Correspondence

Anita Kumar1
Author contributions
Kristie A. Blum2
AK analysed the data and drafted the manuscript. KAB, HF, Henry C. Fung3
MRS, and AY performed the research. AY designed the Mitchell R. Smith4
research study and was the main editor of the manuscript. Paul A. Foster5
PAF contributed to the acquisition, analysis, and interpreta- Anas Younes6
1
tion of the data. All authors read, critically revised, and Memorial Sloan-Kettering Cancer Center, New York, NY, 2Ohio State
approved the final manuscript. University, Columbus, OH, 3Rush University Medical Center, Chicago,
IL, 4Fox Chase Cancer Center, Philadelphia, PA, 5Xencor, Inc.,
Monrovia, CA, and 6MD Anderson Cancer Center, Houston, TX,
Sources of funding
USA.
The clinical trial was funded by Xencor, Inc., Monrovia, CA, E-mail: younesa@mskcc.org
USA.
Keywords: Hodgkin lymphoma, monoclonal antibodies, clinical
trials, therapy, immunotherapy
Conflicts of Interest
PAF is an employee of Xencor, Inc. AK, KAB, HF, MRS, and First published online 1 October 2014
AY have no conflicts of interest to disclose. doi: 10.1111/bjh.13152

References
Ansell, S.M., Horwitz, S.M., Engert, A., Khan,
K.D., Lin, T., Strair, R., Keler, T., Graziano, R.,
Blanset, D., Yellin, M., Fischkoff, S., Assad, A. &
Borchmann, P. (2007) Phase I/II study of an
anti-CD30 monoclonal antibody (MDX-060) in
Hodgkin’s lymphoma and anaplastic large-cell
lymphoma. Journal of Clinical Oncology, 25,
2764–2769.
Bartlett, N.L., Younes, A., Carabasi, M.H., Forero,
A., Rosenblatt, J.D., Leonard, J.P., Bernstein,
S.H., Bociek, R.G., Lorenz, J.M., Hart, B.W. &
Barton, J. (2008) A phase 1 multidose study of
SGN-30 immunotherapy in patients with refrac-
tory or recurrent CD30 + hematologic malig-
nancies. Blood, 111, 1848–1854.
Cartron, G., Dacheux, L., Salles, G., Solal-Celigny,
P., Bardos, P., Colombat, P. & Watier, H.
(2002) Therapeutic activity of humanized anti-
CD20 monoclonal antibody and polymorphism
in IgG Fc receptor FcgammaRIIIa gene. Blood,
99, 754–758.
Cheson, B.D., Pfistner, B., Juweid, M.E., Gascoyne,
R.D., Specht, L., Horning, S.J., Coiffier, B.,
Fisher, R.I., Hagenbeek, A., Zucca, E., Rosen,
S.T., Stroobants, S., Lister, T.A., Hoppe, R.T.,
Dreyling, M., Tobinai, K., Vose, J.M., Connors,
J.M., Federico, M. & Diehl, V. (2007) Revised
response criteria for malignant lymphoma. Jour-
nal of Clinical Oncology, 25, 579–586.
Forero-Torres, A., Leonard, J.P., Younes, A.,
Rosenblatt, J.D., Brice, P., Bartlett, N.L., Bosly,
A., Pinter-Brown, L., Kennedy, D., Sievers, E.L.
& Gopal, A.K. (2009) A Phase II study of
SGN-30 (anti-CD30 mAb) in Hodgkin lym-
phoma or systemic anaplastic large cell lym-
phoma. British journal of haematology, 146,
171–179.
Hammond, PW, Vafa O, Jacinto, J, Vielmetter J,
Karki, S, Yoder S, Lazar, G, Raturi D, Mollet,
M, Zhu M, Kunkel, L & Carmichael D (2005) A
humanized anti-CD30 monoclonal antibody,
XmAb2513, with enhanced In vitro potency
against CD30-positive lymphomas mediated by
high affinity Fc-receptor binding. Blood (ASH
Annual Meeting Abstracts), 106, 1470.
Weng, W.K. & Levy, R. (2003) Two immunoglob-
ulin G fragment C receptor polymorphisms
independently predict response to rituximab in
patients with follicular lymphoma. Journal of
Clinical Oncology, 21, 3940–3947.
Younes, A., Bartlett, N.L., Leonard, J.P., Kennedy,
D.A., Lynch, C.M., Sievers, E.L. & Forero-Tor-
res, A. (2010) Brentuximab vedotin (SGN-35)
for relapsed CD30-positive lymphomas. The
New England Journal of Medicine, 363, 1812–
1821.

