Bone Marrow Transplantation (2015) 50, S37–S39

© 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15

www.nature.com/bmt

ORIGINAL ARTICLE
Unmanipulated haploidentical bone marrow transplantation
and post-transplant cyclophosphamide for hematologic
malignanices following a myeloablative conditioning:
an update
A Bacigalupo, A Dominietto, A Ghiso, C Di Grazia, T Lamparelli, F Gualandi, S Bregante, MT Van Lint, S Geroldi, S Luchetti, R Grasso,
S Pozzi, N Colombo, E Tedone, R Varaldo and AM Raiola

This is a report of 148 patients with hematologic malignancies who received an unmanipulated haploidentical bone marrow
transplant (BMT), followed by post-transplant high-dose cyclophosphamide (PT-CY). All patients received a myeloablative
conditioning consisting of thiotepa, busulfan, fludarabine (n = 92) or TBI, fludarabine (n = 56). The median age was 47 years (17–74);
47 patients were in first remission (CR1), 37 in second remission (CR2) and 64 had an active disease; all patients were first grafts. The
diagnosis was acute leukemia (n = 75), myelodisplastic syndrome (n = 24), myelofibrosis (n = 16), high-grade lytmphoma (n = 15) and
others (n = 18). GVHD prophylaxis consisted in PT-CY on days +3 and +5, cyclosporine (from day 0), and mycophenolate (from
day +1). The median day for neutrophil engraftment was day +18 (13–32). The cumulative incidence of grades II–IV acute GVHD
was 24%, and of grades III–IV GVHD 10%. The incidence of moderate–severe chronic GVHD was 12%. With a median follow-up for
the surviving patients of 313 days (100–1162), the cumulative incidence of transplant-related mortality (TRM) is 13%, and the
relapse-related death is 23%. The actuarial 22 months overall survival is 77% for CR1 patients, 49% for CR2 patients and 38% for
patients grafted in relapse (P o0.001). Major causes of death were relapse (22%), GVHD (2%) and infections (6%). We confirm our
initial results, suggesting that a myeloablative conditioning regimen followed by unmanipulated haploidentical BMT with PT-CY,
results in a low risk of acute and chronic GVHD and encouraging rates of TRM and overall survival, also for patients with active
disease at the time of transplant.

Bone Marrow Transplantation (2015) 50, S37–S39; doi:10.1038/bmt.2015.93

PATIENTS AND METHODS
Clinical characteristics are outlined in Table 1. All patients received a
myeloablative regimen with post-transplant cyclophosphamide, between
august 2010 and January 2014. A total of 64 patients had an active disease
at transplant; 46 were in first remission (CR1) and 39 in second remission
(CR2) hematologic remission. The most common diagnosis was acute
leukemia (n = 76 ), 48 AML (27% with active disease) and 24 ALL (32% with
active disease). Most of the patients with non-Hodgkin lymphoma (60%)
also had advanced disease, as well as patients with myelofibrosis.
Donors
All donor/patient pairs were genotypically haplomismatched. A single
patient, because of HLA homozigosity, had 0 mismatches in the graft vs
host direction and three mismatched in the host vs graft direction.
Conditioning regimen
The myeloablative conditioning regimen was based either on chemother-
apy—thiotepa, busulfan, fludarabine—or TBI and fludarabine: 92 patients
received the thiotepa, busulfan, fludarabine regimen which included
thiotepa 5 mg/kg on days − 6 and − 5 (total dose 10 mg/kg), fludarabine
50 mg/m2 on days − 4, − 3 and − 2 (total dose 150 mg/m2), and busulfan
3,2 mg/kg IV on days − 4, − 3, − 2 (total dose 9,6 mg/kg). Fifty-six patients
received the TBI and fludarabine regimen, which included TBI 3.3 Gy on
days − 8, − 7 and − 6 (total dose 9,9 Gy) (43 patients) or TBI 2Gyx2/day on
each of 3 consecutive days (total dose 12 Gy) (13 patients) plus fludarabine
30 mg/m2 on days − 5, − 4, − 3 and − 2 (total dose 120 mg/m2). For elderly
patients (460 years), or patients in poor clinical condition, the days of
busulfan administration were reduced from 3 to 2 days (total dose 6.4 mg/
kg) (n = 38) or to 1 day (n = 1, a patient over 70).
GvHD prophylaxis
GVHD prophylaxis consisted of cyclophosphamide 50 mg/kg on day +3
and +5, cyclosporine A (CsA) 1 mg/kg given as a continuous IV infusion,
from days 0 to +20, adjusting for blood levels (200–400 ng/ml), and then
orally until the day +180; mycophenolate (15 mg/kg every 12 hours ) from
day +1, to day +28. Pegylated G-CSF 1 vial subcutaneous (s.c) (6 mg) was
given on day +5 to all patients. Mesna (80% of the cyclophosphamide
dose) was administrated in three divided dose from day +3 daily to day +7.
All dosing was based on ideal body weight. Phenytoin, 300 mg per os (p.o.)
was administered on all days of busulfan therapy. Ten advanced patients
received one course of chemotherapy two weeks before the start of the
conditioning regimen.
Stem cell source
Unmanipulated bone marrow was used as a stem cell source for all
patients, and infused intravenously on day 0. Donors underwent bone

