundation (KWF)/Alped’HuZes foundation (grant no.
BCMA axis may render MM cells more dependent on 11108). MC was supported in part by a postdoctoral grant from the
pro-survival IL-6 signaling,5 forcing a scenario where Ramón Areces Foundation.
cells are more sensitive to MCL-1 inhibition.
Interestingly, patients with 1q21 amplification are highly References
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Disclosures: VP received royalty payments related to venetoclax. 11. Braham MVJ, Minnema MC, Aarts T, et al. Cellular immunotherapy
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facilities of the University Medical Center Utrecht (UMCU) and the Cancers. 2020;12(11):3353.

els. One or more serious AE (SAE) were reported in seven

Phase Ib dose-escalation study of the selective, non- patients and consisted of cellulitis (in 2 patients) and lym-
covalent, reversible Bruton’s tyrosine kinase phocytosis, intestinal perforation, myelitis, sepsis, hema-
inhibitor vecabrutinib in B-cell malignancies turia, pleural effusion, and deep vein thrombosis (in 1
patient each). No SAE were considered related to study
Several Bruton’s tyrosine kinase inhibitors (BTKi) have treatment per the investigator. Two patient deaths were
been approved for the treatment of B-cell malignancies associated with AE: perforated bowel in one patient treat-
and are particularly active in chronic lymphocytic ed at the 41 mg BID dose level (who had mantle cell lym-
leukemia (CLL), where they have transformed the treat- phoma [MCL] with bowel involvement), and sepsis in one
ment paradigm. However, the activity of currently avail- patient treated at the 164 mg BID dose level. Neither event
able BTKi (ibrutinib, acalabrutinib, and zanubrutinib) was considered related to study treatment. There were no
requires covalent bond formation with cysteine 481 cardiac events or clinically significant electrocardiogram
(C481) of BTK; hence resistance to covalent BTKi may be findings.
mediated through mutations which remove C481. In order Vecabrutinib showed modest evidence of clinical bene-
to overcome this resistance, and potentially prevent the fit, with one PR observed in a patient with CLL (treated at
proliferation of C481 mutant cells, non-covalent BTKi that the 246 mg BID dose level) and SD in 13 patients (31%; 11
do not require interaction with the C481 residue were CLL, 1 MCL, 1 marginal zone lymphoma). A waterfall plot
developed.1 This phase Ib/II trial investigated the safety of percent change in tumor burden from baseline in all
and clinical activity of vecabrutinib, a reversible, non-cova- patients is displayed in Figure 1. Among 14 patients with
lent inhibitor of BTK that inhibits both wild-type and a best response of PR or SD, six had BTK C481 mutations,
C481-mutated BTK,2 in patients with advanced, BTKi- eight had 17p deletion and/or TP53 mutations, and the
resistant B-cell malignancies. The results of the completed median number of prior therapies was three (range, 2-9).
phase Ib dose-escalation portion of the study demonstrat- Patients with PR or SD remained on study treatment for a
ed that vecabrutinib was well-tolerated up to 410 mg median of 28.5 weeks (range, 6.1-56+ weeks). Eight of
twice daily (BID), the highest dose studied. Evidence of these patients received vecabrutinib for ≥6 months, includ-
clinical benefit was observed, with a best response of paring five who discontinued treatment due to termination of
tial response (PR) in one CLL patient and stable disease the study and not for progressive disease, suggesting some
(SD) in 13 patients. Pharmacokinetics (PK) were approxi- durable clinical benefit.
mately dose-proportional and sustained reductions in Pharmacokinetic data were available for 38 patients.
serum cytokine concentrations were observed at higher Concentration-time profiles and linear regression analysis
dose levels, suggesting BTK inhibition. However, the asso- indicated that both exposure and median steady-state
ciation between vecabrutinib dose, pharmacodynamics Ctrough concentrations generally increased in an approxi-
(PD), and clinical activity was inconsistent and the activity mately dose-proportional manner. Exposure to vecabruti-
observed was considered insufficient for phase II expan- nib was maintained across the ~12 hour dosing interval,
sion in patients with BTKi-resistant CLL. supporting BID dosing, with cycle 1 day 8 trough values at
Patient and disease characteristics are summarized in the dose levels ≥164 mg BID expected to provide >90% inhi-
Online Supplementary Table S1. Thirty-nine patients with bition of BTK signaling based an earlier single-dose phase
histologically confirmed, relapsed/refractory CLL or other I study.3
B-cell malignancies and ≥2 prior lines of standard systemic Pharmacodynamic activity of vecabrutinib was assessed
therapy, including progression during BTKi therapy, were in CLL patients who completed cycle 1 (n=25) via meas-
enrolled and treated across seven dose-levels. The majori- urement of serum cytokine levels. CCL3, CCL4, and TNFa
ty (77%) of patients had CLL. The enrolled population was have previously been shown to be inhibited by other
high-risk, with a median of four prior therapies (range, 2- BTKi.4–6 For all three cytokines, a sustained reduction in
9), 17p deletion or TP53 mutation in 74% of all patients, serum concentration was evident in most patients after
and BTK C481 mutations in 55% of CLL patients. one cycle of vecabrutinib treatment at higher dose levels
Vecabrutinib capsules were administered orally BID at a (246, 328 and 410 mg), suggesting inhibition of BTK activ-
starting dose of 20.5 mg/dose (41 mg total daily dose). ity. Mean reductions at these dose levels ranged from 34-
Vecabrutinib was well-tolerated up to 410 mg BID (820 62% for CCL3, 33-59% for CCL4, and 24-57% for TNFa
mg total daily dose), the highest dose studied. One patient, (Figure 2). In regression analyses, there was a trend for
treated at the 41 mg BID dose level, experienced a dose- greater reduction in serum cytokine levels with increasing
limiting toxicity (DLT), consisting of failure to receive Cmax and AUClast as well as vecabrutinib dose, suggestive of
>80% of planned vecabrutinib doses due to adverse events an exposure-response effect (Online Supplementary Figure
(AE) of grade 3 alanine aminotransferase (ALT) and grade S1). Chemokine reduction was associated with clinical
2 aspartate aminotransferase (AST) elevations. Upon benefit, with decreased serum cytokine levels demonstrat-
expansion of this cohort, no additional DLT were observed ed in all but one patient with a clinical response of PR or
and dose escalation continued. The maximum-tolerated SD. However, the extent of inhibition was generally less
dose of vecabrutinib was not reached. than that observed in BTKi-naïve patients treated with
The most common treatment-emergent AE were ane- ibrutinib (which produced median decreases ≥80% for
mia (31%) and nausea, fatigue, headache and dyspnea CCL3, CCL4 and TNFa),6 consistent with the limited clin-
(21% each). The most common AE considered treatment- ical activity observed for vecabrutinib.
related by the investigator were anemia and fatigue (10% The question arises as to why BTK inhibition by
each). Grade ≥3 AE were mainly hematologic, including vecabrutinib did not translate to a clinical response despite
anemia (23%), neutropenia (13%) and thrombocytopenia strong preclinical evidence and promising early (phase Ia)
(10%) (Online Supplementary Table S2). Grade ≥3 AE con- clinical PK/PD data in healthy subjects, particularly in CLL
sidered treatment-related by the investigator consisted of patients in whom substantial clinical activity has been
leukocytosis in two patients (5.1%) and anemia, neutrope- observed with other non-covalent BTKi. In vitro cellular
nia, and increased AST in one patient (2.6%) each. No assays were performed to evaluate the half maximal
obvious pattern of dose-dependent toxicity was observed, inhibitory concentration (IC50) values and BTK residence
with no grade ≥3 AE observed at the two highest dose lev- time (i.e., the time a BTKi remains bound to BTK) for

Figure 1. Percent change in tumor burden from baseline in patients treated with vecabrutinib. Percent change in tumor burden (sum of the product of the
diameters [SPD]) from baseline at time of best response assessment is shown by patient for all patients who underwent post-treatment imaging-based disease
assessment. Disease type, dose (in mg twice daily [BID]), response assessment per the Investigator, baseline molecular characteristics (Bruton’s tyrosine kinase
[BTK] C481X mutation status, presence of PLCg2 mutation, complex karyotype, TP53 mutation, or 17p deletion), and number of prior regimens received are
indicated for each patient below the graph. U: indicates unknown
vecabrutinib compared with other BTKi to look for poten- brutinib and dasatinib (reported half-lives ranging
tial correlation with outcome (Table 1). IC50 values for between 4 and 14 hours).3,7,9–12 Although no single proper-
vecabrutinib (18.4 nM) and ARQ 531 (32.9 nM) were sim- ty aligned consistently with the observed clinical activity,
ilar, however, ARQ 531 demonstrated greater clinical effi- these attributes provide possible explanations regarding
cacy; conversely, fenebrutinib demonstrated greater in vitro the limited clinical activity observed with vecabrutinib
potency (7.04 nM) but showed limited clinical activity in compared to other reversible BTK inhibitors.
patients previously treated with ibrutinib.7–10 The resi- Overall, vecabrutinib was well-tolerated and demon-
dence time observed for vecabrutinib (15 minutes [min]) strated some evidence of clinical benefit. However, despite
was much shorter than that observed for ARQ 531 (128 dose-proportional PK, the association between vecabruti-
min) and fenebrutinib (557 min) and was also shorter rel- nib dose, PD, and clinical response was inconsistent.
ative to reported values for pirtobrutinib (LOXO-305; 314 Increasing the dose from 246 to 410 mg BID did not uni-
min). Other possible explanations for the limited clinical formly correlate with increased PD activity though there
activity observed are that vecabrutinib is highly protein was an overall trend towards improved inhibition with
bound (98.7%), which may have affected the availability dose. Assessment of clinical activity by dose may have
of the free drug, or that vecabrutinib may not have been been confounded by the impact of baseline patient charac-
consistently distributed from blood to disease sites; either teristics: clinical benefit (i.e., PR or SD lasting >6 months)
of these possibilities may have resulted in levels insuffi- was most commonly observed in patients who were less
cient to provide adequate BTK inhibition. Furthermore, PK heavily pretreated and had better prognostic factors as
properties differ among non-covalent BTKi: the effective identified by Ahn et al.,13 such as lower baseline lactate
agents, pirtobrutinib and ARQ 531, have longer half-lives dehydrogenase levels and wild-type TP53, regardless of
(approximately 20 hours and 55 hours, respectively) than dose. These results suggest that the potency of single-
the agents with limited clinical activity, vecabrutinib, fene- agent vecabrutinib was not sufficient to control disease in

A B
Figure 2. Dose-pharmacodynamics relationship for CCL3, CCL4, and TNFa. Serum

cytokine levels at baseline (cycle [C]1, day [D]1) and after C1 of treatment (predose
on C2D1 [day 29]) were measured by enzyme-linked immunosorbant assay in 25
chronic lymphocytic leukemia (CLL) patients who completed C1. Dot plots show
mean change (with standard deviation) from baseline in CCL3 (A), CCL4 (B), and
TNFa (C) serum levels. BID: twice daily.
Table 1. In vitro assessment of Bruton’s tyrosine kinase (BTK) residence time and half maximal inhibitory concentration values for BTK
engagement for vecabrutinib and other BTK inhibitors.
Vecabrutinib Ibrutinib ARQ 531 Fenebrutinib Dasatinib Pirtobrutinib
BTK WT
IC50 for BTK engagement, nM 18.4 1.65 32.9 7.04 34.8 3.7a
BTK residence time, minutes 15 >1,000 128 557 61 314a
BTK C481S
IC50 for BTK engagement, nM 34.6 229 102 13.1 78.8 8.5a
BTK residence time, minutes 16 31 228 900 120 231a
BTK: Bruton’s tyrosine kinase; BTKi: BTK inhibitor; IC50: half-maximal inhibitory concentration. aIC50 values and residence time values (calculated as the reciprocal of kd
[1/kd]) for pirtobrutinib (LOXO-305) are as reported by Gomez et al.15; WT: wild-type.
refractory patients; however vecabrutinib in combination Shadman,9 Matthew S. Davids,10 John M. Pagel,4 Habte A.
with other agents, including BCL2 inhibitors,14 may result Yimer,11 Renee Ward,12 Gary Acton,12 Pietro Taverna,12 Daniel
in improved efficacy. Based on the dose-escalation results, L. Combs,13 Judith A. Fox,12 Richard R. Furman1 and Jennifer
the activity observed in BTKi-resistant patients at the dose R. Brown10
levels studied was considered insufficient for phase II 1
Weill Cornell Medicine, Department of Medicine, New York,
expansion of this patient cohort. Future directions for NY; 2Moffitt Cancer Center, Tampa, FL; 3Johns Hopkins Sidney
vecabrutinib may include indications such as chronic graft- Kimmel Comprehensive Cancer Center, Baltimore, MD; 4Swedish
versus-host disease or in combination with chimeric anti- Cancer Institute, Seattle, WA; 5Willamette Valley Cancer
gen receptor T-cell therapies, where dual inhibition of BTK Institute/US Oncology, Eugene, OR; 6MD Anderson Cancer
and IL-2 Inducible T-cell Kinase (ITK) may contribute to Center, Houston, TX; 7Moores Cancer Center, University of
clinical outcomes. California San Diego, La Jolla, CA; 8Chao Family Comprehensive
Cancer Center, University of California Irvine, Orange, CA; 9Fred
John N. Allan,1 Javier Pinilla-Ibarz,2 Douglas E. Hutchinson Cancer Research Center, Seattle, WA; 10CLL Center,
Gladstone,3 Krish Patel,4 Jeff P. Sharman,5 William G. Medical Oncology, Dana-Farber Cancer Institute, Boston, MA;
11
Wierda,6 Michael Y. Choi,7 Susan M. O’Brien,8 Mazyar Texas Oncology/US Oncology Research, Tyler, TX; 12Sunesis

Pharmaceuticals, South San Francisco, CA and 13Combs member of the data safetymonitoring board for Ivectys and
Consulting Service, Mountain View, CA, USA Morphosys.
Correspondence: Contributions: JNA, JP-I, DEG, KP, JPS, WGW, MYC, SMO,
JENNIFER R. BROWN - jennifer_brown@dfci.harvard.edu MS, MSD, JMP, HAY, RW, GA, PT, JAF, RRF, and JRB con-
doi:10.3324/haematol.2021.280061 tributed to study concept and study supervision; PT and JAF were
responsible for study administration; JNA, JP-I, DEG, KP, JPS,
Received: September 24, 2021.
WGW, MYC, SMO, MS, MSD, JMP, HAY, RRF, and JRB pro-
Accepted: December 7, 2021. vided patients and conducted the investigation; DC did a formal
Pre-published: December 23, 2021. analysis of the data; DC, PT, and JAF prepared the data presenta-
Disclosures: JNA served as a consultant to AbbVie, Acerta, tion; JA, GA, PT, JAF, and JRB wrote the original draft of the manu-
Ascentage Pharma, AstraZeneca, Bayer, BeiGene, Epizyme, script. All authors reviewed and edited the manuscript.
Genentech, Janssen, Pharmacyclics, Sunesis Pharmaceuticals, TG Acknowledgments: medical writing support was provided by Janis
Therapeutics, and Verastem Oncology; received honoraria from Leonoudakis, PhD, and was funded by Sunesis Pharmaceuticals,
AbbVie, BeiGene, Janssen, Pharmacyclics; and received research Inc., San Francisco, CA.
funding from AstraZeneca, Celgene, Genentech, and Janssen. JP-I
served as a consultant to AbbVie, Bristol-Meyers Squibb, Janssen, Funding: financial support for this study was provided by Sunesis
Novartis, Takeda, Teva, and TG Therapeutics; and served on the Pharmaceuticals, Inc., South San Francisco, CA.
speakers bureau for AbbVie, Bayer, Janssen, Sanofi, and Takeda. Trial registration: clinicaltrials.gov Identifier: NCT03037645; EU
DEG reports no conflict of interest. KP served as a consultant to clinical trials register identifier: EudraCT # 2018-000108-41
AstraZeneca, Celgene, Genentech, Pharmacyclics/Janssen and
Sunesis; received research funding from AstraZeneca; and was a References
member of the Speakers Bureau for AstraZeneca, Celgene,
Genentech, and Pharmacyclics/Janssen. JPS served as a consultant to 1. Gu D, Tang H, Wu J, Li J, Miao Y. Targeting Bruton tyrosine kinase
and received honoraria and research funding from AbbVie, Acerta, using non-covalent inhibitors in B cell malignancies. J Hematol
Oncol. 2021;14(1):40.
AstraZeneca, Genentech, Janssen, Pharmacyclics, and TG
2. Allan JA, Patel K, Mato AR, et al. Preliminary results of a phase 1b/2
Therapeutics. WGW received research funding from AbbVie, Acerta, dose-escalation and cohort-expansion study of the noncovalent,
Cyclcel, Genentech, Gilead Sciences, GSK/Novartis’ Janssen, Juno reversible Bruton’s tyrosine kinase inhibitor (BTKi), vecabrutinib, in
Therapeutics, KITE pharma, Loxo Oncology, Miragen, Oncternal B-cell malignancies. HemaSphere. 2019;3(suppl 1):520.
Therapeutics, Pharmacyclics, Sunesis, and Xencor. MYC served as a 3. Neuman L, Ward R, Arnold D, et al. First-in-human phase 1a study
consultant to AbbVie, Genentech, Gilead, Pharmacyclics, and Rigel; of the safety, pharmacokinetics, and pharmacodynamics of the non-
covalent bruton tyrosine kinase (BTK) inhibitor SNS-062 in healthy
received research funding from AbbVie, Oncternal Therapeutics, subjects. Blood. 2016;128(Suppl):S2032.
Pharmacyclics, and Rigel; and was a member of the Speakers Bureau 4. Ponader S, Chen S-S, Buggy JJ, et al. The Bruton tyrosine kinase
for AbbVie, Genentech, Gilead, and Pharmacyclics. SMO served as inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell sur-
a consultant to AbbVie, Alexion, Amgen, Aptose Biosciences, Astellas, vival and tissue homing in vitro and in vivo. Blood.
Celgene, Eisai, Gilead, GlaxoSmithKline, Janssen, Pfizer, 2012;119(5):1182-1189.
Pharmacyclics, Sunesis, TG Therapeutics, Vaniam Group, and 5. Byrd JC, Harrington B, O’Brien S, et al. Acalabrutinib (ACP-196) in
relapsed chronic lymphocytic leukemia. N Engl J Med.
Verastem Oncology; received honoraria from AbbVie, Janssen, and 2016;374(4):323-332.
Pfizer; and received research funding from Acerta, Gilead, KITE 6. Niemann CU, Herman SEM, Maric I, et al. Disruption of in vivo
pharma, Pfizer, Pharmacyclics, Regeneron, Sunesis, and TG chronic lymphocytic leukemia tumor-microenvironment interac-
Therapeutics. MS served as a consultant to AbbVie, ADC tions by ibrutinib - findings from an investigator-initiated phase II
Therapeutics, AstraZeneca, Atara Biotherapeutics, Genentech, study. Clin Cancer Res. 2016;22(7):1572-1582.
Gilead, Pharmacyclics, Sound Biologics, and Verastem; and received 7. Byrd JC, Smith S, Wagner-Johnston N, et al. First-in-human phase 1
study of the BTK inhibitor GDC-0853 in relapsed or refractory B-
research funding from AbbVie, Acerta Pharma, BeiGene, Celgene, cell NHL and CLL. Oncotarget. 2018;9(16):13023-13035.
Genentech, Gilead, Mustang Bio, Pharmacyclics, Sunesis, TG 8. Crawford JJ, Johnson AR, Misner DL, et al. Discovery of GDC-0853:
Therapeutics, and Verastem. MSD served as a consultant to AbbVie, a potent, selective, and noncovalent Bruton’s tyrosine kinase
Adaptive Biotechnologies, Ascentage Pharma, AstraZeneca, BeiGene, inhibitor in early clinical development. J Med Chem.
BMS, Celgene, Eli Lilly, Genentech, Janssen, MEI Pharma, Merck, 2018;61(6):2227-2245.
9. Herman AE, Chinn LW, Kotwal SG, et al. Safety, pharmacokinetics,
Novartis, Takeda, and Verastem; received research funding from
and pharmacodynamics in healthy volunteers treated with GDC-
Ascentage Pharma, AstraZeneca, BMS, Genentech, MEI pharma, 0853, a selective reversible Bruton’s tyrosine kinase inhibitor. Clin
Novartis, Pharmacyclics, Surface Oncology, TG Therapeutics, and Pharmacol Ther. 2018;103(6):1020-1028.
Verastem; and received honoraria from Research to Practice. JMP 10. Woyach J, Stephens DM, Flinn IW, et al. Final results of phase 1,
served as a consultant to AstraZeneca, Gilead Sciences, and dose escalation study evaluating ARQ 531 in patients with relapsed
Pharmacyclics. HAY was a member of the Speakers Bureau for or refractory B-cell lymphoid malignancies. Blood. 2019;134(Suppl
1):S4298.
Amgen, AstraZeneca, BeiGene, Janssen, Karyopharm, Pharmacylics, 11. Christopher LJ, Cui D, Wu C, et al. Metabolism and disposition of
and Sanofi; and holds publicly-traded stock in Karyopharm. RW dasatinib after oral administration to humans. Drug Metab Dispos.
served as a consultant to Sunesis Pharmaceuticals. GA served as a 2008;36(7):1357-1364.
consultant to Sunesis Pharmaceuticals. PT was employed by Sunesis 12. Mato AR, Shah NN, Jurczak W, et al. Pirtobrutinib in relapsed or
Pharmaceuticals. JAF was employed by Sunesis Pharmaceuticals. refractory B-cell malignancies (BRUIN): a phase 1/2 study. Lancet.
DLC served as a consultant to Sunesis Pharmaceuticals. RRF served 2021;397(10277):892-901.
13. Ahn IE, Tian X, Ipe D, et al. Prediction of outcome in patients with
as a consultant to AbbVie, Acerta Pharma, AstraZeneca, BeiGene, chronic lymphocytic leukemia treated with ibrutinib: development
Genentech, Incyte, Janssen, Loxo Oncology, Morphosys, Oncotracker, and validation of a four-factor prognostic model. J Clin Oncol.
Pharmacyclics, Sanofi, Sunesis, TG Therapeutics, and Verastem. JRB 2021;39(6):576-585.
served as a consultant to AbbVie, Acerta Pharma, AstraZeneca, 14. Jebaraj B, Müller A, Dheenadayalan R, et al. Evaluation of vecabru-
BeiGene, Catapult Therapeutics, Dynamo, Genentech, Gilead, tinib as a model for non-covalent BTK/ITK inhibition for treatment
Juno/Celgene, Kite, Loxo, Novartis, Octapharma, Pfizer, of chronic lymphocytic leukemia. Blood. 2022;139(6):859-875.
15. Gomez EB, Isabel L, Rosendahal MS, Rothenberg SM, Andrews SW,
Pharmacyclics, Sunesis, TG Therapeutics, and Verastem; received Brandhuber BJ. Loxo-305, a highly selective and non-covalent next
honoraria from Janssen and Teva, received research funding from generation BTK inhibitor, inhibits diverse BTK C481 substitution
Gilead, Kite, Loxo, Sun Pharmaceuticals, and Verastem; and was a mutations. Blood. 2019;135(Suppl 1):S4644.