GTF2I-RARA is a novel fusion transcript in a t(7;17) variant of

acute promyelocytic leukaemia with clinical resistance to
retinoic acid

Acute promyelocytic leukaemia (APL) is characterized by the
PML-RARA fusion gene, arising from t(15;17)(q21;q22)
translocation. PML-RARA is a specific molecular marker for
APL and an important determinant for the effective induc-
tion of differentiation by all-trans retinoic acid (ATRA)
(Grignani et al, 1994). We report a novel fusion gene,
GTF2I-RARA, in a variant APL with cryptic t(7;17)(q11;q21),
which manifested insensitivity to ATRA and conventional
chemotherapy.
The patient, a previously healthy 35-year-old male, pre-
sented with a 3-month history of fatigue and skin ecchymosis
for 10 d. Laboratory investigations showed: haemoglobin

904 ª 2014 John Wiley & Sons Ltd

British Journal of Haematology, 2015, 168, 902–919
 Correspondence

level, 61 g/l; platelet count, 12 9 109/l; leucocyte count,
53
7 9 109/l, including 81% abnormal promyelocytes. Coag-
ulopathy was present. A bone marrow (BM) aspirate showed
90% hypergranular promyelocytes with strong myeloperoxi-
dase positivity (Fig 1A, B). The blast cells were positive for
CD13, CD33, CD64 and negative for CD34, HLA-DR, CD7,
CD14 and CD19 by flow cytometry. Reverse transcription
polymerase chain reaction (RT-PCR) analysis of the BM was
negative for PML-RARA, ZBTB16-RARA and NPM1-RARA
(Table S1). FLT3 internal tandem duplication mutation was
not found. Karyotype analysis showed 46,XY,del(7)(q22)[20]
(Fig 1C). Fluorescence in situ hybridization (FISH) con-
firmed the deletion of 7q (Fig 1D) and detected split RARA
signals without PML involvement (Fig 1E). One split RARA
signal was located on the truncated long arm of chromosome
7 (Fig 1F). The diagnosis of a variant APL was made. Hy-
droxycarbamide and low dose ATRA (25 mg/m2) were
immediately initiated following the support with platelet and
plasma infusions. Daunorubicin (45 mg/m2/d for 3 d) was
added on the second day, and DA regimen (daunorubicin
45 mg/m2/d, days 1–3; cytarabine 100 mg/m2/d, days 1–7)
was added to continuous ATRA on day 23. ATRA improved
coagulopathy without morphological differentiation of leu-
kaemia cells. Subsequently, two cycles of salvage treatment,
intermediate dose cytarabine and IAH regimen (idarubicin
12 mg/m2/d, days 1–3; cytarabine 100 mg/m2/d, days 1–7;
homoharringtonine 2 mg/m2/d, days 1–4), failed. Because of
the refractoriness of the disease, arsenic trioxide (0
16 mg/
kg/d) was tried in combination with ATRA (25 mg/m2).
Unfortunately, the patient died of intracranial haemorrhage
on day 146 without achieving remission.
The combination of APL morphology and immunopheno-
type suggested the diagnosis of APL. However, the classic
PML-RARA fusion transcript and the t(15q+;17p) translo-
cation were absent, indicating this case was atypical. FISH
analysis revealed that one split RARA signal was inserted into
the disrupted 7q region, documenting the presence of submi-
croscopic t(7;17)(q?;q21) translocation (Fig 1F).
Adopting rapid 50 amplification of cDNA ends (50 -RACE)
and RT-PCR, we identified GTF2I, the general transcription
factor IIi, as the novel RARA partner (Fig 2A). GTF2I-RARA
results from the fusion between exon 6 of GTF2I and exon 3 of
RARA, and is predicted to encode a 599 amino acid protein
(Fig 2B). No alternative splicing variant was found. The reci-
procal RARA-GTF2I fusion transcript was not detected
(Fig 2C).
Mapped at 7q11
23, GTF2I is ubiquitously expressed and
encodes a phosphoprotein with broad roles in transcription
and signal transduction involving growth factor signalling, cell
cycle regulation, and transforming growth factor, beta 1