Divisione Ematologia e Trapianto di Midollo, IRCCS San Martino, Genova, Italy. Correspondence: Dr A Bacigalupo, Divisione Ematologia e Trapianto di Midollo Osseo IRCCS
San Martino Largo Rosanna Benzi 10, Genova 16132, Italy.
E-mail: andrea.bacigalupo@hsanmartino.it
This article was published as part of a supplement, supported by WIS-CSP Foundation, in collaboration with Gilead, Milteny Biotec, Gamida cell, Adienne Pharma and Biotech,
Medac hematology, Kiadis Pharma and Almog Diagnostic.
 RH: haploidentical BMT and post-transplant cyclophosphamide
A Bacigalupo et al
S38
100%
Table 1. Clinical characteristics of patients

n, Patients 148 80% CR1, n=46

77%
Age; years (range) 47 (17–74)
60%

Survival
Diagnosis CR2, n=39
49%
AML 48
ALL 28 40%
38%
MDS 24
Active disease, n=63
Myelofibrosis 16
Lymphoma 15 20%
Other 18

Disease phase 0%
0 200 400 600 800 1000 1200 1400
CR1 46
Days from transplant
CR2 39
Active disease 63 Figure 1. Actuarial survival of 148 patients receiving a myeloablative
conditioning regimen, followed by haploidentical bone marrow
Stem cell source transplantation and post-transplant cyclophosphamide, cyclospor-
BM 148 ine and mycophenolate for GVHD prophylaxis. Patients are stratified
for disease phase.
Conditioning regimens
FLU-TBI
9.9 Gy n. 43 (P = 0.4). Excluding early deaths (n = 7) and early relapse (n = 3)
12 Gy n. 13 there was only one primary graft failure in a patients with
TBF (BU3) n. 53
TBF (BU2) n. 39
myelofibrosis with a very-large spleen, out of 138 patients (0.7%).
Cell dose x 108/kg (range) 3.37 (1.4–7.8)
Follow up days (range) 301 (3–1332) Graft versus host disease
Abbreviations: BM = bone marrow; BU2 = 2 days of busulfan; BU3 = 3 days Of 148 patients on this study, 147 were alive on day +10 and
of busulfan; FLU = Fludarabine; MDS = myelòodisplastic syndrome; evaluable for acute GVHD. The CI of grades 0–I, II, III–IV was as
TBF = thiotepa, busulfan, fludarabine. follows 82, 14 and 4%. There was a trend for a higher rate of
grades II–IV acute GVHD in patients over 60 as compared with
younger patients (28 vs 14%, P = 0.053).
124 patients were evaluable for chronic GVHD. The CI of chronic
marrow harvest under general anesthesia and the ideal target of
mononuclear cells was 4 × 108 /kg of recipient body weight. GVHD was 20%: again a trend for a higher rate was seen in
patients over 60 years of age (28 vs 18%) (P = 0.1).
Supportive care
Antimicrobial prophylaxis was started during conditioning regimen and
Transplant-related mortality
consisted of acyclovir 500 mg/m2 three times a day, levofloxacine 500 mg a The 4-year CI of transplant-related mortality was 14%. It was not
day and fluconazole 400 mg per days until day +75. Bi-weekly CMV predicted by patients age: 8, 18, 17% for patients aged o 40
monitoring, by PCR or antigenemia, was started on day − 7, until day +100, (n = 51), 41–60 (n = 62) and over 60 (n = 35) (P = 0.2). It was also not
and weekly until day +180. Weekly EBV monitoring by PCR was started on predicted by disease phase: 9, 15, 17% for patients in CR1 (n = 46),
day +15, as described. CR2 (n = 39) and advanced disease (n = 63) (P = 0.4).