ing platelet counts despite the use of PAS during cold stor-
In vitro and in vivo effects of short-term cold storage of age, suggesting persistent microaggregates.2-4 While
platelets in PAS-C numerous in vitro studies on CSP in PAS exist,4-8 only one
study looked at the effect of PAS on in vivo kinetics of
Cold (4°C)-stored platelets (CSP) were the standard of transfused CSP, but lacked fresh comparators.9
care in the 1960s and 1970s but fell out of favor when their In the current study, we investigated CSP in PAS (PAS-
short in vivo survival was discovered. Since then, room CSP) and compared them to CSP in plasma (P-CSP) and
temperature-stored platelets (RSP) have been the standard room temperature-stored platelets in plasma (P-RSP). All
of care. Septic transfusion reactions from bacterially con- platelets were stored for 5 days. We collected a standard
taminated RSP remain the most common transfusion- single apheresis platelet unit from six healthy subjects
transmitted infection. In addition, accumulating data ques- stored in either 100% plasma at 22°C or 4°C, or 65% PAS-
tioning the efficacy and safety of RSP, together with a C (Intersol) and 35% plasma at 4°C. In this study, we
short shelf life, highlight an unmet medical need for an included historical controls for P-CSP and P-RSP,10,11 but all
alternative product. Currently, CSP are being re-evaluated units were collected by apheresis and stored in the same
for bleeding patients, for whom immediate function mat- fashion as described below. Concerns regarding risks for
ters more than long circulation time. An added benefit of healthy human volunteers and their safety, in addition to
CSP is that bacterial growth is markedly reduced at 4°C. the high costs of in vivo radiolabeling studies, made repeat-
However, how to store CSP best is poorly understood. ing control groups that have already been studied and pub-
Some groups reported increased wastage due to aggregates lished both redundant and ethically burdensome for this
in CSP stored in plasma. Platelet additive solutions (PAS) small study.
were developed to reduce transfusion reactions and limit We included PAS-C because it is currently licensed in the
metabolic damage, but one group showed that PAS pre- USA and we previously obtained in vivo data that favored
vented aggregates in CSP.1 Other groups reported decreas- PAS-C over PAS-F (Isoplate) for CSP.9 All three groups
Figure 1. In vitro platelet characteris-

tics. Platelets stored at room tempera-
ture in plasma (P-RSP) (Plasma, 22°C,
solid black bars), platelets stored cold in
plasma (P-CSP) (Plasma, 4°C, striped
B C bars), and platelets stored cold in
platelet additive solution (PAS-CSP)
(PAS-C, 4°C, solid gray bars) were tested
fresh and on day 5 after storage. (A)
Platelet count measured by an ABX
Hemanalyzer, P-RSP (n=18), P-CSP
(n=5), PAS-CSP (n=6). (B) Glucose meas-
ured by a blood gas analyzer, P-RSP
(n=6), P-CSP (n=5), PAS-CSP (n=6). (C)
Lactate measured by a blood gas analyz-
er, P-RSP (n=6), P-CSP (n=5), PAS-CSP
(n=6). Data are shown as percentage of
fresh and as mean + standard error of
mean. *P<0.05, **P<0.01,
***P<0.001. ns = not significant
Figure 2. In vivo platelet characteristics.

A B Healthy human subjects received autolo-
gous radiolabeled fresh platelets or
platelets stored for 5 days in plasma at
room temperature (P-RSP) (Plasma,
22°C, solid black bars), in plasma at
4°C (P-CSP) (Plasma, 4°C, striped bars),
or in plasma additive solution at 4°C
(PAS-CSP) (PAS-C, 4°C, solid gray bars).
(A) Recovery of transfused platelets after
2 hours, P-RSP (n=21), P-CSP (n=5),
PAS-CSP (n=5). (B) Survival of trans-
fused platelets, P-RSP (n=21), P-CSP
(n=5), PAS-CSP (n=5). Data shown as
percentage of fresh, mean + standard
error of mean. ***P<0.001, ns = not
significant

(PAS-CSP, P-CSP, and P-RSP) comprised different cohorts These findings hint at persistent, detectable metabolic
(no matching between groups). The annexin V and P- activity up to 5 days, even though the metabolism is
selectin data were obtained from a separate group of four markedly slowed at 4°C. As expected, platelet in vivo
volunteers whose platelets were collected by apheresis markers decreased significantly, but the recovery of PAS-
and stored in mini-storage bags to clarify a role of these CSP and P-CSP did not differ significantly (Figure 2A). We
platelet activation parameters. All apheresis units were observed a trend for longer survival in PAS-CSP than in P-
collected and bags stored at room temperature were agitat- CSP (Figure 2B). To obtain more insights into the biological
ed as per standard blood banking protocols. Cold-stored health of stored platelets, we studied mitochondrial mem-
units were stored without agitation. We radiolabeled brane potential as an early marker of apoptosis. We
platelets as previously described with minor modifica- observed significantly better-preserved membrane poten-
tions.12 tial in P-CSP than in P-RSP. PAS-CSP and P-CSP values did
The Western Institutional Review Board approved the not differ significantly as percentage of fresh values, but
research, and all human participants gave written informed the absolute data showed significantly better preservation
consent. The study was conducted in accordance with the in PAS-CSP (Figure 3A, and Online Supplementary Figure
Declaration of Helsinki and registered with S2D). All cells responded appropriately to the uncoupler
ClinicalTrials.gov identifier NCT02754414. We assessed CCCP (2-[2-(3-chlorophenyl)hydrazinylyidene]propane-
statistical significance by one way analysis of variance dinitrile) (Online Supplementary Figure S2C, D). There was
(ANOVA) with the Tukey correction for multiple compar- a trend for more caspase activation in P-CSP, but overall,
isons. To minimize biological variability, and following rec- we did not see significant differences in this marker of late
ommendations by Murphy et al., we present the normal- apoptosis (Figure 3B). Adding ABT 737 induced caspase
ized stored data (percentage of fresh values). The absolute activation before and after storage, indicating that platelets
data are shown in Online Supplementary Figures S1-S3. had the capacity to undergo apoptosis (Online
As previously described, CSP counts were significantly Supplementary Figure S2E, F). We did not find significant
lower than P-RSP counts. PAS-CSP counts were signifi- differences in procoagulant activity, but there was a trend
cantly higher than those of P-CSP, corroborating findings to higher levels at room temperature (Figure 3C). Similarly,
from others and our group (Figure 1A).1,11 Consistent with we observed higher P-selectin levels at room temperature,
high metabolic activity at room temperature, glucose lev- but although this was significant when compared to PAS-
els at day 5 were lowest in P-RSP. Post-storage levels of CSP, it was not when compared to P-CSP (Figure 3D).
glucose in PAS-CSP were significantly lower than in P-CSP Integrin activation was greatest in P-CSP, significantly
(Figure 1B). While P-RSP showed the highest lactate lev- more than in P-RSP. The PAS-CSP integrin response was
els, cold storage reduced lactate production, with a trend lower than the P-CSP one, but the difference was not sta-
for lower levels in PAS-CSP than in P-CSP (Figure 1C). tistically significant (Figure 3E-G). The largest difference
A B C D
E F G
Figure 3. In vitro apoptosis and activation parameters. Apoptosis and parameters of platelet activation were measured in fresh platelets and platelets stored
for 5 days in plasma at room temperature (P-RSP) (Plasma, 22 °C, solid black bars), in plasma at 4°C (P-CSP) (Plasma, 4°C, striped bars), or in plasma additive
solution at 4°C (PAS-CSP) (PAS-C, 4 °C, solid gray bars). (A) Platelet mitochondrial membrane potential measured by JC-1 dye red (FL2) to green (FL-1) ratio, P-
RSP (n=7), P-CSP (n=5), PAS-CSP (n=5). (B) Caspase 3,7 activation measured by flow cytometry, P-RSP (n=5), P-CSP (n=5), PAS-CSP (n=5). (C) Procoagulant
activity measured by annexin V binding by flow cytometry. P-RSP (n=4), P-CSP (n=4), PAS-CSP (n=4). (D) a-granule degranulation was measured by CD62P bind-
ing by flow cytometry. P-RSP (n=4), P-CSP (n=4), PAS-CSP (n=4). Platelet aIIbb3 integrin activation was measured by PAC-1 antibody binding by flow cytometry
at baseline (BL) and after stimulation (E) with collagen (C), P-RSP (n=5), P-CSP (n=7), PAS-CSP (n=5). (F) with arachidonic acid (AA), P-RSP (n=5), P-CSP (n=5),
PAS-CSP (n=5). or (G) with ADP (A) P-RSP (n=5), P-CSP (n=5), PAS-CSP (n=5). (A-C) Data are shown as percentage of values for fresh platelets, mean + standard
error of mean. (E-G) Absolute data, mean + standard error of mean. *P<0.05, **P<0.01, ***P≤0.001.

1
between P-CSP and PAS-CSP was after stimulation with Bloodworks Northwest Research Institute, Seattle, WA and
2
arachidonic acid (Figure 3F). University of Washington Medical Center, Department of Medicine,
After only 5 days, we found that PAS prevented a cold- Division of Hematology, Seattle, WA, USA
induced decrease in platelet count, likely by preventing Correspondence:
microaggregates.1,9 One report suggests that aggregate for-
mation is prevented by continuous agitation during cold MORITZ STOLLA - mstolla@bloodworksnw.org
storage.8 We observed a platelet count decrease independ- doi:10.3324/haematol.2021.279865
ently of agitation in preliminary studies (Online Received: August 25, 2021.
Supplementary Figure S3C). In a previous study including
Accepted: December 16, 2021.
over 20 units, we saw one large proteinaceous aggregate in
one single unit, while a study with frequent manipulation Pre-published: December 23, 2021.
and rewarming led to much more frequent detection of Disclosures: MS received research funding from Cerus Corp. and
aggregates (Online Supplementary Figure S3D). Other inves- Terumo BCT
tigators attempted to store cold platelets with repeated Contributions: SLB analyzed the data, established assays, and per-
rewarming episodes (temperature cycling).13 To prevent formed experiments, EP, DB and LYF performed experiments. LF
aggregates, rewarming had to be accompanied by agita- performed apheresis collection procedures. MS designed the study,
tion, an approach we have thus far not incorporated in our
analyzed the data and wrote the manuscript.
studies. Metabolically active platelets under normal stor-
age conditions convert most of the supernatant glucose Acknowledgments: the authors would like to thank Dr. Sherrill
into lactate in P-RSP. Replacing plasma with PAS-C Slichter and the members of the cold-stored platelet interest group
removes sugars but adds acetate, which modifies glucose organized by the Department of Defense for helpful discussions. We
utilization and can suppress lactate generation in RSP. thank Renetta Stevens and Tena Petersen for administrative support.
Accordingly, the level of lactate in PAS-CSP stored for 5 Funding: this project received funding support from the Department
days was lower than that in P-CSP or P-RSP, indicating of Defense, award n.. W81XWH-12-1-0441
that replacing plasma with PAS had a beneficial effect.10,11
We did not observe activation differences between P-CSP References
and PAS-CSP after stimulation with various agonists simi-
1. Getz TM, Montgomery RK, Bynum JA, Aden JK, Pidcoke HF, Cap
lar to what has been described before for PAS/plasma CSP AP. Storage of platelets at 4 degrees C in platelet additive solutions
in aggregometry experiments1 and 100% plasma CSP.11 prevents aggregate formation and preserves platelet functional
However, whether in vitro responses of CSP to agonists responses. Transfusion. 2016;56(6):1320-1328.
predict in vivo hemostasis is not well understood. 2. Johnson L, Tan S, Wood B, Davis A, Marks DC. Refrigeration and
cryopreservation of platelets differentially affect platelet metabo-
Nevertheless, the fact that CSP in PAS and plasma have lism and function: a comparison with conventional platelet storage
similar responses suggests that both media provide the conditions. Transfusion. 2016;56(7):1807-1818.
right environment for platelet function testing in vitro. In 3. Johnson L, Vekariya S, Wood B, Tan S, Roan C, Marks DC.
our study, the mitochondrial membrane potential was best Refrigeration of apheresis platelets in platelet additive solution (PAS-
preserved in P-CSP and best predicted integrin activation, E) supports in vitro platelet quality to maximize the shelf-life.
Transfusion. 2021;61(Suppl 1):S58-S67.
highlighting the need for an intact energy supply for 4. Reddoch-Cardenas KM, Montgomery RK, Lafleur CB, Peltier GC,
platelet activation.10,11 It is likely that our 5-day storage Bynum JA, Cap AP. Cold storage of platelets in platelet additive
time was not enough to induce later stages of apoptosis: a solution: an in vitro comparison of two Food and Drug
recent study of CSP in PAS-F did not find significant differ- Administration-approved collection and storage systems.
Transfusion. 2018;58(7):1682-1688.
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complex apoptotic process and further studies are needed trates. Haematologica. 2019;104(1):207-214.
to explore this in further detail. 6. Reddoch-Cardenas KM, Peltier GC, Chance TC, et al. Cold storage
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Small sample sizes and donor-to-donor variability may integrity by limiting initiation of apoptosis-mediated pathways.
also have affected the outcomes of this study. An addition- Transfusion. 2021;61(1):178-190.
al limitation is the lack of an earlier testing time point 7. Braathen H, Sivertsen J, Lunde THF, et al. In vitro quality and
before the 5-day maximum. PAS may have additional ben- platelet function of cold and delayed cold storage of apheresis
efits by reducing allergic and febrile adverse reactions, platelet concentrates in platelet additive solution for 21 days.
Transfusion. 2019;59(8):2652-2661.
although this has not been systematically studied with 8. Hegde S, Wellendorf AM, Zheng Y, Cancelas JA. Antioxidant pre-
CSP. During storage, the levels of inflammatory mediators vents clearance of hemostatically competent platelets after long-
are lower in CSP than in RSP. Whether PAS further reduces term cold storage. Transfusion. 2021;61(2):557-567.
inflammatory levels in CSP remains to be investigated, but 9. Stolla M, Fitzpatrick L, Gettinger I, et al. In vivo viability of extended
4 degrees C-stored autologous apheresis platelets. Transfusion.
data from RSP support this idea. Most European countries 2018;58(10):2407-2413.
utilize pathogen reduction technology (PRT). Two previ- 10. Zimring JC, Slichter S, Odem-Davis K, et al. Metabolites in stored
ous studies looked into the effects of combining PAS/PRT platelets associated with platelet recoveries and survivals.
with 4°C storage.14,15 The authors found mostly compara- Transfusion. 2016;56(8):1974-1983.
ble results with the notable exception of reduced clot 11. Stolla M, Bailey SL, Fang L, et al. Effects of storage time prolongation
on in vivo and in vitro characteristics of 4 degrees C-stored platelets.
retraction and reduced GPIba-levels with PRT. There may Transfusion. 2020;60(3):613-621.
also be a benefit in promoting coagulation with PRT. 14,15 12. The Biomedical Excellence for Safer Transfusion (BEST)
In summary, we found that PAS had positive effects on Collaborative. Platelet radiolabeling procedure. Transfusion.
in vitro parameters while not negatively affecting in vivo 2006;46(Suppl 3):59S-66S.
13. Vostal JG, Gelderman MP, Skripchenko A, et al. Temperature cycling
kinetics. Our data along with the results from other groups during platelet cold storage improves in vivo recovery and survival
suggest that CSP in PAS are a safe and efficacious product in healthy volunteers. Transfusion. 2018;58(1):25-33.
and could have a practice-changing impact on the blood 14. Agey A, Reddoch-Cardenas K, McIntosh C, et al. Effects of Intercept
banking industry in the coming years. pathogen reduction treatment on extended cold storage of apheresis
platelets. Transfusion. 2020;61(1):167-177.
15. Six KR, Devloo R, Compernolle V, Feys HB. Impact of cold storage
S. Lawrence Bailey,1 Lydia Y. Fang,1 Lynda Fitzpatrick,1 on platelets treated with Intercept pathogen inactivation.
Daire Byrne,1 Esther Pellham1 and Moritz Stolla1,2 Transfusion. 2019;59(8):2662-2671.