(A) (B) (C)

(D) (E) (F)

Fig 1. Morphological and cytogenetic analysis of blast cells. (A) Bone marrow (BM) smear shows abnormal promyelocytes with relative regular
nucleus, scant cytoplasm and abundant azurophilic granules, whereas Auer rods were absent (original magnification 9400). (B) Myeloperoxidase
staining of the patient’s BM sample showed strong positivity (original magnification 9400). (C) Karyotype analysis revealed 46, XY, del(7q22) in
BM blast cells. (D) Metaphase fluorescence in situ hybridization (FISH) confirmed the deletion of the long arm of one chromosome 7 using
probes specific for the chromosome 7 centromere (GLP D7S486 probes, green) and 7q31 (CSP7 probes, red). (E) FISH using PML-RARA dual
colour, dual-fusion translocation probes found RARA rearrangement. The RARA signals are shown in red, while PML signals are shown in green.
Intact RARA and PML are shown as (r) and (p), while the split RARA signals are indicated as (s). (F) The combined application of chromosome
7 probes and PML-RARA dual colour, dual-fusion translocation probes found that one split RARA was translocated to the truncated long arm of
chromosome 7. Signal detection was carried out on metaphases according to the manufacturer’s protocols (Jinpujia, Beijing, China).

ª 2014 John Wiley & Sons Ltd 905

British Journal of Haematology, 2015, 168, 902–919
Correspondence
(A)
(C)
(E)
(TGFB1) signalling (Roy, 2012). The primary structure of
GTF2I characterizes six direct reiterated I-repeats, R1-R6, each
containing a helix-loop-helix motif that is a protein-protein
module (Cheriyath & Roy, 2001). The conserved N-terminal
leucine zipper (LZ, amino acids 23–44) mediates homo- or
heteromeric interaction (Fig 2B). In the GTF2I-RARA fusion
906
(B)
(D)
(F)
protein, the first 195 amino acids of GTF2I, including the LZ
and the first I-repeat (R1, 104-176), are retained (Fig 2B),
providing the possibility of GTF2I-RARA dimerization or
multimerization. It has been reported that RARA chimaeras
acquire their oncogenic potential by forming homodimers
(Sternsdorf et al, 2006). Indeed, our coimmunoprecipi-
ª 2014 John Wiley & Sons Ltd
British Journal of Haematology, 2015, 168, 902–919
 Correspondence