Diagnosis and treatment of GVHD Relapse

The clinical diagnosis of acute GVHD was made according to standard
criteria, and confirmed histologically by skin and/or rectal biopsies. First
line therapy of GVHD was methylprednisolone (MP)1–2 mg/kg day;
second-line therapy was extra-corporeal photopheresis, mycophenolate,
corticosteroids or monoclonal antibodies, as per institutional protocols.
The CI of relapse-related death was 27% and could be predicted
by disease phase: 11% for CR1 patients, 26% for CR2 patients and
40% for patients with active disease at the time of transplant
(P = 0.003).

Statistical analysis Survival

The NCSS 2004 for Windows (Kaysville, UT, USA), was used for Χ2 tables,
rank sum two samples non-matched pair test, cumulative incidence (CI)
rates, and actuarial survival. In calculating the CI of transplant-related
mortality, the competing risk was relapse; when calculating relapse, the
competing risk was transplant-related mortality.
RESULTS
Engraftment
The median number of nucleated bone marrow cells infused was
3.3 × 108/kg (range: 1.4–7.8). The median time to neutrophil
counts of 40.5 × 109 /l was 17 days (range, 13–32 days). There was
no correlation between infused number of nucleated cells and
days of neutrophil engraftment: the median day of engraftment
was 17 days, both for patients receiving less or ⩾ 3.3 × 108 cells/kg
With a median follow-up of over 430 days, the actuarial overall
survival is 77% for CR1 patients, 49 and 38% for CR2 and advanced
patients (P = 0.0001) (Figure 1).
DISCUSSION
Allogeneic transplants from haploidentical family donors, with1–3
or without4–7 T-cell depletion, are being increasingly used in
patients with hematologic malignancies.8
We are now updating our initial experience of unmanipulated
marrow transplants in 50 patients.9 We have continued to use the
same transplant program consisting of haploidentical family
donors, myeloablative regimens, unmanipulated bone marrow
as a stem cell source, and post-transplant cyclophosphamide with
cyclosporine and mycophenolate for GVHD prophylaxis. There are