merase chain reaction (PCR) as previously described.4

Conventional interferon-a 2b versus hydroxyurea for Hematologic and molecular responses were evaluated in
newly-diagnosed patients with polycythemia vera in accordance with the revised response criteria of the
a real world setting: a retrospective study based on European LeukemiaNet (ELN) and International Working
286 patients from a single center Group-Myeloproliferative Neoplasms Research and
Treatment (IWG-MRT).5 Complete hematologic remis-
Polycythemia vera (PV) is a myeloproliferative neo- sion (CHR) was defined as HCT <45% without phleboto-
plasm (MPN) characterized by clonal proliferation of mul- my, white blood cell (WBC) <10×109/L, and platelets
tipotent bone marrow progenitors.1 A clinical trial investi- ≤400×109/L. Complete molecular response (CMR) was
gating the efficacy of pegylated interferon (IFN) for PV defined as indetectable JAK2 V617F mutation. Partial
and essential thrombocytosis is ongoing in China. molecular response (PMR) applied only to patients with
However, as only conventional IFN and hydroxyurea
a JAK2 V617F VAF ≥20% before treatment and was
(HU) are covered by Chinese basic medical insurance,
defined as a ≥50% decrease in allele burden after treat-
these cytoreductive agents are recommended as first-line
ment.5
treatment by the consensus of Chinese experts for the
The clinical and laboratory features of subjects in the
diagnosis and treatment of PV, including low-risk
IFN and HU cohorts are displayed in Table 1. The median
patients.2
treatment duration in the IFN and HU cohorts were 51
As the difference in efficacy between conventional IFN
and HU for newly-diagnosed PV is undefined, we retro- months (interquartile range [IQR], 24–83 months) and 53
spectively analyzed data of 286 newly-diagnosed PV months (IQR, 31–84 months), respectively. The duration
patients who were treated at the Institute of Hematology of exposure to IFN or HU for each patient is shown in the
and Blood Diseases Hospital, Chinese Academy of Online Supplementary Figure S1B and C.
Medical Sciences between June 1, 2007 and February 28, Compared with the HU cohort, a higher proportion of
2020. All patients received conventional IFN-a 2b or HU patients in the IFN cohort achieved CHR (65% vs. 43%;
for at least 6 months. Patients were excluded if they P=0.001), control of HCT (72% vs. 43%; P=0.06), control
changed groups. The flowchart for patient selection is of platelets (88% vs. 77%; P=0.04), and control of WBC
shown in the Online Supplementary Figure S1A. Cases were (89% vs. 72%; P=0.002; Figure 1A) during follow-up.
diagnosed in accordance with the 2016 World Health A higher proportion of low-risk subjects who received
Organization diagnostic definitions.3 IFN achieved CHR compared with those who received
Conventional IFN-a 2b was recommended first for HU (64% vs. 32%; P=0.001; Figure 2A). Consistently,
young (age <60 years old) patients and older patients high-risk subjects who received IFN also had a higher
without contraindications. HU was usually recommended CHR rate than those who received single-agent HU (68%
for other patients. In total, 82 and 204 patients received vs. 47%; P=0.06; Figure 2C).
single-agent conventional IFN-a 2b (IFN cohort) and sin- Interestingly, the median increase in mean corpuscular
gle-agent HU (HU cohort), respectively. Generally, the ini- volume (MCV) from baseline, when patients achieved the
tial dose of conventional IFN-a 2b was 3×106 IU three best control of HCT, was 21.2 fL (IQR, 9.1–31.9 fL) in the
times per week; the initial dose of HU was 20 mg/kg/day. HU cohort, which was much higher than in the IFN
Treatment schedules were adjusted by monitoring periph- cohort (3.9 fL; IQR, −2.8–9.5; P<0.001). Because HCT
eral blood counts with the target of hematocrit (HCT) equals the erythrocyte count multiplied by MCV, some
<45%. patients in the HU cohort might have been phle-
Quantitative measurements of the JAK2 V617F variant botomized with normal RBC.
allele frequency (VAF) were performed by real-time poly- The median duration from starting treatment to achiev-
Table 1. Clinical features of the interferon and hydroxyurea cohorts at baseline.

Variables IFN (N = 82) HU (N = 204) P-value
Age, years 51 (44-57) 61 (52-67) <0.001
Sex, female 50 (61%) 104 (51%) 0.13
Palpable splenomegaly 20 (26%) 52 (27%) 0.83
Disease duration, month; median (range) 0 (0-2) 0 (0-2) 0.31
Baseline hemoglobin, g/L 189 (177-209) 197 (187-210) 0.01
Baseline RBC, ×1012/L 7.0 (6.3-7.6) 7.2 (6.5-7.8) 0.02
Baseline hematocrit, % 58 (54-63) 61 (57-65) 0.003
Baseline WBC, ×109/L 12.6 (9.4-15.1) 13.1 (9.8-18.1) 0.15
Baseline platelet, ×109/L 464 (339-623) 424 (324-572) 0.40
Baseline MCV, fL 84.0 (80.7-89.6) 85.4 (79.8-89.9) 0.99
JAK2 V617F mutation 77 (94%) 191 (94%) 0.93
Baseline JAK2 V617F VAF, %, (n=209)* 56 (35-73） 59 (33-73) 0.62
Abnormal cytogenetics, % (n/N) 4% (2/49) 4% (4/114) 1.00
Thrombosis pretreatment (n=406) 23 (29%) 69 (35%) 0.36
Thrombosis risk stratification (n=406) <0.001
Low risk 52 (65%) 59 (30%)
High risk 28 (35%) 141 (70%)
Follow-up from start of treatment, months 52 (35-91) 55 (33-84) 0.82
Data are presented as median (interquartile range [IQR]) or n (%), unless otherwise indicated. IFN: interferon; HU: hydroxyurea; RBC: red blood cell; WBC: white blood cell;
MCV: median corpuscular volume; VAF: variant allele frequency; JAK2 V617F VAF in JAK2 V617F-mutated patients.

A B
C D
Figure 1. Comparison of hematologic and molecular responses between the interferon and hydroxyurea cohorts. (A) Hematologic responses, (B) complete
hematologic remission (CHR) rates over time, (C) molecular responses, and (D) dynamics of JAK2 V617F variant allele frequencies (VAF) over time are compared
between the interferon (IFN) and hydroxyuera (HU) cohorts. In (D) the horizontal lines indicate median values; bars represent minimum and maximum values;
boxes represent values included between the 25% and 75% percentiles. (E) JAK2v V617F VAF waterfall plot in the IFN (n=22) and HU (n=31) cohorts; the y-axis
indicates the absolute change of the JAK2 V617F VAF from baseline to the best molecular response; each bar represents a patient; dotted lines represent medi-
an changes of the JAK2 V617F VAF in each group. IFN: conventional (non-pegylated) interferon; HCT: hematocrit; PLT: platelet; WBC: white blood cell; PMR: partial
molecular response; Pts: patients. *P<0.05; **P<0.01; ***P<0.001.

ing CHR was 11 months (IQR, 7–23 months) in the IFN <10% was higher in the IFN (65%) than in the HU (33%)
cohort, which was shorter than in the HU cohort (19 cohort (P=0.007; Figure 1C). Among patients with base-
months; IQR, 10–47 months; P=0.001). Among the line JAK2 VAFs ≥20%, 95% (19/20) achieved PMR in the
patients who achieved CHR, two (4%) and seven (7%) IFN and 59% (17/29) in the HU cohorts (P=0.007; Figure
were lost during follow-up in the IFN and HU cohorts, 1C). The median change in JAK2 V617F VAF from base-
respectively. Compared with the HU cohort, CHR rates in line to the best molecular response in the IFN and HU
the IFN cohort were higher throughout the treatment cohorts was −58% (IQR, −69% to −34%) and −30%
duration and became significantly better after 3 years of (IQR, −51% to –0.4%) (P=0.001; Figure 1E). Finally, the
continuous treatment (88% vs. 44%; P<0.001; Figure 1B), JAK2 V617F VAF in the IFN cohort was significantly lower
which was consistent with the results of the PROUD-PV than in the HU cohort after 3 years of continuous treat-
and CONTINUATION-PV studies, which used pegylated ment (Figure 1D).
IFN.6 Because the IFN cohort was younger than the HU
In addition to hematologic responses, the IFN cohort cohort, we compared treatment responses between
also showed better molecular responses than the HU patients in the IFN and HU cohorts matched for age and
cohort. In total, 31 and 40 patients had data regarding sex. The baseline peripheral blood counts, JAK2 V617F
molecular responses in the IFN and HU cohorts, respec- allele burdens, follow-ups, and thrombosis risk stratifica-
tively. The median JAK2 V617F VAF at baseline were not tions were balanced between the two matched cohorts
significantly different between the IFN and HU cohorts (Online Supplementary Figure S2A). The CHR rate (66%
(68% [IQR, 51–78%] vs. 62% [IQR, 40–70%]; P=0.21). [44/67] vs. 34% [23/67]; P=0.001; Online Supplementary
Only one patient in the IFN cohort achieved CMR. The Figure S2B), control of HCT rate (73% [49/67] vs. 54%
percentage of patients who obtained a JAK2 V617F VAF [36/67]; P=0.03; Online Supplementary Figure S2B), and
A B
C D
Figure 2. Comparison of hematologic and molecular responses between the interferon and hydroxyurea cohorts stratified by thrombosis risk. Hematologic (A)
and molecular (B) responses of low-risk patients. Hematologic (C) and molecular (D) responses of high-risk patients. IFN: interferon; HU: hydroxyurea; RBC: red
blood cell; WBC: white blood cell; HCT: hematocrit; PLT: platelet; VAF: variant allele frequency; IQR: interquartile range; *JAK2 V617F VAF in JAK2 V617F-mutated
patients; CHR: complete hematologic remission; PMR: partial molecular response. *P<0.05; **P<0.01; ***P<0.001.

PMR rate (95% [18/19] vs. 62% [8/13]; P=0.029; Online low- and high-risk patients, and only a few patients who
Supplementary Figure S2C) were significantly higher in the were tested for molecular responses, which are all sources
IFN cohort than in the HU cohort when matched for age of potential bias.
and sex. When patients were stratified by thrombosis- Dan Liu,1,2* Zefeng Xu,1,2* Peihong Zhang,1,3 Tiejun Qin,1,2
risk, the CHR rate in the IFN cohort was higher than in Xiujuan Sun,1 Shiqiang Qu,1,2 Lijuan Pan,1,2 Jiao Ma,1,3
the HU cohort for age- and sex-matched low-risk (63% Wenyu Cai,1,3 Jinqin Liu,1 Huijun Wang,1,3 Qi Sun,1,3
[24/38] vs. 26% [9/35]; P=0.002) and high-risk (70% Zhongxun Shi,1,2 Huijun Huang,1,2 Gang Huang,4 Robert
[19/27] vs. 45% [14/31]; P=0.067) patients. Peter Gale,5 Bing Li,1,2 Raajit K. Rampal 6 and Zhijian
In total, 14 of 82 subjects (17%) discontinued IFN treat- Xiao1,2,3
ment for the following reasons: normalized peripheral 1
State Key Laboratory of Experimental Hematology, National
blood counts (n=8, 57%), adverse effects (n=2, 14%), dis- Clinical Research Center for Blood Diseases, Institute of Hematology
ease progression (n=1, 7%), and unknown reasons (n=3, and Blood Diseases Hospital, Chinese Academy of Medical Sciences
21%). Fever was the most common adverse effect of IFN, and Peking Union Medical College, Tianjin, China; 2MDS and
which was reported in 23% (14/62) of patients, followed MPN Center, Institute of Hematology and Blood Diseases Hospital,
by bone pain in 11% (7/62) of patients. Chinese Academy of Medical Sciences and Peking Union Medical
Post-treatment thrombotic events occurred in two (2%) College, Tianjin, China; 3Hematologic Pathology Center, Institute of
and six (3%) patients in the IFN and HU cohorts. The
Hematology and Blood Diseases Hospital, Chinese Academy of
thrombosis rates were 0.5% (95% confidence interval
Medical Sciences and Peking Union Medical College, Tianjin,
[CI]: 0–1.1) patients per year for the IFN cohort and 0.7%
China; 4Divisions of Experimental Hematology and Cancer Biology,
(95% CI: 0.2–1.2) for the HU cohort. These rates were
Cincinnati Children's Hospital Medical Center, Cincinnati, OH,
much lower than those published in a previous study
USA; 5Hematology Section, Division of Experimental Medicine,
(2.62%; 95% CI: 2.34–2.94 patients per year).7 In our
Department of Medicine, Imperial College London, London, UK and
study, the thrombosis rate in the low-risk cohort (95% CI: 6
Leukemia Service, Department of Medicine, Memorial Sloan
0.6% [0.2–0.9]) was lower than that of low-risk PV
Kettering Cancer Center, New York, NY, USA.
patients treated by phlebotomy, as reported by Barbui et
al. (95% CI: 2.0% [1.5-2.5]).8 *DL and ZX contributed equally as co-first authors.
The lower incidence of thrombosis in this study com- Correspondence:
pared with previous studies might be related to racial dif- BING LI - libing@ihcams.ac.cn
ferences in thromboembolism between Asian and RAAJIT K RAMPAL - rampalr@mskcc.org
Western populations. This is related to differences in ZHIJIAN XIAO - zjxiao@ihcams.ac.cn
genetic polymorphisms and environmental factors, such
as obesity and healthcare facilities.9,10 For instance, a study doi:10.3324/haematol.2021.280080
reported that Japanese patients with paroxysmal noctur- Received: September 24, 2021.
nal hemoglobinuria (PNH) had a significantly lower inci-
dence of thrombosis than American PNH patients.11 Accepted: December 24, 2021.
Moreover, the ECLAP study and a matched study of 951 Prepublished: January 6, 2022.
patients with PV reported a benefit-risk profile of HU
Disclosures: RPG is a consultant to BeiGene Ltd., Fusion Pharma
therapy over phlebotomy with respect to the lower rate of
LLC, LaJollaNanoMedical Inc., Mingsight Pharmaceuticals Inc.,
arterial thrombosis.12,13 Our findings suggested that early
and CStone Pharmaceuticals; is an advisor to Antegene Biotech LLC;
intervention with cytoreductive treatments for low-risk
medical director of FFF Enterprises Inc.; is a partner of AZAC Inc.; is
subjects rather than phlebotomy might also correlate with
a member of the Board of Directors of the Russian Foundation for
lower thrombosis rates.
Cancer Research Support; and member of the Scientific Advisory
Thrombosis-free survival rates were not significantly
Board of StemRad Ltd. RKR has received grants and personal fees
different between the IFN and HU cohorts (P=0.81), simi-
lar results were found when adjusted by age (P=0.73; from Constellation, Incyte, Celgene/BMS, and Stemline, and person-
Online Supplementary Figure S3A). There was no significant al fees from Promedior, CTI, Blueprint, Jazz Pharmaceuticals,
difference in overall survival (P=0.99; Online Supplementary Galecto, Pharmaessentia, AbbVie, and Novartis. All other authors
Figure S3B) or myelofibrosis-free survival (P=0.98; Online declare no conflicts of interest.
Supplementary Figure S3C) between the IFN and HU Contributions: ZJX designed the study; DL and ZFX collected and
cohorts when adjusted by age. A previous retrospective interpreted the data and performed statistical analysis; PHZ and QS
study of PV patients reported that IFN reduced the risk of contributed to analysis of bone marrow histology; TJQ, SQQ, LJP,
mortality and transformation into myelofibrosis compared WYC, JQL, HJW, XJS, MJ, and QYG contributed to recruiting
with HU or phlebotomy.14 The different conclusions that patients and collecting data; DL wrote the manuscript with contribu-
we report might be due to the relatively short follow-up in tions from ZJX, ZFX, BL, GH, RPG, RKR, ZXS, and HJH. All
our study. Finally, there was no significant difference in authors reviewed the manuscript during its preparation and approved
thrombosis-free survival (P=0.40), overall survival the final version of the manuscript.
(P=0.55), or myelofibrosis-free survival (P=0.26) between Funding: this study was supported in part by National Natural
patients who achieved PMR or not. Science Funds (No. 81530008, 81870104, and 82070134), Tianjin
A recent meta-analysis reported that CHR rates, throm- Natural Science Funds (18JCZDJC34900, 16JCQNJC11400, and
botic complications, and treatment discontinuations 19JCQNJC09400), and CAMS Initiative Fund for Medical
owing to adverse events were not significantly different Sciences (No. 2016-I2M-1-001, 2020-I2M-C&T-A-020, and
between pegylated and conventional IFN.15 In our study, 2020-I2M-C&T-B-090).
conventional IFN-a 2b was a good choice for PV, showing
better efficacy than HU and acceptable tolerance.
In conclusion, this study found that the hematologic References
and molecular responses of newly-diagnosed PV to con-
1. Grinfeld J, Nangalia J, Baxter EJ, et al. Classification and personal-
ventional IFN-a 2b were better than to HU. There are lim- ized prognosis in myeloproliferative neoplasms. N Engl J Med.
itations to this study, such as it being a retrospective study 2018;379(15):1416-1430.
from a single center with a short follow-up, a mixture of 2. Leukemia and Lymphoma Group, Chinese Society of Hematology,

Chinese Medical Association. [Chinese expert consensus on the Hematol. 2017;92(1):E5-E6.

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Xue Ye Xue Za Zhi. 2016;4(4):265-268. system dysfunction and thrombophilia in Asia. Ann Lab Med.
3. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the 2013;33(1):8-13.
World Health Organization classification of myeloid neoplasms 10. Zakai NA, McClure LA. Racial differences in venous thromboem-
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4. Tashi T, Swierczek S, Kim SJ, et al. Pegylated interferon Alfa-2a and 11. Nishimura JI, Kanakura Y, Ware RE, et al. Clinical course and flow
hydroxyurea in polycythemia vera and essential thrombocythemia: cytometric analysis of paroxysmal nocturnal hemoglobinuria in the
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2018;32(8):1830-1833.
12. Barbui T, Vannucchi AM, Finazzi G, et al. A reappraisal of the ben-
5. Barosi G, Mesa R, Finazzi G, et al. Revised response criteria for
efit-risk profile of hydroxyurea in polycythemia vera: a propensity-
polycythemia vera and essential thrombocythemia: an ELN and
matched study. Am J Hematol. 2017;92(11):1131-1136.
IWG-MRT consensus project. Blood. 2013;121(23):4778-4781.
6. Gisslinger H, Klade C, Georgiev P, et al. Ropeginterferon alfa-2b 13. Barbui T, De Stefano V, Ghirardi A, et al. Different effect of hydrox-
versus standard therapy for polycythaemia vera (PROUD-PV and yurea and phlebotomy on prevention of arterial and venous throm-
CONTINUATION-PV): a randomised, non-inferiority, phase 3 trial bosis in polycythemia vera. Blood Cancer J. 2018;8(12):124.
and its extension study. Lancet Haematol. 2020;7(3):e196-e208. 14. Abu-Zeinah G, Krichevsky S, Cruz T, et al. Interferon-alpha for
7. Tefferi A, Rumi E, Finazzi G, et al. Survival and prognosis among treating polycythemia vera yields improved myelofibrosis-free and
1545 patients with contemporary polycythemia vera: an interna- overall survival. Leukemia. 2021;35(9):2592-2601.
tional study. Leukemia. 2013;27(9):1874-1881. 15. Bewersdorf JP, Giri S, Wang R, et al. Interferon alpha therapy in
8. Barbui T, Vannucchi AM, Carobbio A, et al. The effect of arterial essential thrombocythemia and polycythemia vera - a systematic
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Mann-Whitney test, as appropriate. The cut-off date for