Fig 2. Molecular analysis and functional assay of the GTF2I-RARA fusion transcript. (A) Sequence analysis of the GTF2I-RARA transcript at the
junction site (bold arrowhead). The DNA and amino acid sequences of the GTF2I and RARA are indicated in bold and normal font, respectively.
(B) Schematic diagram of RARA, GTF2I and GTF2I-RARA fusion protein. A black line indicates the break point. DBD, DNA-binding domain;
LBD+DD, ligand-binding domain and dimerization domain; LZ, leucine zipper; NLS, nuclear localization signal; BR, basic region; R1-R6, I-repeat
domains. (C) Long-distance reverse transcription polymerase chain reaction (RT-PCR) of GTF2I-RARA, GTF2I and RARA transcripts, and nested
PCR for the detection of RARA-GTF2I transcripts. (D) GTF2I-RARA interacts with itself or GTF2I, but not RARA. FLAG-tagged GTF2I-RARA
was co-transfected into 293 cells with either empty vector or HA-tagged expression vector, as shown in the table. Immunoblotting (IB) of the
whole cell lysates (WCL) confirmed successful transfection and expression (left). Cell lysates were immunoprecipitated (IP), and then analysed by
IB (right). Only HA-tagged GTF2I-RARA and GTF2I could be detected after immunoprecipitation with anti-FLAG antibodies. (E) Subcellular
localization and co-localization of GTF2I-RARA. Two hundred and ninety three cells were transiently transfected with GTF2I-RARA-FLAG and
one of GTF2I-RARA-HA, GTF2I-HA, and RARA-HA. FLAG-tagged GTF2I-RARA was stained with Alexa Fluor 594 (red, upper panels), HA-
tagged proteins were stained with Alexa Fluor 488 (green, middle panels), and the nuclei were visualized with 40 , 6-diamidino-2-phenylindole
(DAPI: blue, lower panels). The bottom panels show merged Alexa 594, Alexa 488, and DAPI staining: yellow fluorescence in the composite
images indicates co-localization. (F) GTF2I-RARA responds poorly to ATRA. RXRA-FLAG, 49 RARE pGL3 reporter vector, and pRL-TK were
co-transfected together with one of MOCK, RARA, PML-RARA, ZBTB16-RARA, and GTF2I-RARA into 293 cells. The relative luciferase activity
of each group in response to various concentrations of RA is shown in the histogram. RA-induced luciferase activity of GTF2I-RARA and
ZBTB16-RARA was much lower than MOCK, RARA, and PML-RARA. The error bars represent the mean of an experiment performed in tripli-
cate; *P < 0
05.