Bone Marrow Transplantation (2015) S37 – S39 © 2015 Macmillan Publishers Limited

several differences in this protocol, as compared with the
Baltimore original study:5 firstly, the use of full dose TBI or two
alkylating agents (thiotepa and busulfan), which results in
significant control of the underlying disease, also when active at
the time of transplant. Secondly, the timing of cyclophosphamide
on days +3 and +5, rather than +3 and +4: this was adopted to
allow for a days rest between two cyclophosphamide infusions,
after a fully myeloablative regimen. Thirdly, the timing of
cyclosporine and mycophenolate, starting on day 0 – before,
rather than after cyclophosphamide.
The results in 148 consecutive patients are encouraging, in
terms of engraftment, GVHD, transplant mortality, as well as in
terms of relapse-related mortality. We were particularly impressed
with the lack of correlation between the number of grafted cells
and day of neutrophil engraftment: there was only one true
primary graft failure (0.7%). The rate of GVHD, both acute and
chronic, was low, and 80% of patients were off cyclosporine at
1 year. There was a trend for a higher risk of acute GVHD in patient
over the age of 60. Transplant mortality at 4 years was also low,
and was not predicted by patients age, which is rather unusual for
an allogeneic transplant program.
Finally relapse, one of the remaining major problems of
allogeneic transplants: the relapse rate in CR1 and CR2, were
similar to what one would expect with a conventional transplant.
Also the 40% relapse-related death in advanced patients is not
different from what we have seen with sibling or unrelated grafts.
In conclusion, haploidentical bone marrow transplants are an
alternative to unrelated or cord grafts, especially in patients with
advanced disease, who have little time available to wait in suitable
hematologic or clinical conditions.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
© 2015 Macmillan Publishers Limited
RH: haploidentical BMT and post-transplant cyclophosphamide
A Bacigalupo et al
S39
ACKNOWLEDGEMENTS
The study was partly supported by Associazione Italiana Ricerca contro il Cancro
Milano and Associazione Italiana contro le Leucemie Genova.
REFERENCES
1 Aversa F, Tabilio A, Velardi A, Cunningham I, Terenzi A, Falzetti F et al. Treatment of
high-risk acute leukemia with T-cell-depleted stem cells from related donors with
one fully mismatched HLA haplotype. N Engl J Med 1998; 339: 1186–1193.
2 Aversa F, Terenzi A, Tabilio A, Falzetti F, Carotti A, Ballanti S et al. Full haplotype-
mismatched hematopoietic stem-cell transplantation: a phase II study in patients
with acute leukemia at high risk of relapse. J Clin Oncol 2005; 23: 3447–3454.
3 Handgretinger R, Lang P. The history and future prospective of haplo-identical
stem cell transplantation. Cytotherapy 2008; 10: 443–451.
4 Lu DP, Dong L, Wu T, Huang XJ, Zhang MJ, Han W et al. Conditioning including
antithymocyte globulin followed by unmanipulated HLA-mismatched/haploiden-
tical blood and marrow transplantation can achieve comparable outcomes with
HLA-identical sibling transplantation. Blood 2006; 107: 3065–3073.
5 Luznik L, O'Donnell PV, Symons HJ, Chen AR, Leffell MS, Zahurak M et al.
HLA-haploidentical bone marrow ransplantation for hematologic malignancies
using nonmyeloablative conditioning and high-dose, posttransplantation cyclo-
phosphamide. Biol Blood Marrow Transplant 2008; 14: 641–650.
6 Di Bartolomeo P, Santarone S, De Angelis G, Picardi A, Bavaro P, Di Bartolomeo E,
Olioso P et al. Unmanipulated bone marrow transplantation from haploidentical
related donors for patients with high risk hematologic malignancies. Blood 2013;
121: 849–857.
7 Raj K, Pagliuca A, Bradstock K, Noriega V, Potter V, Streetly M et al. Peripheral blood
hematopoietic stem cells for transplantation of hematological diseases from rela-
ted, haploidentical donors after reduced-intensity conditioning. Biol Blood Marrow
Transplant 2014; 20: 890–895.
8 Passweg JR, Baldomero H, Bregni M, Cesaro S, Dreger P, Duarte RF et al.
Hematopoietic SCT in Europe: data and trends in 2011. Bone Marrow Transplant
2013; 48: 1161–1167.
9 Raiola AM, Dominietto A, Ghiso A, Di Grazia C, Lamparelli T, Gualandi F et al.
Unmanipulated Haploidentical Bone Marrow. Transplant and Posttransplantation
Cyclophosphamide for Hematologic Malignancies after Myeloablative Condition-
ing. Biol Blood Marrow Transplant 2013; 19: 117–122.
Bone Marrow Transplantation (2015) S37 – S39