Daratumumab with or without chemotherapy in this analysis was March 31, 2021 and data were analyzed
relapsed and refractory acute lymphoblastic with STATA 12.1 software (Stata Corporation, College
leukemia. A retrospective observational Campus Station, TX, USA).
ALL study We included 20 patients (85% males) in the study.
Thirteen had T-ALL, four had B-ALL, one had mixed phe-
The anti-CD38 antibody daratumumab, currently notype acute leukemia and two had lymphoblastic lym-
approved for the treatment of patients with multiple phoma (B-lineage in 1, T-linage in the other); 11 patients
myeloma, is also being explored for patients with acute
were treated front-line according to the GIMEMA
lymphoblastic leukemia (ALL), whose blasts commonly
LAL1913 intensive pediatric-like protocol.11 The patients’
express high levels of CD38.1 Patients with relapsed or
characteristics are summarized in Table 1.
refractory (R/R) disease, as well as those with positive
Daratumumab was administered at a median time of
measurable residual disease (MRD) have consistently
shown unfavorable outcomes and, especially for T-lin- 13 months after diagnosis and patients had received a
eage ALL, therapeutic options beyond first-line treatment median of three prior lines of therapy. The median age of
remain limited. Preclinical studies have demonstrated the patients at the start of daratumumab treatment was
that daratumumab has significant activity in human 35 years (range, 8-73) and three patients were below the
xenograft models of ALL, both as a single agent and in age of 18. Nine patients had already undergone an allo-
combination with chemotherapy.2-4 However, the clinical geneic HCT, with a median time from transplantation to
experience is so far very limited. A few case reports have daratumumab treatment of 6 months (range, 2-20). At
suggested that daratumumab has anti-leukemic activity the start of daratumumab treatment, 80% of patients had
in R/R and MRD-positive ALL cases,5-10 but the small a bone marrow relapse, either isolated or with concomi-
numbers of patients and the positive-outcome bias, due
to the propensity to publish mainly positive results, pre- Table 1. Patients’ characteristics.
vent any robust conclusions on the clinical impact of this Variable N. or median % or range
drug in ALL.
We hereby report a retrospective, observational study Male sex 17 85
performed in patients with R/R or MRD-positive ALL Type of ALL 18 80
who received daratumumab in Italy in order to provide T 13 65
further information on the safety and efficacy of this drug ETP 4 20
in a real-life context. In this study we included adult and B 4 20
pediatric patients with R/R or MRD-positive T- or B-lin- MPAL 1 5
eage ALL or lymphoblastic lymphoma, who received at
Type of LBL 2 10
least one dose of daratumumab between December 2018
T 1 5
and December 2020 at 17 Italian hematology centers.
B 1 5
Data were retrospectively collected in an anonymous
database. This study was performed in the context of the Firstline treatment
Campus ALL national framework and in agreement with LAL-1913 11 55
the Declaration of Helsinki. Patients received daratu- Hyper-CVAD 3 15
mumab either off-label or in the context of a compassion- AIEOP-BFM 4 20
ate use program, kindly supported by Janssen-Cilag Spa. Others (EWALL, BFM) 2 10
Daratumumab was administered at the approved Allo-HCT before daratumumab 9 45
schedule for multiple myeloma (i.e., 16 mg/kg weekly for Lines of treatment before
8 doses, then every 2 weeks for 8 doses, then monthly daratumumab 3 1-4
until disease progression), either alone or in combination
with chemotherapy. The co-primary endpoints were Age at daratumumab start, years 35 8 - 73
overall response rate and overall survival of patients after Below 18 years 3 15
daratumumab. Additional endpoints were safety and Disease status at daratumumab
bridge to allogeneic hematopoietic cell transplant (HCT) start
after daratumumab. Complete response was defined as a Isolated BM relapse 8 40
bone marrow blast count <5% without evidence of Extramedullary and BM 8 40
extramedullary manifestations, partial response was Extramedullary and MRD positivity 2 10
defined as a bone marrow blast count ≥5% and <25% Extramedullary only 1 5
with a reduction of leukemic involvement of at least CR, MRD-positive 1 5
50%. For lymphoblastic lymphoma, the Lugano criteria Disease characteristics at
were applied. MRD was monitored either by flow daratumumab start
cytometry and/or by real-time quantitative polymerase White blood cells, x109/L 3.34 0.1 - 39
chain reaction in centralized laboratories. The overall Platelets, x109/L 34 1 - 233
response rate was defined as the proportion of patients Peripheral blood blasts, % 14 0 - 98
who obtained a partial response, a complete response or, Bone marrow blasts, % 45 0 - 100
only for patients who were MRD positive, MRD negativ- ECOG at daratumumab start 2 0-4
ity. Any systemic anti-neoplastic treatment started at
diagnosis or with R/R disease counted as a line of thera- Concomitant chemotherapy 9 45
py, except for allogeneic HCT. Time from diagnosis to
Survival was estimated using the Kaplan-Meier daratumumab, months 13 7 - 28
method and overall survival was calculated from the date ALL: acute lymphoblastic leukemia; ETP: early-T-precursor; MPAL: mixed phenotype
of the first daratumumab infusion to the date of death or acute leukemia; LBL: lymphoblastic lymphoma; allo-HCT: allogeneic hematopoiet-
ic cell transplant; BM: bone marrow; MRD: measurable residual disease; CR: com-
the last follow-up. The association of baseline variables plete response; ECOG: performance status according to the Eastern Cooperative
with response was explored using the Fisher exact, c2 or Oncology Group scale.

tant extramedullary disease. Extramedullary sites To our knowledge, this is the largest series of ALL
involved were the lymph nodes in four patients, the cen- patients treated with daratumumab reported so far and
tral nervous system in three, the mediastinum in two, the further underlines the potential activity of this drug in
breast in one and the gut in one. The median perform- R/R and MRD-positive cases.
ance status according to the Eastern Cooperative While the advent of monoclonal antibodies and
Oncology Group (ECOG) was 2. Daratumumab was chimeric antigen receptor T-cell therapy is progressively
administered alone in 11 cases (in 1 case after a short changing the therapeutic scenario in B-lineage ALL, the
dexamethasone pre-phase), while nine patients received approved treatment options for R/R and MRD-positive T-
concomitant chemotherapy (Online Supplementary Table ALL remain unsatisfactory, as highlighted by the large
S1). prevalence of T-lineage diseases in our cohort (14/20
The overall response rate was 20%, with two patients total patients). Nelarabine has been confirmed to be
achieving a MRD-negative complete response, one a active in this setting,12 but responses are short-lived and
complete response with persistent MRD and one a partial half of the patients are resistant to the drug. More recent-
response (Table 2). Patients responded after two to six ly, the AKR1C activated prodrug OBI342413 and the BCL-
infusions of daratumumab and the median time to 2 inhibitor venetoclax have been tested in R/R T-ALL,
response was 4 weeks. Three of the four responses were and the combination of venetoclax and navitoclax with
observed in patients with T-ALL, who were treated with chemotherapy appears particularly promising.14
daratumumab as a single agent. Two patients (both with However, data on these new agents are still immature
T-ALL) were alive at the last follow-up, one patient died and new therapeutic approaches are urgently needed.
after relapse and one died of treatment-related complica- Following preclinical data and a few positive case
tions after allogeneic HCT. The characteristics of the reports, daratumumab has started to be used in patietns
responding patients are summarized in Online with advanced ALL without other therapeutic options,
Supplementary Table S2. Four patients (2 responders, 2 but data from unselected cohorts are lacking. In our
refractory) proceeded to allogeneic HCT after daratu- series, in which we included all patients who received at
mumab.
Next, we explored the potential factors associated with
response. Patients with a bone marrow hematologic Table 3. Predictors of response to daratumumab.
relapse (P=0.013), lower platelet count (P=0.019) and
higher circulating blast percentage (P=0.034) were less
Number (%) or median (range)
likely to respond, while those with a better ECOG per-
Variable* Responders Non-responders P=
Sex 1
formance status (P=0.019) and who had received fewer
Male 4 (23.5) 13 (76.5)
prior lines of therapy (P=0.022) responded better (Table
Female 0 (0) 3 (100)
3). Consistently, bone marrow MRD positivity, with or
without extramedullary involvement, was associated Age, years 34 (25 - 45) 35.5 (8 - 73) 0.92
with a better overall response rate, without however the T-lineage 3 (21.4) 11 (78.6) 1
difference reaching statistical significance (P=0.088). B-lineage 1 (20) 4 (80) 1
Finally, we evaluated the potential association of CD38
expression on lymphoblasts with response. Among the Lymphoblastic lymphoma 1 (50) 1 (50) 0.37
18 evaluable cases, CD38 positivity and mean fluores- Extramedullary disease 2 (18.2) 9 (81.8) 1
cence intensity did not differ significantly between BM MRD° 2 (66.7) 1 (33.7) 0.088
responders and non-responders (median 96.5% vs.
BM relapse 1 (6.2) 15 (93.8) 0.013
95.6%, P=0.9 and 16,800 vs. 12,800; P=0.51, respectively)
At the last follow-up, all but one patient had stopped Previous allo-HCT 2 (22.2) 7 (77.8) 1
treatment and two patients remained alive and in com- Previous lines of treatment 0.022
plete remission. The median overall survival of the whole 1 2 (100) 0 (0)
cohort was 4 weeks, with a 3-month overall survival rate 2 1 (25) 3 (75)
of 25% (Online Supplementary Figure S1). No unexpected 3 1 (9.1) 10 (90.9)
toxicities were observed and there was only one grade 2 4 0 (0) 3 (100)
infusion reaction. White blood cells, x109/L 3.36 (3 - 4.3) 4.66 (0.1 - 39.4) 0.91
Hemoglobin, g/dL 10 (10 - 11) 9.5 (8 - 13) 0.25
Table 2. Outcome after daratumumab treatment.
Platelets, x109/L 151 (70 - 233) 27 (1 - 199) 0.019
Outcome N. or median % or range
PB blasts, % 0 (0 - 0) 24 (0 - 98) 0.034
Response to daratumumab BM blasts, % 2 (0 - 78) 50 (1 - 100) 0.099
Responders 4 20
ECOG score 0.019
CR, MRD-negative 2 10
0 2 (100) 0 (0)
CR, MRD-positive 1 5
1 2 (40) 3 (60)
PR 1 5
2 0 4 (100)
Non-responders 16 80
3 0 7 (100)
Stable disease 2 10
4 0 1 (100)
Progressive disease 12 60
Not evaluable 2 10 Concomitant
chemotherapy 1 (11.1) 8 (88.9) 0.37
Allo-HCT post-daratumumab 4 20
*Disease status and patients’ characteristics evaluated at the time of starting dara-
in CR/PR 2 10 tumumab therapy. °Includes the patient in complete remission with isolated meas-
Treatment duration, weeks 2 2-120 urable residual disease positivity and those with extramedullary relapse and meas-
urable residual disease positivity in the bone marrow. Bold values denote statistical
Discontinued treatment 19 95 significance at the P<0.05 level. BM: bone marrow; MRD: measurable residual dis-
CR: complete remission; MRD: measurable residual disease; PR: partial remission; ease; Allo-HCT: allogeneic hematopoietic cell transplant; ECOG: performance status
Allo-HCT: allogeneic hematopoietic cell transplant. according to the Eastern Cooperative Oncology Group scale.

1
least one dose of the drug, we observed a relatively low Divisione di Ematologia, Dipartimento di Oncologia, AOU Città
overall response rate of 20% and limited survival. della Salute e della Scienza, Torino; 2UOC di Ematologia, Azienda
However, most patients were heavily pre-treated, with a Ospedaliera Universitaria Integrata di Verona, Verona; 3Dipartimento
poor ECOG performance status and a high disease bur- di Medicina, Sezione di Ematologia, Università di Verona, Verona;
4
den. Indeed, several of these patients would be excluded Dipartimento di Biotecnologie Molecolari e Scienze per la Salute,
from any clinical trial. Responses were obtained rapidly, Università di Torino, Torino; 5IRCCS Azienda Ospedaliero-
after two to six infusions of the drug, either alone or in Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna;
6
combination with chemotherapy, and interestingly also Università di Brescia, Unità Operativa di Ematologia e Centro
in cases with extramedullary disease. Although limited Trapianti, ASST Spedali Civili di Brescia, Brescia; 7Unità Operativa
by the small numbers, we could analyze potential predic- di Ematologia e Trapianto, Ospedale Vito Fazzi, Lecce; 8Unità
tive factors of response to daratumumab. We observed Operativa di Ematologia e Trapianto, Ospedale dell'Angelo, Mestre;
9
that patients with a high ALL burden (i.e., those with a Unità Operativa Complessa di Ematologia, Azienda Ospedaliero-
bone marrow hematologic relapse and circulating blasts) Universitaria di Modena, Modena; 10UO Ematologia ed Immunologia
were unlikely to benefit from the treatment, while dara- Clinica, Azienda Ospedale-Università Padova, Padova; 11Unità
tumumab proved to be effective in patients with a good Operativa di Ematologia, Fondazione IRCCS Policlinico San Matteo,
performance status and less advanced disease. These Pavia; 12Unità Operativa di Ematologia e Trapianto, AOU-Ospedale
findings are in agreement with the current literature, S. Maria della Misericordia, Perugia; 13UOC Ematologia Fondazione
with positive case reports mostly describing patients IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milano;
14
treated for MRD positivity or with low disease burden Dipartimento di Diagnostica per Immagini, Radioterapia Oncologica
and in good clinical conditions. Indeed, a better selection ed Ematologia, Fondazione Policlinico Universitario A. Gemelli IRCCS,
of patients, as well as earlier use of the compound (e.g., Roma; 15Sezione di Ematologia, Dipartimento di Scienze Radiologiche
in MRD-positive cases) appear crucial to obtain meaning- ed Ematologiche, Università Cattolica del Sacro Cuore, Roma; 16Unità
ful results. We also evaluated CD38 expression on lym- di Trapianto Allogenico di Cellule Staminali, Dipartimento di
phoblasts before the start of daratumumab therapy and Oncologia, A.O.U. Città della Salute e della Scienza, Torino;
17
found no significant association with response. Sample Ematologia, Dipartimento di Biomedicina e Prevenzione, Università di
investigation was not centralized and so this exploratory Roma Tor Vergata, Roma; 18Unità Operativa di Ematologia e
analysis was limited by the heterogeneity of antibodies Trapianto di Midollo Osseo, IRCCS Ospedale San Raffaele, Milano;
19
and analytical techniques employed by different flow Unità Operativa Complessa di Ematologia, AOUS, Università di
cytometry laboratories. Siena, Siena; 20Unità Operativa di Oncologia ed Ematologia
We observed a patient who responded to daratumum- Pediatrica, Ospedale Pediatrico Meyer, Firenze; 21Unità Operativa di
ab despite high disease burden, but in this case the anti- Ematologia Pediatrica, Università di Milano-Bicocca, Fondazione
body was used in combination with chemotherapy. MBBM, Monza; 22Laboratorio Analisi, AO S. Croce e Carle, Cuneo;
23
Recently, a case report outlined the feasibility of this UOC di Ematologia, AORN Cardarelli, Napoli and 24Ematologia,
approach,15 which is currently being tested in a clinical Dipartimento di Medicina Traslazionale e di Precisione, Università
trial evaluating daratumumab in combination with Sapienza, Roma, Italy
chemotherapy in younger ALL patents (NCT03384654). Correspondence:
This strategy might be the best option in the presence of
a full-blown relapse, but the best chemotherapy regimen MARCO CERRANO - cerranomarco@gmail.com
to combine with daratumumab remains to be defined. ROBIN FOÀ - rfoa@bce.uniroma1.it
Combinations with innovative drugs with promising doi:10.3324/haematol.2021.279851
activity in ALL, such as venetoclax or bortezomib,16 could Received: September 6, 2021.
also be tested following the experience in multiple
myeloma,17 as these patients are often chemorefractory. Accepted: Junuary 4, 2022.
We could confirm the safety of daratumumab in the Pre-published: January 13, 2022.
setting of ALL, with a lower than expected rate of infu- Disclosures: no conflicts of interest to disclose
sion reactions compared to those occurring in multiple
Contributions: MC, MB, SC and RF designed the study. MC
myeloma and no unexpected toxicities. Although limited
assembled and analyzed the data. MO collected data and contributed
by its retrospective nature and the heterogeneity of the
to their analysis. BC analyzed flow cytometry data. MC, MB, SC
patients, our study provided data that could help to
and RF drafted the manuscript. All authors contributed to data collec-
design new clinical trials aimed at testing daratumumab
tion, revised the manuscript, and accepted its final version.
in ALL and to selecting patients who may benefit more
from its use. Acknowledgments: we thank Janssen-Cilag Spa Italy for providing
In conclusion, our data confirm the potential activity daratumumab for compassionate use.
and safety of daratumumab in R/R and MRD-positive Funding: the work was partly supported by the Associazione
ALL, and suggest that this compound should be possibly Italiana per la Ricerca sul Cancro (AIRC), Metastases Special
used earlier, rather than after several lines of salvage Program, N° 21198, Milan, Italy (to RF).
treatment. Further studies are needed to clarify whether
daratumumab could be another game-changer in this dis-
ease. References
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Marco Cerrano,1 Massimiliano Bonifacio,2,3 Matteo Olivi,1,4 expressed in pediatric or adult relapsed/refractory T-ALL: implica-
Antonio Curti,5 Michele Malagola,6 Michelina Dargenio,7 tions for immunotherapy. Blood Adv. 2020;4(19):4665-4668.
Anna Maria Scattolin,8 Cristina Papayannidis,5 Fabio 2. Bride KL, Vincent TL, Im S-Y, et al. Preclinical efficacy of daratumum-
ab in T-cell acute lymphoblastic leukemia. Blood. 2018;131(9):995-
Forghieri,9 Carmela Gurrieri,10 Ilaria Tanasi,2,3 Patrizia 999.
Zappasodi,11 Roberta La Starza,12 Nicola Stefano Fracchiolla,13 3. Vogiatzi F, Winterberg D, Lenk L, et al. Daratumumab eradicates
Patrizia Chiusolo,14,15 Luisa Giaccone,4,16 Maria Ilaria Del minimal residual disease in a preclinical model of pediatric T-cell
Principe,17 Fabio Giglio,18 Marzia Defina,19 Claudio Favre,20 acute lymphoblastic leukemia. Blood. 2019;134(8):713-716.
4. Naik J, Themeli M, de Jong-Korlaar R, et al. CD38 as a therapeutic
Carmelo Rizzari,21 Barbara Castella,22 Giovanni Pizzolo,2,3 target for adult acute myeloid leukemia and T-cell acute lymphoblas-
Felicetto Ferrara,23 Sabina Chiaretti24 and Robin Foà24 tic leukemia. Haematologica. 2019;104(3):e100-e103.