tation results confirmed the self-association of GTF2I-RARA
(Fig 2D). Additionally, GTF2I-RARA heterodimerizes with
GTF2I, suggesting that it may contribute to leukaemogenesis
by dysregulating the GTF2I-mediated signalling pathway.
Double-labelling immunofluorescence experiments showed
the co-localization of FLAG-tagged GTF2I-RARA with either
HA-tagged GTF2I-RARA or GTF2I, but not with RARA,
consistent with coimmunoprecipitation data (Fig 2E). In
GTF2I-RARA-expressing cells, two patterns of GTF2I-RARA
localization were observed: diffuse nuclear distribution with a
micropunctate pattern, and aggregation in the cytoplasm as
macrogranules (Fig 2E, upper panel). This is clearly distinct
from the microspeckled pattern reported for PML-RARA.
Apart from PML-RARA, 8 other RARA fusion genes have
been reported in a small number of APL cases. The corre-
sponding RARA partners are ZBTB16, NPM1, NUMA1,
STAT5B, PRKAR1A, FIP1L1, BCOR, and NABP1 (Redner,
2002; Won et al, 2013). All these chimaeras possess identical
RARA sequences and mainly act as dominant negative regu-
lators on RARA/RXR pathways. GTF2I-RARA shares a com-
mon RARA portion and negatively regulates the retinoic acid
response element (RARE) on luciferase assays (Fig 2F). It has
been reported that ZBTB16-RARA and STAT5B-RARA vari-
ants are resistant to ATRA (Redner, 2002; Strehl et al, 2013).
The explanation for ATRA resistance was ascribed to aber-
rant recruitment of co-repressors by the ZBTB16 or STAT5B
portion, which did not dissociate in response to pharmaco-
logical concentrations of ATRA (He et al, 1998; Alexander
et al, 2002). Consistent with the patient’s clinical resistance
to ATRA, GTF2I-RARA responds poorly to retinoic acid,
similar to ZBTB16-RARA on luciferase assays (Fig 1F). As a
transcription regulator, the inhibitory role of GTF2I was
mediated by its ability to recruit co-repressor complexes
(Roy, 2012). It is possible that GTF2I-RARA contributes to
ATRA resistance by recruiting corepressors through the N-
terminal GTF2I portion.
Although rare, t(7;17)(q11;q21) was reported in a chronic
myeloid leukaemia (CML) patient after chemotherapy,
radiotherapy and imatinib treatment (Bumm et al, 2003).
However, the t(7;17)(q11;q21) clonality did not constitute a
haematopoietic advantage and the patient did not manifest
APL phenotypes. Whether the patient had GTF2I-RARA
transcript or another RARA rearrangement was unknown.
We think that t(7;17)(q11;q21) in the CML patient might be
an secondary event and was not related to APL leukaemo-
genesis, while GTF2I-RARA is an initiating event and respon-
sible for the pathogenesis of this variant APL.
In summary, we identified a novel GTF2I-RARA fusion
transcript in a case of variant APL with cryptic t(7;17)(q11;
q21) translocation. It represents a new subtype of APL that is
resistant to retinoic acid differentiation induction therapy.
Further studies are required to elucidate the pathogenesis of
GTF2I-RARA APL.
Supplementary data, including Supplementary Methods
(Data S1) and Table S1, is available at British Journal of
Haematology’s website.
Acknowledgements
This work was supported in part by Scientific Research Pro-
gramme for Public Interests from the Health Ministry of
China (project No. 201202017), a Clinical Research Pro-
gramme from the Health Ministry of China (key project
2012-2014), and Project ‘Famous Clinical Doctors in
Xiang-Ya Medical College of Central South University’
(2012-2014).
Author contribution
G.S.Z diagnosed the case, designed and performed research,
analysed data and wrote the paper; J.L performed research,
collected and analysed data and wrote the paper; Y.Z finished
parts of experiments; L.X performed cytogenetic and flow
cytometric analysis; H.Y.Z provided clinical care and data
collection; S.F.L was partly involved in experimental studies;
L.H.B performed morphological analysis; G.B.Z contributed

ª 2014 John Wiley & Sons Ltd 907

British Journal of Haematology, 2015, 168, 902–919
Correspondence

some study suggestions and reviewed the manuscript. All Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sci-
authors gave their final approval of the manuscript. ences, Beijing, China
E-mail: zgsllzy@163.com

Conflict of interest
Keywords: variant acute promyelocytic leukaemia, t(7; 17) translo-
The authors declare no conflict of interests. cation, GTF2I, RARA fusion gene, retinoic acid resistance

Ji Li1 First published online 4 October 2014

Hai-Ying Zhong1 doi: 10.1111/bjh.13157
Yang Zhang2
Le Xiao1
Li-Hong Bai1
Su-Fang Liu1 Supporting Information
Guang-Biao Zhou3 Additional Supporting Information may be found in the
Guang-Sen Zhang1 online version of this article:
1
Department of Haematology/Institute of Molecular Haematology, the
Data S1. Methods.
Second Xiang-Ya Hospital, Central South University, 2Department of Table S1. Hematological malignancies related fusion genes
Oncology, the Second Xiang-Ya Hospital, Central South University,
tested in our patient.
Changsha, Hunan, and 3Division of Carcinogenesis and Targeted
Therapy for Cancer, State Key Laboratory of Biomembrane and