5. Ofran Y, Ringelstein-Harlev S, Slouzkey I, et al. Daratumumab for apy and bridge to allogeneic stem cell transplant in 118 adult patients
eradication of minimal residual disease in high-risk advanced relapse with relapsed/refractory T-cell acute lymphoblastic leukemia/lym-
of T-cell/CD19/CD22-negative acute lymphoblastic leukemia. phoma. A CAMPUS ALL study. Am J Hematol. 2020;95(12):1466-
Leukemia. 2020;34(1):293-295. 1472.
6. Mirgh S, Ahmed R, Agrawal N, et al. Will daratumumab be the next 13. Evans K, Duan J, Pritchard T, et al. OBI-3424, a novel AKR1C3-acti-
game changer in early thymic precursor-acute lymphoblastic vated prodrug, exhibits potent efficacy against preclinical models of
leukaemia? Br J Haematol. 2019;187(2):e33-e35. T-ALL. Clin Cancer Res. 2019;25(14):4493-4503.
7. Ganzel C, Kharit M, Duksin C, Rowe JM. Daratumumab for 14. Pullarkat VA, Lacayo NJ, Jabbour E, et al. Venetoclax and navitoclax
relapsed/refractory Philadelphia-positive acute lymphoblastic in combination with chemotherapy in patients with relapsed or
leukemia. Haematologica. 2018;103(10):e489-e490. refractory acute lymphoblastic leukemia and lymphoblastic lym-
8. Zhang Y, Xue S, Liu F, Wang J. Daratumumab for quick and sustained phoma. Cancer Discov. 2021;11(6):1440-1453.
remission in post-transplant relapsed/refractory acute lymphoblastic
15. Fulcher J, Berardi P, Christou G, Villeneuve PJA, Bredeson C, Sabloff
leukemia. Leuk Res. 2020;91:106332.
M. Nelarabine-containing regimen followed by daratumumab as an
9. Cerrano M, Castella B, Lia G, et al. Immunomodulatory and clinical
effects of daratumumab in T-cell acute lymphoblastic leukaemia. Br effective salvage therapy and bridge to allogeneic hematopoietic
J Haematol. 2020;191(1):e28-e32. stem cell transplantation for primary refractory early T-cell precursor
10. Ruhayel SD, Valvi S. Daratumumab in T-cell acute lymphoblastic lymphoblastic leukemia. Leuk Lymphoma, 2021;62(9):2295-2297.
leukaemia: a case report and review of the literature. Pediatr Blood 16. Kaspers GJL, Niewerth D, Wilhelm BAJ, et al. An effective modestly
Cancer. 2021;68(5):e28829. intensive re-induction regimen with bortezomib in relapsed or
11. Chiaretti S, Messina M, Della Starza I, et al. Philadelphia-like acute refractory paediatric acute lymphoblastic leukaemia. Br J Haematol,
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(n=90) were subjected to repeated analysis of T-cell

T-cell immune responses following vaccination immunity against SARS-CoV-2 specific peptides. Baseline
with mRNA BNT162b2 against SARS-CoV-2 in characteristics of the patients and controls are shown in
patients with chronic lymphocytic leukemia: Table 1. Patient groups included i) previously untreated
results from a prospective open-label clinical trial (n=14), ii) individuals with ongoing ibrutinib therapy
(n=14), iii) individuals who had stopped ibrutinib ≥2
The Coronavirus disease 2019 (COVID-19) pandemic months ago due to remission as part of another study
has seriously affected patients with chronic lymphocytic addressing intermittent ibrutinib treatment (n=10), iv) or
leukemia (CLL).1 Fatalities exceeding 30% has been individuals who had received CD20 monoclonal anti-
reported among hospitalized patients in international body (mAb)-containing chemoimmunotherapy 6-30
surveys,2,3 and in consecutively identified CLL patients.4 months ago (of which 11 received their last dose >12
Additionally, many patients with CLL do not achieve months ago) (n=14). Cellular and humoral immunity was
seroconversion upon mRNA vaccination.5,6 We recently measured at day (d) 0 (baseline), d10 (10 days after first
confirmed those observations in the course of a prospec- dose), d21 and d35 (21 days after first dose, day of second
tive clinical trial involving the BNT1622b2 mRNA vac- dose). Seroconversion and antibody titres were analyzed
cine.7 The study included five equally sized cohorts of as reported7 with d35 data available in 48 of the 52 pre-
patients with different types of immunocompromised defined patients who were analyzed for T-cell immunity.
disorders (total n=449) and 90 healthy controls. Sixty- Additionally, 41 of 90 healthy controls in the trial7 were
three percent of patients within the specific CLL cohort preselected for T- cell analysis (Table 1).
(n=90) seroconverted after two vaccine doses. The low- The ELISpot assay was applied as previously
est seroconversion rate was found in patients on ibrutinib described,11 using plates and reagents from a human IFN-
followed by those who had stopped Bruton’s tyrosine g ELISpot kit (3420-2APT-2, Mabtech). Briefly, 2.5x105
kinase inhibitor (BTKi)-therapy.7 Even though T-cell peripheral blood mononuclear cells (PBMC)/well were
responses may occur in most patients with CLL following seeded and supplemented with 0.15 µg/mL of co-stimu-
SARS-CoV-2 infection,4 there is still limited data on T-cell latory anti-CD28/CD49d (347690, BD Biosciences). Cells
immunity following vaccination of many immunocom- were stimulated for 20 hours (h) with a peptide pool cov-
promised patient groups including CLL. In the first report ering the SARS-CoV-2 spike glycoprotein (0.5 µg/mL,
on hematological malignancies, nine of 18 patients devel- LB01792; peptides&elephants) and equivalent dimethyl
oped SARS-CoV-2-specific T-cell reactivity.8 Similar sulfoxide (DMSO) in unstimulated wells. Spot-forming
results were seen in a large patient group applying a units (SFU) were counted using the IRIS (Mabtech) auto-
whole blood interferon-g (IFN-g) release assay.9 mated reader system. Data points are presented as back-
An early study10 of T-cell responses identified IFN-g as ground corrected SFU/106 cells, calculated by subtracting
a key cytokine produced by spike-specific CD4+ and the mean value of the corresponding duplicate unstimu-
CD8+ T cell in BNT162b1 mRNA-vaccinated individuals. lated wells from the mean value of duplicate spike stim-
We report here on T-cell immunity in patients with CLL ulated wells. Negative values after background correction
from our above mentioned prospective clinical trial7 using are set to 1. The threshold for positive response corre-
IFN-g ELISpot, a validated quantitative assay to measure sponds to the average SFU/106 cells of unstimulated wells
T-cell responses against SARS-CoV-2-specific peptides.4,11 + 2 standard deviations (30 SFU/106 cells). Data were
Inclusion criteria and monitoring of our vaccine clinical excluded when unstimulated wells had >100 SFU/106
trial has been described earlier.7 Patients with a previous cells.
history or signs of COVID-19, or who tested positive for All analyses were prespecified as per protocol. Data is
SARS-CoV-2 or had spike protein-specific antibodies at summarized using descriptive statistics such as counts,
baseline were excluded. No patient with CLL developed percentages, medians and range. Categorical variables are
break-through infection during the study or early follow- presented as cross-tabulations and distributional differ-
up. Fifty-two predefined patients from the CLL cohort ences were tested using the Chi-squared test.
Table 1. Baseline characteristics and immunological results 2 weeks after the second dose (d35) of the mRNA BNT162b2 vaccine in relation
to chronic lymphocytic leukemia patient subgroup and healthy controls.
Entire Indolent Previous Previous Ongoing Healthy
cohort untreated CD20 mAb ibrutinib ibrutinib controls
N=52 N=14 N=14 N=10 N=14 N=41
Age, years 70 71 71 71 70 52
median (range) (23–86) (49–82) (56–84) (54–86) (23–84) (25–79)
Male sex 36 6 13 6 11 19
n (%) (69%) (43%) (93%) (60%) (79%) (46%)
Seroconverted d35 29/48 11/13 11/12 5/9 2/14 41/41
n (%) (60%) (85%) (92%) (56%) (14%) (100%)
Ab titres d35, U/mL* 24.6a 81.6 42.4 35.5 0.4 2696
median (range) (0.4–3,320)a (0.4–3,320) (0.4–1,343) (0.4–559) (0.4–170) (766–14,269)
ELISpot d35, SFU/106 PBMC 10 6 10 53 5 48
median (range) (1–1,096) (1–526) (1–490) (2–1,096) (2–70) (1-1,526)
Positive T-cell response 15/52 4/14 2/14 7/10 2/14 24/41
n (%) (29%) (29%) (14%) (70%) (14%) (59%)
*Ab baseline = 0.4 U/mL; seropositive >0.8 U/mL. an = 48 (with d35 data). Ab: antibodies; ELISpot: enzyme-linked immunospot; SFU: spot forming units; PBMC: peripheral
blood mononuclear cells; d35: day 35.
1000 haematologica | 2022; 107(4)

Significance between time points with missing values significant increase in SARS-CoV-2-specific immunoglob-
was assessed using Kruskal-Wallis test with Dunn’s post- ulin G (IgG) after vaccination on d10 in either group. At
test. Correlation analysis was done using non-parametric d21 and at d35, both groups had a significant response
Spearman rank correlation. P-values <0.05 were consid- compared to baseline (CLL: d0 vs. d21, P<0.05, d0 vs.
ered significant. Graphs and associated statistical tests d35, P<0.0001; controls: d0 vs. d21, P<0.0001; d0 vs. d35,
were performed in Prism v.9 (GraphPad Software Inc.). P<0.0001).
Seroconversion rates were in line with our full clinical Subgroup analysis revealed that seroconversion
trial report7 and are summarized in Table 1 and Figure occurred in 11 of 13 (85%) of previously untreated
1A. Seroconversion occurred in 29 of 48 patients (60%) patients, 11 of 12 (92%) of previously CD20 mAb-treated
(4/52 patients had missing serology data at d35) com- patients, five of nine (56%) of previously ibrutinib-treat-
pared to 41 of 41 (100%) of controls. The time kinetics of ed and two of 14 (14%) of patients with ongoing ibruti-
seroconversion are shown in Figure 1A. Only 4% and nib therapy. The difference between patients on or off
18% of patients had seroconverted at d10 and d21, ibrutinib was significant (P=0.036). The median antibody
respectively, followed by 60% at d35. Patients’ and con- titer at d35 in each subgroup as above was 81.6 U/mL
trols’ responses showed similar kinetics. There was no (range, 0.4-3,320), 42.4 U/mL (range, 0.4-1,343), 35.5
A B
C D
Figure 1. Humoral and cellular immune response in chronic lymphocytic leukemia patients. Longitudinal assessment (day 0, 10, 21, 35 post-vaccination) of
SARS-CoV-2-specific immunglobulin G (IgG) (A) and IFN-g T cells (B) after spike glycoprotein stimulation, with summarized number and frequency of patients
tested positive. Subgroup analysis of chronic lymphocytic leukemia (CLL) patients at day 35 (C) and correlation day 35 (D). The dashed line indicates positive
threshold for SARS-CoV-2-specific IgG and IFN-g spot forming units (SFU)/106 cells, 0.8 U/mL and 30 SFU/106 cells respectively. The dotted line represents the
lower limit of detection of both assays. Each dot represents one patient. Rs=Spearman r value. Kruskal-Wallis test with Dunn’s correction for multiple compar-
isons. *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001.
haematologica | 2022; 107(4) 1001

U/mL (range, 0.4-559) and 0.4 U/mL (0.4-170) respective- tology patients whereas we found it only in three of 52
ly. This compared to a median antibody titer of 2,696 patients with CLL (6%). A third dose is currently
U/ml (range, 766-14,269) in controls (Table 1). explored in CLL and its effect on T-cell immunity, even
Longitudinal assessment of T-cell immunity against though its additional effect on T cells was limited in solid
SARS-CoV-2 spike peptides (ELISpot) is shown in Figure tumors.15 CLL remain as a group of special concern in the
1B and summarized in Table 1. At d35, 15 of 52 patients ongoing pandemic.
(29%) had a specific T-cell response (P<0.05 vs. baseline,
Figure 1B) compared to 24 of 41 (59%) in controls Lisa Blixt,1,2* David Wullimann,3* Soo Aleman,4,5 Jeanette
(P<0.01). Pre-existing spike-cross-reactive T cells12 was Lundin,1,2 Puran Chen,3 Yu Gao,3 Angelica Cuapio,3 Mira
observed at baseline in five of 50 patients; four patients Akber,3 Joshua Lange,3 Olga Rivera-Ballesteros,3 Marcus
showed no vaccine response and one patient showed a Buggert,3 Hans-Gustaf Ljunggren,3 Lotta Hansson1,2 and
marginal increase in T-cell response (mean spot count Anders Österborg1,2 for the COVAXID clinical study group***
from 44 to 70 SFU/106 PBMC at d35). A positive T-cell 1
Department of Hematology, Karolinska University Hospital Solna;
response was observed in nine of 50 tested patients 2
Deptartment of Oncology-Pathology, Karolinska Institutet; 3Center for
(18%) at d10 and in six of 51 (12%) at d21. Infectious Medicine, Department of Medicine Huddinge, Karolinska
CLL subgroup results are shown in Figure 1C and Table Institutet; 4Department of Infectious Diseases, Karolinska University
1. IFN-g positivity was observed in seven of ten patients Hospital and 5Department of Medicine Huddinge, Infectious Diseases,
at d35 who were off ibrutinib, whereas only two of 14 Karolinska Institutet, Stockholm, Sweden
patients on ibrutinib developed T-cell immunity (P<0.01).
The corresponding numbers were four of 14 among pre- *LB and DW contributed equally as co-first authors
viously untreated patients and two of 14 if previously Correspondence:
treated with CD20 mAb (P<0.05 and P<0.01, respective- LOTTA HANSSON- lotta.hansson@regionstockholm.se
ly, vs. patients off ibrutinib). doi:10.3324/haematol.2021.280300
Finally, we analyzed correlation between seroconver-
sion and T-cell response (Figure 1D). A weak but signifi- Received: November 9, 2021.
cant correlation was observed (r=0.2861, P=0.049). Accepted: January 5, 2022.
Fifteen patients (29%) were double-negative i.e., neither Prepublished: January 20, 2022.
mounted a T-cell response nor seroconverted, whereas
nine (18%) came out positive in both assays. Twenty Disclosures: MB is a consultant for Oxford Immunotech. All other
patients (39%) were positive in serology only and only authors have no conflicts of interest to disclose..
three patients (2 in the off ibrutinib group) had an IFN-g Contributions: LH, AÖ, MB and HGL contributed to conceptual-
response in the absence of seroconversion. Most double- ization, funding acquisition and discussion of data; DW, PC, YG, AC,
negative patients (11/15) were found among patients on JL, ORB and MA contributed to sample processing throughout the
ibrutinib. Double-positive patients were most frequent in COVAXID clinical trial; DW performed experiments and analyzed
those off ibrutinib (4/9). Of the 20 seroconverted patients data; LB, LH and AÖ recruited CLL study participants, conducted
with no T-cell response, one patient was found in the investigation through recruitment of the study participants and conduct-
ongoing ibrutinib and one in the previously ibrutinib ed management of participants during the trial and analyzed data; SA
treated group, while eight were previously untreated and was the PI of the COVAXID clinical trial, contributed to conceptualiza-
ten were previously treated with CD20 mAb. tion, funding acquisition and discussion of data; JL recruited CLL study
Following natural COVID-19 infection, durable immu- participants including those off BTK inhibitor-treatment and discussed
nity including both antibodies and T cells seem to occur data; LB, DW, AÖ, LH, MB and HGL wrote the original draft of the
in most healthy individuals13 as well as in patients with manuscript. All authors reviewed and edited revisions of the manuscript
CLL.4 Most healthy individuals mount T-cell responses and had final responsibility for the decision to submit for publication.
following mRNA vaccination.14 This was reported also in Acknowledgements: we thank all patients who donated blood for
patients with solid tumors.15 Lower numbers were this study and Leila Relander and Sonja Sönnert-Husa for technical
recently reported in patients with hematological malig- assistance.
nancies.8,9 The present study shows that, compared to Funding: this study was supported by grants from the SciLifeLab
healthy controls, half as many of patients with CLL National COVID-19 Research Program, financed by the Knut and
developed IFN-g T-cell response (28% vs. 59%) after two Alice Wallenberg Foundation, the Swedish Research Council, Region
doses of mRNA vaccine. A limitation of the present T-cell Stockholm, the Swedish Blood Cancer Foundation and Karolinska
assay11 is capturing of only IFN-g positive cells e.g., miss- Institutet.
ing-out on other cytokine-secreting antigen-specific T
cells. Despite this, we were able to capture both temporal Clinical trial information: The COVAXID clinical trial (EudraCT
and group dynamic changes of the SARS-CoV-2 spike- no.2021-000175-37) was approved by the Swedish Medical Product
specific T-cell response, and were able to make compari- Agency (ID 5.1-2021-5881) and the Swedish Ethical Review
son of patients with CLL with healthy controls. CLL sub- Authority (ID 2021-00451)
group results were driven by patients who were off ibru- ***The COVAXID Clinical Study Group: Peter Bergman, MD,
tinib (7/10 responded) whereas other CLL sub-groups Dept. of Infectious Diseases, Karolinska University Hospital and
had few T-cell responders. However, the data must be Dept. of Laboratory Medicine, Clinical Microbiology, Karolinska
viewed with caution due to the open-label trial design Institutet, Stockholm; Ola Blennow, MD, Dept. of Infectious Diseases,
and the small numbers within each subset. Thus, our Dept. of Transplantation, Karolinska University Hospital and Dept. of
subgroup analysis should be confirmed in extended stud- Clinical Science, Intervention and Technology, Karolinska Institutet,
ies. Even though our healthy controls were younger Stockholm; Lotta Hansson, MD, Dept. of Hematology, Karolinska
(median age 52 years) age did not impact on their T-cell University Hospital Solna and Dept. of Oncology-Pathology,
response (data not shown). Double-negativity was found Karolinska Institutet, Stockholm; Stephan Mielke, MD, Dept of
in most patients on ibrutinib who remain of major con- Laboratory Medicine, Biomolecular and Cellular Medicine, Karolinska
cern, suggesting that temporary cessation of BTKi may be Institutet and Dept. of Cellular Therapy and Allogeneic Stem Cell
explored onwards in future studies. Of note, Ehmsen et Transplantation (CAST), Karolinska University Hospital Huddinge,
al.9 found T-cell responses in 26% of seronegative hema- Stockholm; Piotr Nowak, MD, Dept. of Infectious Diseases,
1002 haematologica | 2022; 107(4)

Karolinska University Hospital and Dept. of Medicine Huddinge, chronic lymphocytic leukemia: clinical outcome and B- and T-cell
Infectious Diseases, Karolinska Institutet, Stockholm, and Laboratory immunity during 13 months in consecutive patients. Leukemia.
2022;36(2):476-481.
for Molecular Infection Medicine Sweden MIMS, Umeå University,
5. Herishanu Y, Avivi I, Aharon A, et al. Efficacy of the BNT162b2
Umeå; Puran Chen, MD, Dept. of Medicine Huddinge, Center for mRNA COVID-19 vaccine in patients with chronic lymphocytic
Infectious Medicine, Karolinska Institutet, Stockholm; Gunnar leukemia. Blood. 2021;137(23):3165-3173.
Söderdahl, MD, Dept. of Transplantation, Karolinska University 6. Roeker LE, Knorr DA, Thompson MC, et al. COVID-19 vaccine effi-
Hospital and Dept. of Clinical Science, Intervention and Technology, cacy in patients with chronic lymphocytic leukemia. Leukemia.
Karolinska Institutet, Stockholm; Gordana Bogdanovic, MD, Dept. of 2021;35(9):2703-2705.
7. Bergman P, Blennow O, Hansson L, et al. Safety and efficacy of
Clinical Microbiology, Karolinska University Hospital, Stockholm; mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of
Anders Österborg, MD, Dept. of Hematology, Karolinska University immunocompromised patients and healthy controls in a prospective
Hospital Solna and Dept. of Oncology-Pathology, Karolinska open-label clinical trial. EBioMedicine. 2021;9;74:103705.
Institutet, Stockholm; C. I. Edvard Smith, MD, Dept. of Laboratory 8. Monin L, Laing AG, Munoz-Ruiz M, et al. Safety and immunogenic-
Medicine, Karolinska Institutet, Stockholm; Marcus Buggert, PhD, ity of one versus two doses of the COVID-19 vaccine BNT162b2 for
Dept. of Medicine Huddinge, Center for Infectious Medicine, patients with cancer: interim analysis of a prospective observational
study. Lancet Oncol. 2021;22(6):765-778.
Karolinska Institutet, Stockholm; Hans-Gustaf Ljunggren, MD, Dept. 9. Ehmsen S, Asmussen A, Jeppesen SS, et al. Antibody and T cell
of Medicine Huddinge, Center for Infectious Medicine, Karolinska immune responses following mRNA COVID-19 vaccination in
Institutet, Stockholm; Per Ljungman, MD, Dept. of Medicine patients with cancer. Cancer Cell. 2021;39(8):1034-1036.
Huddinge, Hematology, Karolinska Institutet and Dept. of Cellular 10. Sahin U, Muik A, Derhovanessian E, et al. COVID-19 vaccine
Therapy and Allogeneic Stem Cell Transplantation (CAST), BNT162b1 elicits human antibody and TH1 T cell responses. Nature.
Karolinska University Hospital Huddinge, Stockholm; Soo Aleman, 2020;586(7830):594-599.
11. Sekine T, Perez-Potti A, Rivera-Ballesteros O, et al. Robust T cell
MD, Dept. of Infectious Diseases, Karolinska University Hospital and immunity in convalescent individuals with asymptomatic or mild
Dept. of Medicine Huddinge, Infectious Diseases, Karolinska Institutet, COVID-19. Cell. 2020;183(1):158-168.
Stockholm, Sweden 12. Loyal L, Braun J, Henze L, et al. Cross-reactive CD4(+) T cells
enhance SARS-CoV-2 immune responses upon infection and vacci-
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haematologica | 2022; 107(4) 1003