References
Alexander, B.M., Christian, W., Alexandra, G.,
Kunkel, H., Heinzel, T., Ruthardt, M., Groner,
B. & Grez, M. (2002) The Stat5-RARa fusion
protein represses transcription and differentia-
tion through interaction with a corepressor
complex. Blood, 99, 2647–2652.
Bumm, T., M€ uller, C., Al-Ali, H.K., Krohn, K.,
Shepherd, P., Schmidt, E., Leiblein, S., Franke,
C., Hennig, E., Friedrich, T., Krahl, R., Nieder-
wieser, D. & Deininger, M.W. (2003) Emergence
of clonal cytogenetic abnormalities in Ph- cells
in some CML patients in cytogenetic remission
to imatinib but restoration of polyclonal hema-
topoiesis in the majority. Blood, 101, 1941–
1949.
Cheriyath, V. & Roy, A.L. (2001) Structure-func-
tion analysis of TFII-I. Roles of the N-terminal
end, basic region, and I-repeats. Journal of Bio-
logical Chemistry, 276, 8377–8383.
Grignani, F., Fagioli, M., Alcalay, M., Longo, L.,
Pandolfi, P.P., Donti, E., Biondi, A., Lo Coco,
F., Grignani, F. & Pelicci, P.G. (1994) Acute
promyelocytic leukemia: from genetics to treat-
ment. Blood, 83, 10–25.
He, L.Z., Guidez, F., Tribioli, C., Peruzzi, D., Rut-
hardt, M., Zelent, A. & Pandolfi, P.P. (1998)
Distinct interactions of PML-RARalpha and
PLZF-RARalpha with co-repressors determine
differential responses to RA in APL. Nature
Genetics, 18, 126–135.
Redner, R.L. (2002) Variations on a theme: the
alternate translocations in APL. Leukemia, 16,
1927–1932.
Roy, A.L. (2012) Biochemistry and biology of the
inducible multifunctional transcription factor
TFII-I: 10 Years Later. Gene, 492, 32–41.
Sternsdorf, T., Phan, V.T., Maunakea, M.L.,
Ocampo, C.B., Sohal, J., Silletto, A., Galimi, F.,
Le Beau, M.M., Evans, R.M. & Kogan, S.C.
(2006) Forced retinoic acid receptor alpha ho-
modimers prime mice for APL-like leukemia.
Cancer Cell, 9, 81–94.
Strehl, S., K€
onig, M., Boztug, H., Cooper, B.W.,
Suzukawa, K., Zhang, S.J., Chen, H.Y., Attarba-
schi, A. & Dworzak, M.N. (2013) All-trans reti-
noic acid and arsenic trioxide resistance of acute
promyelocytic leukemia with the variant
STAT5B-RARA fusion gene [letter]. Leukemia,
27, 1606–1610.
Won, D., Shin, S.Y., Park, C.J., Jang, S., Chi,
H.S., Lee, K.H., Lee, J.O. & Seo, E.J. (2013)
OBFC2A/RARA: a novel fusion gene in variant
acute promyelocytic leukemia. Blood, 121,
1432–1435.

Is the continued use of UK plasma sourced cryoprecipitate

justified?

In their recent report in this journal, Idris et al (2014) con-
cluded that, “cryoprecipitate is an effective and safe method
of increasing plasma fibrinogen level in hypofibrinogenae-
mic patients”. They retrospectively reviewed the medical
and laboratory records of 89 patients who received cryopre-
cipitate, supplied by the UK National Health Service Blood
and Transplant service, at Addenbrookes Hospital in Cam-
bridge, UK. The mean increase in fibrinogen concentration
after receiving an average of 2
04 cryoprecipitate pooled
units (1 unit is prepared from five blood donations) was
0
79 g/l, from a baseline of 1
01 g/l to a 24 h post-infusion
level of 1
80 g/l.
In this study, efficacy was only assessed in terms of
increasing fibrinogen concentration. Given that cryoprecipi-
tate is known to contain fibrinogen, it is not surprising that
patients receiving cryoprecipitate experience a rise in plasma

908 ª 2014 John Wiley & Sons Ltd

British Journal of Haematology, 2015, 168, 902–919