Neural stem cells residing in the SVZ divide slowly and

Acute lymphoblastic leukemia cells are able to give rise to rapidly proliferating cells, called transit ampli-
infiltrate the brain subventricular zone stem cell fying progenitors, which then differentiate to neuro-
niche and impair neurogenesis blasts. These immature neuronal progenitors further
migrate along a pathway, called the rostral migratory
In pediatric acute lymphoblastic leukemia (ALL), the stream, towards the olfactory bulb where they differenti-
most common hematologic malignancy in childhood, ate into olfactory interneurons and integrate in the exist-
central nervous system (CNS) relapse is a major clinical ing neuronal circuitry involved in odor discrimination.6
problem, accounting for about one-third of the relapses.1
Nevertheless, the neurogenic niches, apart from their
Since the early autopsy studies, CNS leukemia has been
ability to support and maintain neural stem cells, can also
described primarily as a leptomeningeal disease which
serve as refuge for neoplastic cells, and the migratory
can be accompanied by infiltration of different brain
parenchyma areas by ALL cells.2,3 The CNS is therefore mechanisms of neural stem cells can be utilized by tumor
considered to act as a sanctuary for ALL, but the specific cells;7 therefore, in this study we investigated whether
neural microenvironments in which leukemic cells can the SVZ can also harbor ALL cells.
stay for prolonged periods of time as extramedullary min- A xenograft model of non-obese diabetic/severe com-
imal residual disease and be responsible for CNS relapses bined immunodeficiency/IL-2Rgnull (NSG) mice injected
are still poorly defined. Recently, we and others have with the Nalm-6 human pre-B ALL cell line was used to
reported that the choroid plexus stroma and the lep- analyze the presence of leukemia cells in the brain SVZ.
tomeningeal stromal cell network are two of those neural When symptoms of CNS involvement (such as hind limb
microenvironments able to lodge ALL cells and promote paralysis) appeared, animals were deeply anesthetized
their survival and acquisition of quiescence and chemore- and transcardially perfused immediately before the SVZ
sistance.4,5 were carefully dissected and dissociated. Flow cytometry
Neurogenic niches, such as the subventricular zone analysis of cells recovered from dissociated SVZ revealed
(SVZ) and the subgranular zone, are areas of the brain in that all mice with leukemic infiltration in the CNS
which neurogenesis takes place throughout life. showed CD19+ human blasts in this brain location
Concretely, the SVZ is located along the walls of the lat- (Figure 1A), and these ALL cells could represent up to
eral brain ventricles and represents the largest neurogenic 30% of total leukemic cells invading the nervous
niche in the postnatal and adult mammalian brain. parenchyma (Figure 1B). Similar results were obtained
A B
C D E
Figure 1. Acute lymphoblastic leukemia cells infiltrate the subventricular zone neurogenic niche. (A) Percentage of CD19+ leukemia cells detected in subven-
tricular zones (SVZ) from mice xenografted with pre-B acute lymphoblastic leukemia (ALL) (n=8). When disease symptoms were evident, mice were deeply anes-
thetized and transcardially perfused with cold 0.1 M phosphate-buffered saline (pH 7.4) to clear circulating leukemia cells prior to euthanasia. Brains were then
removed, washed several times with cold Dulbecco phosphate-buffered saline and SVZ were carefully microdissected, dissociated and processed for flow cytom-
etry. Representative dot plots show, after gating out the non-neurogenic cell populations, the expression of human CD19 (hCD19) versus murine CD24 (mCD24),
a neuroblast cell marker, in SVZ from leukemic and healthy mice. (B) Percentages (mean ± standard deviation [SD]) represent the leukemic cells present in the
SVZ or the brain parenchyma out of total CD19+ cells infiltrated into the brain. (C) Proportion of CD19+ leukemia cells detected by flow cytometry in SVZ from
mice xenografted with ALL cell lines REH and RS4;11 (n=4-8). (D) Percentages of CD19+ leukemia cells expressing Ki-67 in the SVZ and meninges from
xenografted mice (n=3). Representative histograms show the expression of Ki-67 antigen in CD19+ leukemic cells found in the SVZ and the meninges (*P≤0.05;
t-test). (E) Mice were injected with Nalm-6 cells and after successful engraftment and randomization, the leukemic mice were intraperitoneally treated with
methotrexate (5 mg/kg) or saline twice a week for 4 weeks (n=6). Percentages of CD19+ cells present in the SVZ and the rest of the brain, including the
meninges, were determined by flow cytometry (**P≤0.01; t-test).
1004 haematologica | 2022; 107(4)

with mice xenografted with other ALL cell lines, REH tor EGFR. The population of neural stem cells was
and RS4;11 (Figure 1C). However, no correlation was defined as GLAST+CD24-/lowCD9high and further classified
seen between the degree of leukemic infiltration in brain by EGFR expression and GLAST intensity into quiescent,
parenchyma and the proportion of ALL cells present in primed quiescent and activated neural stem cells. Transit
the SVZ. Immunofluorescence studies in brain cryosec- amplifying progenitors were defined as GLAST-CD24-
/low
tions from xenografted mice showed that leukemic cells EGFR+ cells, and the GLAST-CD24high population
could be seen, apart from in the SVZ niche, also along the included EGFR+ proliferating (NB1 or early) and EGFR-/low
rostral migratory stream (Online Supplementary Figure S1). migrating (NB2 or late) neuroblasts. As can be seen in
These data indicate that the SVZ can provide a favorable Figure 2A, the percentage of total neural stem cells was
microenvironment in which ALL cells can survive and be notably increased in xenografted mice, with quiescent
maintained over time. Supporting this, the study of the neural stem cells being the main subset responsible for
leukemia proliferation rate using Ki-67 staining showed this rise. Concomitantly, the proportion of transit ampli-
that ALL cells found in the SVZ niche exhibit a much fying progenitors and late neuro-blasts was reduced in
lower proliferative activity than those leukemic cells iso- these animals. These results suggest that SVZ neurogen-
lated from the meninges (Figure 1D). Furthermore, esis is impaired in leukemia-bearing mice at the expense
leukemic cells infiltrating the SVZ niche were shown to of an increase in quiescence, and the effect appears to be
have higher chemoresistance after methotrexate treat- a direct consequence of the leukemic cell infiltration in
ment of xenografted mice (Figure 1E). the SVZ since the most affected animals were those
The above results showed that leukemic invasion of showing the highest numbers of CD19+ cells in the neu-
the SVZ neurogenic niche is a common event in the rogenic niche. Figure 2B shows that the percentages of
xenograft model of ALL, so we analyzed the effects of CD19+ leukemia cells correlated directly with the accu-
this infiltration on the differentiation of neural stem cells. mulation of quiescent neural stem cells, and inversely
The proportion of the different SVZ populations was with the proportions of late neuroblasts (Figure 2C). In
determined by flow cytometry using a combination of line with these data, and since the generation of new
multiple specific cell markers, as we previously olfactory bulb neurons from the SVZ is required for novel
described.8 Non-neurogenic cells were first discarded odor discrimination,9 xenografted mice displayed altered
from the study, and the remaining neurogenic lineage cell olfactory discrimination capacities (Figure 2D).
pool was subdivided according to the expression of the To analyze the effects of leukemia on neural precursors
glial marker GLAST, the neuroblast marker CD24, the directly, we first generated SVZ neurospheres, floating
tetraspanin CD9 and the proliferation-associated recep- cellular aggregates clonally derived from neurosphere-ini-
B C D
Figure 2. Subventricular zone cell populations are affected by the presence of acute lymphoblastic leukemia cells. (A) Bars represent the percentages (mean
± standard deviation [SD]) of total, quiescent (q), primed quiescent (p) and activated (a) neural stem cells (NSC), as well as transit amplifying progenitors (TAP)
and proliferating (NB1) and migrating (NB2) neuroblasts present in the subventricular zones (SVZ) from leukemic (gray) and healthy (black) mice (n=8). All these
neurogenic cell populations were defined according to the expression of the glial marker GLAST, the neuroblast marker CD24, the tetraspanin CD9 and the pro-
liferation-associated receptor EGFR (*P ≤ 0.05; t-test). (B,C) The percentages of CD19+ cells found in the SVZ are represented as a function of the corresponding
(B) increases in the proportion of quiescent NSC and (C) decreases in the proportion of migrating EGFR- neuroblasts. P values of the Pearson correlation are
provided. (D) Olfactory habituation-dishabituation tests of healthy (black circles) and leukemic (gray triangles) mice were performed at week 3, before the typical
disease symptoms (including rough hair, lethargy, hunched-back posture, loss of motor functions and hind limb paralysis) were observed. Exploration time (in
seconds) of successive cotton swabs soaked in octanal (O), heptanal (H), or anisole (A) is shown. After exposure to octanal-soaked swabs, healthy mice reacted
to heptanal- and anisole-soaked swabs; however leukemic mice displayed lower olfactory exploration and no reaction to the new odor stimuli. Asterisks represent
statistically significant differences (*P≤0.05; t-test). ALL: acute lymphoblastic leukemia.
haematologica | 2022; 107(4) 1005

tiating neural stem cells which constitute an ideal system were re-plated in fresh growth medium without
to evaluate modifications in proliferation and self-renew- leukemia-derived factors and neurosphere formation was
al. Single cells dissociated from neurospheres were either evaluated. In these cultures, the numbers of secondary
cultured with medium conditioned by leukemic cells or neurospheres were not altered but significant changes in
co-cultured with ALL cells using transwell inserts. In sphere diameters could be newly detected, indicating
both cases, although no change in the number of new that leukemia cells can reduce activation without altering
neurospheres was found after 10 days (Figure 3A), a sig- self-renewal (Figure 3D, E).
nificant reduction in neurosphere sizes could be clearly ALL cells have been reported to be able to induce a pro-
observed (Figure 3B), suggesting that leukemia-derived inflammatory microenvironment in different locations.4,10
factors limit growth but not survival of neurosphere cells. The expression of pro-inflammatory factors was there-
In agreement, the inhibition of the expansion potential of fore analyzed in the SVZ of healthy and leukemic mice.
neurospheres in the presence of leukemia cells was As shown in Figure 3F, the levels of IL-1b, IL-6 and TNF-
detected throughout the culture period using MTS prolif- a cytokines as well as CCL2 and CXCL10 chemokines
eration assays (Figure 3C). To analyze whether ALL- were notably upregulated in the leukemia-invaded SVZ
mediated effects included effects on self-renewal, cells niches. All these inflammatory mediators have been
obtained from neurospheres that had been grown in the described as negative regulators of neurogenesis.11
presence of soluble factors secreted by leukemic blasts However, we have recently reported that TNF-a, which
A B C
D E
Figure 3. Acute lymphoblastic leukemia cells impair neurogenesis. (A) Number

of subventricular zone (SVZ) primary neurospheres grown in neural stem cell
(NSC) complete medium previously conditioned by Nalm-6 leukemic cells for 48
h (CM; grey bars) or unconditioned growth medium (CTL; black bars). Data rep-
resent mean ± standard deviation (SD) from three independent experiments.
F (B) Percentage of small (<60 mm diameter), medium (60-180 mm diameter) and
large (>180 mm diameter) primary neurospheres grown in conditioned or
unconditioned NSC complete medium. Data represent mean ± SD from three
independent experiments (*P≤0.05, **P≤0.01; t-test). (C) Proliferation of neu-
rospheres was measured by MTS assays after 1, 3 and 6 days of culture in
unconditioned (black circles) or leukemia-conditioned medium (gray squares).
Data represent mean ± SD from three independent experiments (***P≤0.001;
Mann–Whitney test). (D) Percentages of small, medium and large secondary
neurospheres generated after dissociation of primary neurospheres, grown in
conditioned and unconditioned medium, and replated in fresh NSC complete
medium. Data represent mean ± SD from three independent experiments
(*P≤0.05; t-test). (E) Micrographs show floating neurospheres grown in uncon-
ditioned and leukemia-conditioned medium. Scale bar: 100 mm. (F) The expres-
sion of the indicated cytokines and chemokines was analyzed by reverse tran-
scriptase quantitative polymerase chain reaction in SVZ cells from healthy
(black bars) and leukemic mice (gray bars). Fold induction relative to cells from
healthy SVZ is shown, and the mean ± SD of four independent experiments is
presented (*P≤0.05; Mann–Whitney test). ALL: acute lymphoblastic leukemia.
1006 haematologica | 2022; 107(4)

underwent one of the highest increases in expression, Funding: this work was supported by grants RTI2018-093899-B-
reduces neuroblast generation because it induces a tran- I00 and SAF2017-86690-R (Spanish Ministry of Economy and
sient activation of neural stem cells followed by their Competitiveness), RD16/0011/0002 and CB06/05/0086 (Institute of
entry into quiescence.8 Transgenic mice overexpressing Health Carlos III, Spain), B2017/BMD-3692 AvanCell-CM
IL-6 in astrocytes exhibit reduced cycling of neural stem (Community of Madrid), and Beca I-UnoEntreCienMil (Uno Entre
cells in the subgranular zone niche, suggesting that it acts Cien Mil Foundation). LMFS was supported by a pre doctoral fellow-
as a negative regulator of proliferation,12 and IL-1b is ship (CT45/15 CT46/15) from the Complutense University of
reportedly secreted by choroid plexus cells to the cere-
Madrid. POS is supported by a pre-doctoral fellowship (CT63/19-
brospinal fluid and induces the upregulation of VCAM-1
CT64/19) from the Complutense University.
levels in SVZ neural stem cells, reducing their prolifera-
tion and preventing lineage progression.13 Importantly,
the levels of IL-1b, IL-6, TNF-a, CCL2 and CXCL10 have References
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haematologica | 2022; 107(4) 1007

CASE REPORTS
transfusion support. However, the patient refused hospi-
Immune-mediated thrombotic thrombocytopenic tal admission and was discharged. Seven days later, on
purpura following administration of Pfizer-BioNTech admission to our center, the complete blood count again
COVID-19 vaccine showed severe anemia (hemoglobin 5.6 g/dL) and throm-
bocytopenia (platelet count 23x109/L) with a normal
Coronavirus disease 2019 (COVID-19) is an infectious white blood cell count. Markers of hemolysis were pres-
disease caused by a recently discovered coronavirus ent, including increased reticulocytes, increased lactate
called severe acute respiratory syndrome coronavirus 2 dehydrogenase (1905 U/L, normal values [n.v:] 0-248),
(SARS-CoV-2). The disease affected well over a hundred increased unconjugated bilirubin (5.5 mg/dL, n.v: 0.30-
million people all over the world and mortality rates 1.20) and reduced haptoglobin (<7 mg/dL) (Table 2).
were greater in the elderly population. The quick devel- Coagulation and renal function tests were within normal
opment of safe and effective vaccines represents an limits. Both direct and indirect antiglobulin tests were
important step in the management of the ongoing pan- negative. Examination of a peripheral blood smear
demic and, so far, four vaccines have been approved by revealed an increased number of schistocytes (10% per
the European Medicines Agency, including mRNA field). The PLASMIC score (6 points) classified the
(Pfizer-BioNTech and Moderna) and adenovirus-based
patient as being at high risk of severe ADAMTS13 defi-
vaccines (AstraZeneca and Johnson & Johnson).
ciency.6 Tumor markers and infectious screening for hep-
Immune-mediated thrombotic thrombocytopenic pur-
atitis B virus, hepatitis C virus, human immunodeficiency
pura (TTP) is a rare disease (annual incidence between
virus, cytomegalovirus and Epstein-Barr virus resulted
1.5 and 6.0 cases per million)1 characterized by microan-
negative. Autoimmunity screening revealed the presence
giopathic hemolytic anemia, thrombocytopenia and
of anti-nuclear antibodies (titer 1:640, n.v. <1:80). A rapid
ischemic end-organ injury due to microvascular platelet-
ADAMTS13 test was performed demonstrating marked-
rich thrombi. The formation of microvascular thrombi is
caused by a deficiency of ADAMTS13, a von Willebrand ly reduced activity (below 10%) with a high titer of anti-
factor-cleaving protease, due to the presence of anti- ADAMTS13 antibodies according to enzyme-linked
ADAMTS13 autoantibodies.2 Cases of immune-mediated immunosorbent assay (ELISA) in serum (40 U/mL, n.v.
TTP following the administration of vaccines have been 12-15), thus confirming the diagnosis of immune-mediat-
previously described and recently reviewed (Table 1).3 ed TTP. The patient was promptly started on intravenous
However, only one case of newly diagnosed immune- methylprednisolone 1 mg/kg and daily sessions of plas-
mediated TTP following COVID-19 vaccination has been ma exchange in combination with the humanized anti-
reported in the literature: it occurred in a 62-year-old von Willebrand factor nanobody, caplacizumab.
female patient 37 days after receiving the adenovirus- Caplacizumab was administered intravenously at the
based AstraZeneca vaccine.4 Additinoally, a case of dose of 10 mg before the first plasma-exchange, followed
immune-mediated TTP relapse was recently described in by 10 mg subcutaneous injections after each plasma-
a 48-year-old female patient after the second dose of the exchange. The patient was transfused with several units
Pfizer-BioNTech vaccine.5 Here we report two cases of of concentrated red blood cells. After an initial clinical
immune-mediated TTP following the first dose of the benefit with resolution of hematuria and early signs of
Pfizer-BioNTech COVID-19 vaccine. Informed consent hematologic recovery with a platelet count of 30x109/L,
was obtained from the patients regarding the report of the patient died after only 2 days of treatment, probably
their clinical scenarios. due to a sudden cardiovascular event.
Case 1. In April 2021, an 83-year-old female patient Case 2. In June 2021, a 30-year-old woman, a b-tha-
was admitted to the emergency room with severe anemia lassemia carrier, was admitted to the emergency room
and macrohematuria, in the absence of fever, neurologi- because of the appearance of diffuse petechiae, intense
cal signs and renal impairment. On clinical examination, headache and fatigue. The patient had received her first
diffuse petechiae and venipuncture hematomas were dose of the Pfizer-BioNTech COVID-19 vaccine 18 days
observed. The patient suffered from undifferentiated before the admission. On admission, a complete blood
connective tissue disease treated with low-dose steroids count revealed the presence of anemia (hemoglobin 8.9
and steroid-induced diabetes mellitus. The woman had g/dL) and thrombocytopenia (platelet count 11x109/L),
been administered the first dose of Pfizer-BioNTech with a normal white blood cell count (9.2x109/L). Total
COVID-19 vaccine 14 days prior to the admission. One body computed tomography was negative. A peripheral
week before the admission, the patient was treated blood smear showed the presence of schistocytes (5-10%
briefly at another center because of fatigue and the per field). Investigations for hemolysis were positive,
appearance of petechiae. A complete blood count while both direct and indirect Coombs tests were nega-
revealed grade 3 anemia (hemoglobin 6.1 g/dL) and tive. Coagulation, hepatic and renal function tests were
thrombocytopenia (platelet count 46x109/L), requiring all within normal limits (Table 3). Tumor markers,
Table 1. Cases of immune-mediated thrombotic thrombocytopenic purpura following the administration of vaccines.
Vaccine type n Age (years) Sex Time of development Literature
th
Rabies 1 28 Male 14 day Kadikoylu et al., 2014
Pneumococcal 1 68 Female 15th day Kojima et al., 2014
Influenza 1 Unknown Unknown Unknown Ramakrishnan et al., 1998
Influenza 1 54 Male 5th day Dias and Gopal, 2009
H1N1 1 56 Male 13th day Hermann et al., 2010
Influenza 1 23 Female 14th day Brown et al., 1973
Source: Yavaşoğlu I.Vaccination and thrombotic thrombocytopenic purpura. Turkish J Hematol. 2020;37(3):218-219.
1008 haematologica | 2022; 107(4)

Case Reports
Table 2. Case 1: clinical and laboratory characteristics at diagnosis. Table 3. Case 2: clinical and laboratory characteristics at diagnosis.
Characteristic Characteristic
Age 83 years Age 30 years
Sex Female Sex Female
Comorbidities UCTD, DM Comorbidities None
Medications Low-dose steroids, insulin Medications None
Time from vaccination to admission, days 14 Time from vaccination to admission, days 18
Hemoglobin, g/dL 5.6 (n.v. 12-16) Hemoglobin, g/dL 8.9 (n.v. 12-16)
Platelets, x109/L 23 (n.v. 130-400) Platelets, x109/L 11 (n.v. 130-400)
9
WBC, x10 /L 9.260 (n.v. 4.8-10.8) WBC, x109/L 9.2 (n.v. 4.8-10.8)
INR 1.14 (n.v. 0.8-1.2) INR 1.03 (n.v. 0.8-1.2)
aPTT, sec 24 (n.v. 24-36) aPTT, sec 26 (n.v. 24-36)
Fibrinogen, mg/dL 141 (n.v. 170-400) Fibrinogen, mg/dL 156 (n.v. 170-400)
Reticulocytes, % 28 (n.v. 0-25) Reticulocytes, % 29 (n.v. 0-25)
LDH, U/L 1905 (n.v. 0-248) LDH, U/L 900 (n.v. 0-248)
Haptoglobin, mg/dL <7 (n.v. 36-145) Haptoglobin, mg/dL <7 (n.v. 36-145)
Unconjugated bilirubin, mg/dL 5.5 (n.v. 0.30-1.20) Unconjugated bilirubin, mg/dL 2.5 (n.v. 0.30-1.20)
Creatinine, mg/dL 0.88 (n.v. 0.51-0.95) Creatinine, mg/dL 0.90 (n.v. 0.51-0.95)
UCTD: undifferentiated connective tissue disease; DM: steroid-induced diabetes n.v.: normal values; WBC: white blood cell count; INR: International Normalized
mellitus; n.v.: normal values; WBC: white blood cell count; INR: International Ratio; aPTT: activated partial thromboplastin time; LDH: lactate dehydrogenase.
Normalized Ratio; aPTT: activated partial thromboplastin time; LDH: lactate dehy-
drogenase.
autoimmune and infectious screening resulted negative. disease onset or its flare were described after COVID-19
The PLASMIC score (6 points) classified the patient as vaccination,9-11 including cases of post-vaccination
high risk A rapid ADAMTS13 test revealed reduced immune thrombocytopenic purpura.12,13 A lot of atten-
activity (below 10%), while a high titer of anti-ADAMTS tion has recently been given to the thrombotic risk of
13 antibodies was confirmed by ELISA (77.6 U/mL; n.v. COVID-19 vaccination. In particular, a new syndrome
12-15). The woman was treated promptly with daily ses- called vaccine-induced immune thrombotic thrombocy-
sions of plasma exchange in combination with capla- topenia (VITT) following administration of the aden-
cizumab and intravenous methylprednisolone 1 mg/kg, ovirus-based vaccine AstraZeneca has been described.
and responded well to treatment. Her platelet count nor- This syndrome is characterized by thrombosis at unusual
malized on day 5 (platelet count, 158x109/L) with a sites, thrombocytopenia and the presence of high levels
hemoglobin value of 8.1 g/dL. Daily plasma exchange of antibodies to platelet factor 4 (PF4) in the absence of
was continued for 8 consecutive days and she was dis- heparin treatment.14 In our cases, another disease charac-
charged on day 8. On day 14 and on day 30 ADAMTS13 terized by an increased thrombotic risk developed fol-
activity was 0 U/mL (n.v. 0.4-1.3), while anti- lowing administration of an mRNA COVID-19 vaccine.
ADAMTS13 antibody titer progressively reduced, being Furthermore, the clinical cases we have described con-
47 U/mL and 30 U/mL on day 14 and on day 30, respec- firm that even a single administration of vaccine can
tively. The patient continued therapy with caplacizumab induce the development of autoimmune manifestations
for 30 days after stopping daily plasma-exchange treat- especially in predisposed subjects. The consequences of
ment. developing antibodies against ADAMTS13 can be very
To the best of our knowledge, this is the first report of serious and even fatal. It is, therefore, always necessary
newly diagnosed immune-mediated TTP following the to take a thorough history before the administration of
first dose of COVID-19 Pfizer-BioNTech vaccine. COVID-19 vaccines, and careful clinical surveillance in
Immune-mediated TTP was not mentioned among the the post-vaccine period must be taken into consideration
adverse events in the pivotal study leading to the in patients with autoimmune diseases or a clinical or fam-
approval of this vaccine.7 Given the immune origin of the ily history leading to the suspicion of an autoimmune
TTP and the short latency period between the COVID- tendency.
19 vaccine and disease onset, a hypothesized temporal
association is plausible. Although many cases of Gaetano Giuffrida,1* Annalisa Condorelli,1,2*
immune-mediated TTP following vaccinations have been Mary Ann Di Giorgio,1,2 Uros Markovic,1-3
reported previously, including those following influenza, Roberta Sciortino,1,2 Daniela Nicolosi1 and
pneumococcal, rabies and a recently published COVID- Francesco Di Raimondo1
19 adenovirus vector-based vaccines,3-8,10 the underlying *These authors contributed equally to the work
mechanism is still unknown. In our first case, the patient 1
Division of Hematology, AOU "Policlinico G. Rodolico-San
also suffered from undifferentiated connective tissue dis- Marco", Via Santa Sofia 78, 95124 Catania, Italy; 2Postgraduate
order, an autoimmune disease that might have been a School of Hematology, University of Catania, Italy and 3Unità
predisposing factor to post-vaccination TTP. In the litera- Operativa di Oncoematologia e BMT Unit, Istituto Oncologico del
ture, there is evidence of vaccine-induced autoimmunity, Mediterraneo, Viagrande, Italy
adjuvant-induced autoimmunity and antibody cross-
reaction in both experimental models as well as human Correspondence: ANNALISA CONDORELLI -
patients.8 Furthermore, other cases of immune-mediated condorelli.1312@gmail.com
haematologica | 2022; 107(4) 1009

Case Reports
doi:10.3324/haematol.2021.279535 bocytopenic purpura after COVID-19 vaccine. Transfus Apher Sci.

2021:103145.
Received: June 30, 2021. 6. Oliveira DS, Lima TG, Benevides FLN, et al. Plasmic score applica-
Accepted: July 21, 2021. bility for the diagnosis of thrombotic microangiopathy associated
with ADAMTS13-acquired deficiency in a developing country.
Pre-published: August 12, 2021. Hematol Transfus Cell Ther. 2019;41(2):119-124.
Disclosures: no conflicts of interest to disclose. 7. Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the
BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020;
Ethical statement: informed consent was obtained from the patients
383(27):2603-2615
regarding the report of their clinical scenario. 8. Guimarães LE, Baker B, Perricone C, Shoenfeld Y. Vaccines, adju-
Contributions: AC, MDG, UM, RS wrote the original draft; DN vants and autoimmunity. Pharmacol Res. 2015;100:190-209.
performed diagnostic tests; GG and FDR revised the paper critically 9. Watad A, De Marco G, Mahajna H, et al. Immune-mediated disease
and approved the final version for submission. flares or new-onset disease in 27 subjects following mRNA/DNA
SARS-CoV-2 vaccination. Vaccines. 2021;9(5):435.
10. Ïremli BG, Şendur SN, Ünlütürk U. Three cases of subacute thyroidi-
References tis following SARS-CoV-2 vaccine: post-vaccination ASIA syn-
drome. J Clin Endocrinol Metab. 2021;106(9):2600-2605.
1. Miesbach W, Menne J, Bommer M, et al. Incidence of acquired 11. Condorelli A, Markovic U, Sciortino R, Di Giorgio MA, Nicolosi D,
thrombotic thrombocytopenic purpura in Germany: a hospital level Giuffrida G. Immune thrombocytopenic purpura cases following
study. Orphanet J Rare Dis. 2019;14(1):260. COVID-19 vaccination. Mediterr J Hematol Infect Dis. 2021;
2. Sukumar S, Lämmle B, Cataland SR. Thrombotic thrombocytopenic 13(1):e2021047.
purpura: pathophysiology, diagnosis, and management. J Clin Med. 12. Helms JM, Ansteatt KT, Roberts JC, et al. Severe, refractory immune
2021;10(3):536. thrombocytopenia occurring after SARS-CoV-2 vaccine. J Blood
3. Yavaşoğlu I. Vaccination and thrombotic thrombocytopenic purpu- Med. 2021;12:221-224.
ra. Turkish J Hematol. 2020;37(3):218-219. 13. Candelli M, Rossi E, Valletta F, De Stefano V, Francheschi F. Immune
4. Yocum A, Simon EL. Thrombotic thrombocytopenic purpura thrombocytopenic purpura after SARS-CoV-2 vaccine. Br J
after Ad26.COV2-S vaccination. Am J Emerg Med. 2021:441.e3- Haematol. 2021;194(3):547-549.
441.e4. 14. Cines DB, Bussel JB. SARS-CoV-2 vaccine-induced immune throm-
5. Sissa C, Al-Khaffaf A, Frattini F, et al. Relapse of thrombotic thrombotic thrombocytopenia. N Engl J Med. 2021;384(23):2254-2256.
1010 haematologica | 2022; 107(4)

Case Reports
results in short stature, skeletal abnormalities, primary

VEXAS syndrome in a female patient with constitu- ovarian failure, and several other end-organ complica-
tional 45,X (Turner syndrome) tions.13 As with acquired X chromosome mosaicism, con-
stitutional loss of the X chromosome may also predispose
VEXAS (vacuoles, E1 enzyme, X-linked, autoinflamma- to female VEXAS. In this case report, we present the first
tory, somatic) syndrome is a newly-defined autoinflam- case of female VEXAS syndrome diagnosed in a patient
matory disorder that arises from somatic mutations with constitutional 45,X (Turner syndrome).
affecting UBA1, a major E1 enzyme that initiates ubiqui- A 67 year-old female was referred with a diagnosis of
tinylation and is important for the maturation of myelodysplastic syndrome with multilineage dysplasia
autophagic vacuoles.1,2 Specifically, these mutations (MDS-MLD), originally low-risk by IPSS (score 0) and
occur in hematopoietic stem cells and in the erythroid very low-risk by IPSS-R (score 1), which was diagnosed
and granulocyte precursors in the bone marrow.1 This 36 months prior. Peripheral blood counts at relevant
results in an autoinflammatory syndrome characterized timepoints are demonstrated in Table 1. Her bone mar-
by recurrent fevers, cytopenias, chondritis, vasculitis, pul-
row cytogenetics demonstrated 45,X (20/20) and a tar-
monary inflammation, and neutrophilic dermatoses. This
geted capture panel demonstrated variants of uncertain
inflammatory syndrome is typically treatment-refractory,
significance in DNMT3A (VAF 28.0%) and SMC3
with patients being persistently steroid-dependent, and is
(48.0%). The patient’s medical history was notable for
often fatal: 40% of patients were deceased at the time of
relapsing polychondritis, diagnosed five years previous,
inclusion in the original study.1 There is also a strong
and constitutional 45,X (Turner syndrome) that was diag-
association with hematologic malignancy. In the original
nosed in her teenage years after a failure of pubertal
series, 24% of patients developed a low-grade myelodys-
plastic syndrome (MDS) while 20% had multiple myelo- development. Her relapsing polychondritis had presented
ma or a monoclonal gammopathy.1 Patients who with a migratory inflammatory arthritis and auricular
progress to MDS tend to have more prominent thrombo- inflammation. This was treated initially with prednisone;
cytopenia and neutropenia, and one of the most com- she had trialed multiple steroid-sparing agents
monly co-occurring somatic mutations appears to be in (methotrexate, azathioprine, cyclosporine) but was
DNMT3A.3-5 Interestingly, the bone marrow morphology unable to wean from steroids. She had remained depend-
demonstrates a characteristic cytoplasmic vacuolation ent on a low dose of prednisone (10mg) from the time of
restricted to the erythroid and granulocyte precursors.6 her original diagnosis; her disease had remained stable
The UBA1 gene lies on the X-chromosome, making with a combination of steroids and etanercept. In addi-
VEXAS an X-linked syndrome. Consistent with this, the tion, she had recently been found to have a monoclonal
majority of reported cases have occurred in biological gammopathy of unclear significance (MGUS), with a very
males. The presence of a second UBA1 allele in females is faint (unquantifiable) IgG lambda monoclonal band on
thought to mitigate the impact of the presence of the immunofixation.
mutant allele should it arise.1 Part of the reason the sec- Her low-risk MDS was observed until she developed
ond allele is protective against VEXAS in women is relat- transfusion dependence 24-months after her initial diag-
ed to the fact that UBA1 does not undergo X-inactivation; nosis. This was managed with erythropoietin injections,
in contrast, other disease-causing mutations in genes on which reduced her transfusion requirements for eight
the X-chromosome that do undergo inactivation (e.g., months’ time. After failing erythropoietin, a repeat bone
PIGA mutations in Paroxysmal Nocturnal marrow biopsy was performed (32 months); this marrow
Haemoglobinuria) manifest disease at similar rates in was hypercellular (95%) and demonstrated more promi-
males and females.7,8 However, VEXAS has been nent trilineage dysplasia than her previous diagnostic
described in a small number of women (N=4).9-11 These marrow. Interestingly, her bone marrow morphology
women were observed to have acquired monosomy X demonstrated multiple vacuolated erythroid and granulo-
due to age-related mosaicism in the X chromosome.12 In cytic precursor cells (Figure 1A). The number of blast cells
contrast to acquired mosaicism of the X chromosome, was unchanged (2% of total nucleated cells), hence her
Turner syndrome refers to a constitutional loss of one X diagnosis was rendered as persistence of MDS-MLD. Her
chromosome (i.e., 45,X karyotype). This occurs in cytogenetics were unchanged (45,X [20/20]) (Figure 1B)
between 1 in 2,000 to 1 in 2,500 live female births and and her targeted capture panel re-demonstrated variants
Table 1. Peripheral blood counts at relevant timepoints throughout the disease course of a female VEXAS patient with Myelodysplastic syn-
drome (MDS)
Parameter MDS diagnosis MDS progression Transplant referral On treatment (HMA)
Timepoint (months) 0 32 36 43
Hemoglobin (g/L) 107 101 92 188
Mean cell volume (fL) 109.1 101 99 100
Platelets (x109/L) 137 35 40 102
White blood cell (x109/L) 6.60 5.30 6.90 2.60
Neutrophils (x109/L) 5.60 3.98 5.10 1.50
Lymphocytes (x109/L) 0.80 1.06 0.90 1.00
Monocytes (x109/L) 0.10 0.05 0.30 0.10
Eosinophils (x109/L) 0.10 0.11 0.00 0.10
Basophils (x109/L) 0.00 0.00 0.10 0.00
BM blasts 1% 2% NA 1%
Karyotype 45,X 45,X NA 45,X
Abbreviations: VEXAS: vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic; HMA: hypomethylating agent; BM: bone marrow.
haematologica | 2022; 107(4) 1011

Case Reports
A B
Figure 1. Representative diagnostic features of a female patient with constitutional 45,X and VEXAS syndrome (A) A diagnostic bone marrow aspirate was col-
lected and stained with Giemsa-Wright. There is trilineage dysplasia, consistent with the known diagnosis of myelodysplastic syndrome with multilineage dys-
plasia. There is notable vacuolation of the erythroid and granulocytic precursors, indicated by the black arrows. (B) Karyotyping with G-banding demonstrated X
monosomy, consistent with the patient’s reported history of constitutional 45,X (Turner syndrome). No clonal evolution was identified. A single X-chromosome
is indicated by the black arrow. (C) Sanger sequencing for the UBA1 locus performed on peripheral blood demonstrated the presence of a somatic UBA1
p.Met41Thr mutation (c.122T>C) with approximately equal allele frequency as the reference allele, confirming the diagnosis of VEXAS syndrome; the Sanger
chromatogram is demonstrated.
of uncertain significance in DNMT3A (VAF 30.0%) and underlying a diagnosis of VEXAS in a female. In contrast,
SMC3 (VAF 43.8%). With the deterioration in her periph- the underlying reason our patient was predisposed to
eral counts, her IPSS-R score was now intermediate-risk developing VEXAS was the presence of a constitutional
(score 3.5) and her IPSS score intermediate-1 (score 0.5). 45,X karyotype from birth, which was diagnosed in her
Given the recently described association between low- teenage years after a failure of pubertal development.
risk MDS/MGUS, bone marrow vacuolation, and a There are other mechanisms that can lead to the func-
steroid-dependent autoinflammatory syndrome resem- tional loss of one X chromosome, such as uniparental dis-
bling relapsing polychondritis (VEXAS syndrome) caused omy and skewed X-inactivation; in the future we may
by somatic mutations in the X-linked E1 enzyme UBA1, see these reported as an underlying risk factor in female
further diagnostic testing was pursued by referral of this VEXAS patients.
patient to the National Institutes of Health (NIH). Sanger While clinicians are increasingly aware of VEXAS syn-
sequencing was performed at the NIH; genomic DNA drome as a diagnostic entity, it is still largely character-
was prepared from peripheral blood using the Maxwell ized as an X-linked disorder affecting only males.
16 Blood DNA purification kit (Promega). Coding exons However, as our case demonstrates, it is important to be
of UBA1 were sequenced using the BigDye Terminator aware that VEXAS can affect females and further investi-
v1.1 Cycle Sequencing Kit (Applied Biosystems), and gations should be pursued in the appropriate context for
sequencing data analyzed using Sequencher (Gene female patients. In our case, the diagnosis of Turner syn-
Codes) and 4Peaks (Mekentosj). drome was established long before the development of
Sanger sequencing results were positive for the pres- her VEXAS syndrome. The underlying risk factor for
ence of a somatic UBA1 p.Met41Thr mutation female VEXAS may not always be obvious, however, as
(c.122T>C) with approximately equal allele frequency as in patients with acquired X chromosome mosaicism. It is
the reference allele, (Figure 1C) confirming the diagnosis important to be alert for the presence of disorders that
of VEXAS given her X monosomy. With her transfusion result in the inactivation of the X-chromosome when
dependence, the increase in IPSS-R score to intermediate, assessing female patients with a possible X-linked disor-
and the persistent steroid requirement due to VEXAS, it der, such as VEXAS syndrome.
was recommended that the patient be initiated on
hypomethylating agent therapy with 5-azacitidine. She Ryan J. Stubbins,1,2 Eric McGinnis,3 Bhupinder Johal,4 Luke
tolerated 5-azacitidine well and achieved transfusion YC Chen,2 Lorena Wilson,5 Daniela Ospina Cardona5 and
independence with an improvement in both hemoglobin Thomas J. Nevill1,2
and platelets (43 months). Human leukocyte antigen 1
Leukemia/BMT Program of BC, BC Cancer, Vancouver, BC,
(HLA) typing was initiated for the patient and her siblings
Canada; 2Division of Hematology, Department of Medicine,
and an unrelated donor search activated. Given her rela-
University of British Columbia, Vancouver, BC, Canada; 3Department
tively young age, a sibling-donor allogeneic hematopoiet-
of Pathology and Laboratory Medicine, University of British Columbia,
ic stem cell transplant is planned as a definitive therapy
Vancouver, BC, Canada; 4Department of Pathology, Kelowna
for both her MDS and VEXAS syndrome.
General Hospital, Kelowna, BC, Canada and 5National Human
Although VEXAS syndrome primarily affects males as
Genome Research Institute, National Institutes of Health, Bethesda,
an X-linked disease, it is important for the clinician to
MD, USA
realize that there are several factors that can lead VEXAS
(and other X-linked diseases) to manifest in females. Correspondence:
Mosaicism of the X chromosome is an age-related phe- RYAN J. STUBBINS- ryan.stubbins1@bccancer.bc.ca
nomenon that primarily affects the inactivated X chro- doi:10.3324/haematol.2021.280238
mosome; it has been shown to occur in 0.11% of 50 year-
old females, with this increasing to 0.45% of 75 year- Received: October 25, 2021.
olds.12 This has been the most commonly reported reason Accepted: December 3, 2021.
1012 haematologica | 2022; 107(4)

Case Reports
Pre-published: December 16, 2021. 4. Georgin-Lavialle S, Terrier B, Guedon AF, et al. Further characteriza-
tion of clinical and laboratory features occurring in VEXAS syndrome
Disclosures: no conflicts of interest to disclose. in a large-scale analysis of multicenter case-series of 116 French
Contributions: RJS and TJN conceived the study; RJS, EM, BJ, LW patients. Br J Dermatol., October 10, 2021,
https://doi.org/10.1111/bjd.20805, [Epub ahead of print].
and DOC collected data and images; RJS wrote the manuscript. All
5. van der Made CI, Potjewijd J, Hoogstins A, et al. Adult-onset autoin-
authors read, critically assessed, and approved the final manuscript. flammation caused by somatic mutations in UBA1: A Dutch case
Acknowledgments: we thank Dr. David Beck and the National series of patients with VEXAS. J Allergy Clin Immunol.
Institutes of Health for their collaboration and assistance with this man- 2022;149(1):432-439.e4.
6. Dehghan N, Marcon KM, Sedlic T, Beck DB, Dutz JP, Chen LYC.
uscript. Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS)
Funding: RJS is supported by grants from the Leukaemia syndrome: fevers, myalgia, arthralgia, auricular chondritis, and ery-
Lymphoma Society of Canada (20200LFC-439884), Canadian thema nodosum. Lancet. 2021;398(10300):621.
7. Carrel L, Clemson CM, Dunn JM, et al. X inactivation analysis and
Institutes of Health Research, and the University of British Columbia DNA methylation studies of the ubiquitin activating enzyme E1 and
Clinician Investigator Program. PCTAIRE-1 genes in human and mouse. Hum Mol Genet.
Data-sharing statement: data is available, upon request, from the 1996;5(3):391-401.
8. Luzzatto L, Risitano AM, Notaro R. Mutant UBA1 and Severe Adult-
corresponding author. Onset Autoinflammatory Disease. N Engl J Med. 2021;384(22):2164.
Trial registration and ethics: informed consent was obtained from the 9. Arlet JB, Terrier B, Kosmider O. Mutant UBA1 and severe adult-onset
patient for this case report. autoinflammatory disease. N Engl J Med. 2021;384(22):2163.
10. Tsuchida N, Kunishita Y, Uchiyama Y, et al. Pathogenic UBA1 vari-
ants associated with VEXAS syndrome in Japanese patients with
References relapsing polychondritis. Ann Rheum Dis., March 31, 2021,
https://doi.org/10.1136/annrheumdis-2021-220089 [Epub ahead of
1. Beck DB, Ferrada MA, Sikora KA, et al. Somatic mutations in UBA1 print].
and severe adult-onset autoinflammatory disease. N Engl J Med. 11. Barba T, Jamilloux Y, Durel CA, et al. VEXAS syndrome in a woman.
2020;383(27):2628-2638. Rheumatology (Oxford). 2021;60(11):e402-e403.
2. Lenk SE, Dunn WA, Trausch JS, Ciechanover A, Schwartz AL. 12. Machiela MJ, Zhou W, Karlins E, et al. Female chromosome X
Ubiquitin-activating enzyme, E1, is associated with maturation of mosaicism is age-related and preferentially affects the inactivated X
autophagic vacuoles. J Cell Biol. 1992;118(2):301-308. chromosome. Nat Commun. 2016;7:11843.
3. Obiorah IE, Patel BA, Groarke EM, et al. Benign and malignant 13. Gravholt CH, Viuff MH, Brun S, Stochholm K, Andersen NH. Turner
hematologic manifestations in patients with VEXAS syndrome due syndrome: mechanisms and management. Nat Rev Endocrinol.
to somatic mutations in UBA1. Blood Adv. 2021;5(16):3203-3215. 2019;15(10):601-614.
haematologica | 2022; 107(4) 1013

Case Reports
recurrence to date (2 years).

A case series of primary cutaneous B-cell lymphomas The second case was an 83-year-old Caucasian male
with atypical presentations: diagnostic and therapeutic presenting with an erythematous, asymptomatic growth
challenges on the forehead that had increased in size to 6 x 7 cm
over 6 months (Figure 2A). The patient had no systemic
Primary cutaneous B-cell lymphomas (PCBCL) are symptoms of fever, night sweats, weight loss, or lym-
defined as B-cell lymphomas of the skin without nodal, phadenopathy. Punch biopsy of the lesion demonstrated
bone marrow, or visceral involvement at the time of diag- a dense lymphocytic neoplasm composed of centroblasts
nosis.1 They represent approximately 25% of primary and immunoblasts positive for CD10, CD20, and BCL6
cutaneous lymphomas.1,2 The histopathological diagnosis but negative for MUM1. Small-sized reactive lympho-
of PCBCL can be challenging in certain instances in cytes were BCL2-positive (Figure 2B, D-H).
which overlapping features are present. Nevertheless, Immunoglobulin heavy chain gene-rearrangement stud-
identification of the correct subtype of PCBCL is impera- ies revealed a monoclonal population. Blood flow cytom-
tive for determining the prognosis and avoiding inappro- etry analysis and PET scan were negative for blood or
priate aggressive treatments which could lead to unnec- systemic involvement. This case was also initially diag-
essary morbidity.3 Here we present a series of three cases nosed as DLBCL, but a secondary histopathological
that highlight the distinguishing features between two review combined with clinical correlation led to a diag-
subtypes of PCBCL. nosis of PCFCL with diffuse pattern. Radiation therapy
The first case was a 57-year-old African American man provided complete regression of the tumor without
with multiple pruritic nodules on his abdomen that recurrence to date (1.5 years).
appeared and rapidly progressed in size 6 months prior to The third case was a 72-year-old man with a history of
presentation (Figure 1A). Review of systems was nega- chronic kidney disease and heart failure who presented
tive for pain, weight loss, night sweats or fever. Blood with a tender pink tumor on his scalp that abruptly
flow cytometry analysis was normal, and positron emis- appeared as a small papule but rapidly grew in size to 4
sion tomography (PET) scan was negative for metaboli- x 4 cm over 1 month (Figure 3A). His clinical history was
cally active lymph nodes or systemic disease. Skin biopsy negative for fever, lymphadenopathy, fatigue, night
demonstrated sheets of large atypical lymphoid aggre- sweats, and systemic symptoms. Punch biopsy revealed
gates with centroblast morphology extending into the a sheet-like diffuse dense infiltrate composed of cells
entire thickness of the dermis with an accompanying sig- with immunoblastic morphology and high mitotic activi-
nificant reactive small-sized lymphocytic infiltrate (Figure ty (Figure 3B). The atypical lymphocytes stained positive
1B). The large cells expressed CD20 and BCL6 but were for CD10, CD20, BCL2, BCL6, and MUM1 (Figure 3D-H)
negative for CD10, BCL2, and MUM-1 on immunostain- with a more than 80% proliferative population based on
ing (Figure 1D-H). Ki67 stain showed a proliferation rate Ki-67 positivity. Fluorescent in-situ hybridization (FISH)
of 30-40%. B-cell receptor clonality assay (ClonoSeq) was positive for a BCL6 gene rearrangement (18% of
identified two dominant immunoglobulin heavy chain nuclei) and negative for rearrangement of MYC, CCND1,
sequences. An initial diagnosis of diffuse large B-cell lym- and BCL2. A dominant immunoglobulin heavy chain
phoma (DLBCL) was considered, but a complete sequence present in 99% of all nucleated cells was iden-
histopathological review with clinical correlation led to a tified by immunosequencing (ClonoSeq). PET scan
final diagnosis of primary cutaneous follicle center lym- revealed a hypermetabolic scalp lesion with no evidence
phoma (PCFCL) with diffuse pattern. Three large lesions of lymph node or systemic involvement, and blood flow
were excised and local radiation therapy provided total cytometry analysis was normal. The patient was diag-
regression of the rest of the abdominal tumors without nosed with primary cutaneous diffuse large B-cell lym-
A B C
D E F G H
Figure 1. Primary cutaneous follicle center lymphoma identified by various features. (A) Three firm, ill-defined, pink tumors, 2 to 3 cm in size, with surrounding
erythematous plaques and significant induration on the mid abdomen. (B, C) Dense lymphocytic infiltrate of small to medium sized lymphocytes with condensed
nuclei (hematoxylin & eosin: [B] 40x, [C] 100x) that are (D) CD10-negative, (E) CD20-positive, (F) BCL2-negative, (G) BCL6-positive, and (H) MUM1-negative.
1014 haematologica | 2022; 107(4)

Case Reports
A B C
D E F G H
Figure 2. A different presentation of primary cutaneous follicle center lymphoma. (A) A single firm erythematous tumor on the right of the forehead. (B, C)
Dense infiltrate of medium-sized centroblasts and immunoblasts in the dermis (hematoxylin & eosin: [B] 40x, [C] 100x) that are (D) CD10-positive, (E) CD20-
positive, (F) BCL2-negative on large cells and BCL2-positive on reactive cells, (G) BCL6-positive, and (H) MUM1-negative.
phoma, leg type (PCDLBCL, LT). Combination sis of any subtype of PCBCL. BCL-2 is not expressed by
chemotherapy with rituximab plus cyclophosphamide, malignant cells in PCFCL, but it may be present in reac-
doxorubicin, vincristine, and prednisone (R-CHOP) was tive T cells.3 In the second case of the series presented
not initiated due to the patient’s poor ejection fraction here, BCL-2 was originally called positive but upon fur-
(48%). Instead, given his comorbidities and life expectan- ther evaluation it was clear that only reactive cells
cy, he received radiation therapy with complete resolu- expressed BCL-2. FISH studies can be utilized in cases of
tion of the lesion, confirmed with PET scan. Due to his PCDLBCL, LT, and we found a positive BCL6 gene
aggressive diagnosis, the patient was closely monitored rearrangement in our case.
by oncology without recurrence for 2 years until he died Overall, these cases demonstrate the architectural, his-
from a cardiac arrest due to his comorbidities. tomorphological, and immunohistochemical features
The three cases presented here highlight the challenge that can distinguish PCFCL from PCDLBCL, LT and high-
of distinguishing between PCFCL and PCDLBCL-LT, two light the diagnostic challenges that arise as a result of
of the three main subtypes of PCBCL. The first two cases overlapping characteristics. The clinical impact of this
of PCFCL were originally diagnosed as DLBCL without overlap is most acutely felt in PCDLBCL, LT, because of
specification. The correct diagnosis of diffuse PCFCL was its more aggressive course and the fact that radiation
made after a secondary histological consultation along therapy alone is generally considered inadequate.7,8
with clinical correlation.4,5 The third case illustrates the While there is currently no evidence-based standard of
fact that a PCDLBCL, LT can present on the scalp. care, most cases of PCDLBCL, LT are treated as systemic
For the first two cases, the absence of a follicular pat- DLBCL, with R-CHOP chemoimmunotherapy, often
tern and diffuse sheets of atypical large cells gave an ini- with central nervous system prophylaxis, due to the high
tial impression of DLBCL while the lack of expression of risk of central nervous system dissemination.9 The addi-
MUM1 and the presence of a reactive infiltrate clearly tion of radiation therapy to chemoimmunotherapy was
pointed to the diagnosis of PCFCL.5,6 Although all cases found to be important in a recent case series.10 Despite
showed a diffuse infiltrate on histology, their histomor- historical data showing that the outcomes of patients
phology, immunophenotype, pattern of molecular aber- with PCDLBCL,LT have improved since the introduction
ration on FISH analysis, and clinical presentation distin- of modern chemoimmunotherapy, outcomes remain rel-
guished the correct diagnosis. This underscores the atively poor.11 In addition, many patients are unfit for
importance of recognizing PCFCL with diffuse pattern to chemotherapy, due to age or comorbidities. In the cohort
avoid overcalling DLBCL and the resulting unnecessary reported by Grange et al. in 2014 about 50% of the
aggressive treatment. patients were older than 80 years. At the moment, front-
Histomorphology should be investigated in detail to line radiation therapy, especially for localized, unifocal
arrive at the correct diagnosis among cases of PCBCL.3 disease, is an acceptable option for elderly and frail
Although diffuse sheets of cells were seen in all cases, a patients, and some patients, including the third case pre-
close evaluation of cellular morphology clearly distin- sented here, have durable responses and long-progres-
guishes DLBCL from other subtypes. Large cells with sion free survival.12,13
multiple mitotic figures and nuclear atypia in PCDLBCL, The clinical and histological findings of B-cell lym-
LT contrast directly with the smaller cells and condensed phomas can vary widely. The clinical picture in the first
nuclei seen in PCFCL cases (Figures 1C, 2C, and 3C).6 case reminds readers that it is possible to have multiple
Immunohistochemistry is an essential tool in diagnos- lesions in PCFCL, including multifocal lesions, although it
ing subtypes of PCBCL. MUM-1 positivity precludes is often thought to present as a solitary lesion. The sec-
PCFCL and must be investigated before making a diagno- ond case emphasizes that histological and immunohisto-
haematologica | 2022; 107(4) 1015

Case Reports
A B C
D E F G H
Figure 3. Primary cutaneous diffuse large B-cell lymphoma, leg type is defined by features seen in this figure. (A) A well-defined tumor on the left parietal scalp
with (B, C) diffuse proliferation of large polygonal lymphocytes with high mitotic activity, large nuclei, and little cytoplasm (hematoxylin & eosin: [B] 40x, [C] 100x)
that are (D) CD10-positive, (E) CD20-positive, (F) BCL2-positive, (G) BCL6-positive and (H) MUM1-positive.
chemical results must be assessed together, without rely-

ing on one over the other. Case three cautions physicians References
that PCDLBCL, LT can occur elsewhere on the body
while having overlapping histological features with 1. Suárez AL, Pulitzer M, Horwitz S, Moskowitz A, Querfeld C,
PCFCL. Due to the complexity of cutaneous lymphomas, Myskowski PL. Primary cutaneous B-cell lymphomas: part I. Clinical
features, diagnosis, and classification. J Am Acad Dermatol.
it is imperative for physicians to work together in a mul- 2013;69(3):329.
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matopathology and radiation oncology in order to pro- insights: primary cutaneous lymphomas, version 2.2020. J Natl
vide the best care for these patients. Compr Canc Netw. 2020;18(5):522-536.
3. Willemze R, Cerroni L, Kempf W, et al. The 2018 update of the
WHO-EORTC classification for primary cutaneous lymphomas.
Emily Correia,1 Jisun Cha,1 Shalini Krishnasamy,1,2 Megan Blood. 2019;133(16):1703-1714.
O’Donnell,1 Wenyin Shi,3 Pierluigi Porcu2 and Neda 4. Malachowski SJ, Sun J, Chen PL, Seminario-Vidal L. Diagnosis and
management of cutaneous B-cell lymphomas. Dermatol Clin.
Nikbakht1 2019;37(4):443-454.
1 5. Kodama K, Massone C, Chott A, Metze D, Kerl H, Cerroni L.
Department of Dermatology and Cutaneous Biology, Thomas
Primary cutaneous large B-cell lymphomas: clinicopathologic fea-
Jefferson University, Philadelphia, PA; 2Division of Hematologic tures, classification, and prognostic factors in a large series of
Malignancies and HSCT, Department of Medical Oncology, Sidney patients. Blood. 2005;106(7):2491-2497.
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 6. Felcht M, Klemke CD, Nicolay JP, et al. Primary cutaneous diffuse
and 3Department of Radiation Oncology, Thomas Jefferson University, large B-cell lymphoma, NOS and leg type: Clinical, morphologic and
prognostic differences. J Dtsch Dermatol Ges. 2019;17(3):275-285.
Philadelphia PA, USA. 7. Senff NJ, Hoefnagel JJ, Neelis KJ, et al. Results of radiotherapy in 153
Correspondence: primary cutaneous B-cell lymphomas classified according to the
WHO-EORTC classification. Arch Dermatol. 2007;143(12):1520-
NEDA NIKBAKHT - neda.nikbakht@jefferson.edu 1526.
8. Haverkos B, Tyler K, Gru AA, et al. Primary cutaneous B-cell lym-
PIERLUIGI PORCU - pierluigi.porcu@jefferson.edu phoma: management and patterns of recurrence at the
doi:10.3324/haematol.2021.279992 Multimodality Cutaneous Lymphoma Clinic of The Ohio State
University. Oncologist. 2015;20(10):1161-1166.
Received: September 13, 2021. 9. Sundriyal D, Arya L, Srivastava R, Walia M, Sehrawat A.
Accepted: December 2, 2021 Leptomeningeal relapse in primary ccutaneous DLBCL: implications
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Pre-published: December 16, 2021. 2021;4(1):e1295.
10. Kraft RM, Ansell SM, Villasboas JC, et al. Outcomes in primary cuta-
Disclosures: no conflicts of interest to disclose neous diffuse large B-cell lymphoma, leg type. J Clin Oncol.
Contributions: EC performed the literature review, wrote the manu- 2021;39(15_suppl):e19547.
11. Grange F, Joly P, Barbe C, et al. Improvement of survival in patients
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the manuscript, and led discussions of the manuscript; SK and MOD France. JAMA Dermatol. 2014;150(5):535-541.
wrote parts of the manuscript; WS wrote part of the manuscript and 12. Graham PM, Richardson AS, Schapiro BL, Saunders MD, Stewart
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script, edited it, helped with the histology, and led discussions of the JAAD Case Rep. 2018;4(4):305-309.
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Nagel Memorial Research Grant. Haematol. 2020;191(3):e83-e86.
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Associated with USPI, Unione Stampa Periodica Italiana.

Landmark Paper in Hematology

787 Not all mismatches are equal: importance of alloreactivity direction

Acute Myeloid Leukemia

Haematologica 2022; vol. 107 no. 4 - April 2022

Bone Marrow Transplantation

Cell Therapy and Immunotherapy

Chronic Lymphocytic Leukemia

Plasma Cell Disorders

Platelet Biology & its Disorders

Red Cell Biology & its Disorders

Haematologica 2022; vol. 107 no. 4 - April 2022

Letters to the Editor

Haematologica 2022; vol. 107 no. 4 - April 2022

The origin of a name that reflects Europe’s cultural roots.

Scientific Latin haematologicus (adjective) = related to blood

Scientific Latin haematologica (adjective, plural and neuter,

Modern English The oldest hematology journal,

haematologica | 2022; 107(4) 781

JOURNAL New England Journal of Medicine. 1948;238(23):787-793.

782 haematologica | 2022; 107(4)

haematologica | 2022; 107(4) 783

emptive intervention, as opposed to salvage at the time of Disclosures

784 haematologica | 2022; 107(4)

Thrombotic thrombocytopenic purpura and other immune-mediated blood disorders follow-

Table 1. Main features of vaccine-induced, immune mediated thrombocytopenias.

haematologica | 2022; 107(4) 785

786 haematologica | 2022; 107(4)

Not all mismatches are equal: importance of alloreactivity direction

E-mail: NADA HAMAD - nada.hamad@svha.org.au

Figure 1. Competing risks

haematologica | 2022; 107(4) 787

Figure 2. Competing risks regres-

Figure 3. Competing risks regres-

788 haematologica | 2022; 107(4)

Across the trial period, the use of anti-thymocyte globulin Disclosures

haematologica | 2022; 107(4) 789

Ferrata Storti Foundation The mitochondrial anti-apoptotic dependencies

790 haematologica | 2022; 107(4)

haematologica | 2022; 107(4) 791

792 haematologica | 2022; 107(4)

Figure 3. The anti-apoptotic map of hematologic

haematologica | 2022; 107(4) 793

794 haematologica | 2022; 107(4)

gen stimulation and CXCL12, both converging on BTK, Multiple myeloma

haematologica | 2022; 107(4) 795

T-cell prolymphocytic leukemia anti-apoptotic proteins in sustaining Hodgkin lymphoma

796 haematologica | 2022; 107(4)

haematologica | 2022; 107(4) 797

798 haematologica | 2022; 107(4)

haematologica | 2022; 107(4) 799

800 haematologica | 2022; 107(4)

lymphocytic leukemia. Leukemia. 2017;31 ABT-199. Cancer Discov. 2014;4(9):1074- 2020;4(17):4217-4231.

haematologica | 2022; 107(4) 801