Acute lymphoblastic leukemia associated with ...

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1422 ACKNOWLEDGEMENTS This work was undertaken at University College London Hospitals/University College London, which received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding Scheme. The funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

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KJ Thomson1,2, I Kayani2, K Ardeshna2, EC Morris2, R Hough1,2, A Virchis2, AH Goldstone2, DC Linch1,2 and KS Peggs1,2 1 University College London Cancer Institute, London, UK and 2 Department of Haematology, University College London, University College London Hospitals NHS Trust, London, UK E-mail: [email protected]

REFERENCES 1 Viviani S, Zinzani PL, Rambaldi A, Brusamolino E, Levis A, Bonfante V et al. ABVD versus BEACOPP for Hodgkin’s lymphoma when high-dose salvage is planned. N Engl J Med 2011; 365: 203–212. 2 Linch DC, Winfield D, Goldstone AH, Moir D, Hancock B, McMillan A et al. Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: results of a BNLI randomised trial. Lancet 1993; 341: 1051–1054. 3 Schmitz N, Pfistner B, Sextro M, Sieber M, Carella AM, Haenel M et al. Aggressive conventional chemotherapy compared with high-dose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin’s disease: a randomised trial. Lancet 2002; 359: 2065–2071. 4 Sweetenham JW, Carella AM, Taghipour G, Cunningham D, Marcus R, Della VA et al. High-dose therapy and autologous stem-cell transplantation for adult patients with Hodgkin’s disease who do not enter remission after induction chemotherapy: results in 175 patients reported to the European group for blood and marrow transplantation. Lymphoma working party. J Clin Oncol 1999; 17: 3101–3109. 5 Sureda A, Arranz R, Iriondo A, Carreras E, Lahuerta JJ, Garcia-Conde J et al. Autologous stem-cell transplantation for Hodgkin’s disease: results and prognostic

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factors in 494 patients from the grupo Espanol de Linfomas/Transplante Autologo de Medula Osea Spanish cooperative group. J Clin Oncol 2001; 19: 1395–1404. Jabbour E, Hosing C, Ayers G, Nunez R, Anderlini P, Pro B et al. Pretransplant positive positron emission tomography/gallium scans predict poor outcome in patients with recurrent/refractory Hodgkin lymphoma. Cancer 2007; 109: 2481–2489. Moskowitz AJ, Yahalom J, Kewalramani T, Maragulia JC, Vanak JM, Zelenetz AD et al. Pretransplantation functional imaging predicts outcome following autologous stem cell transplantation for relapsed and refractory Hodgkin lymphoma. Blood 2010; 116: 4934–4937. Peggs KS, Hunter A, Chopra R, Parker A, Mahendra P, Milligan D et al. Clinical evidence of a graft-versus-Hodgkin’s-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005; 365: 1934–1941. Sureda A, Robinson S, Canals C, Carella AM, Boogaerts MA, Caballero D et al. Reduced-intensity conditioning compared with conventional allogeneic stem-cell transplantation in relapsed or refractory Hodgkin’s lymphoma: an analysis from the lymphoma working party of the European group for blood and marrow transplantation. J Clin Oncol 2008; 26: 455–462. Peggs KS, Sureda A, Qian W, Caballero D, Hunter A, Urbano-Ispizua A et al. Reduced-intensity conditioning for allogeneic haematopoietic stem cell transplantation in relapsed and refractory Hodgkin lymphoma: impact of alemtuzumab and donor lymphocyte infusions on long-term outcomes. Br J Haematol 2007; 139: 70–80. Peggs KS, Kayani I, Edwards N, Kottaridis P, Goldstone AH, Linch DC et al. Donor lymphocyte infusions modulate relapse risk in mixed chimeras and induce durable salvage in relapsed patients after T-cell-depleted allogeneic transplantation for Hodgkin’s lymphoma. J Clin Oncol 2011; 29: 971–978. Meignan M, Gallamini A, Haioun C. Report on the first international workshop on Interim-PET-Scan in lymphoma. Leuk Lymphoma 2009; 50: 1257–1260. Barrington SF, Qian W, Somer EJ, Franceschetto A, Bagni B, Brun E et al. Concordance between four European centres of PET reporting criteria designed for use in multicentre trials in Hodgkin lymphoma. Eur J Nucl Med Mol Imaging 2010; 37: 1824–1833. Moskowitz CH, Matasar MJ, Zelenetz AD, Nimer SD, Gerecitano J, Hamlin P et al. Normalization of pre-ASCT, FDG-PET imaging with second-line, non-cross resistant, chemotherapy programs improves event-free survival in patients with Hodgkin lymphoma. Blood 2011; 119: 1665–1670.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Acute lymphoblastic leukemia associated with RCSD1–ABL1 novel fusion gene has a distinct gene expression profile from BCR–ABL1 fusion Leukemia (2013) 27, 1422–1424; doi:10.1038/leu.2012.332

De Braekeleer et al.1 published the first case of acute lymphoblastic leukemia (ALL) associated with a t(1;9)(q24;q34) and showed colocalization of bacterial artificial chromosome (BAC) clones containing the RCSD1 and ABL1 genes, suggesting the presence of a yet undescribed fusion gene. Mustjoki et al.2 identified a second patient. Sequencing of the PCR products showed that the first three exons of RCSD1 were fused to ABL1, starting at exon 4. We obtained biological samples from patients diagnosed in Finland, United Kingdom, Kuwait and Ecuador. Five patients (three males and two females) were then included in this study (Table 1). Their age ranged from 11 to 40 years. All had B-cell ALL. Immunophenotyping showed the blasts to be CD10 þ , CD19 þ ,

CD22 þ , CD24 þ and CD79a þ . One patient, Pt 4, was lost to follow-up soon after diagnosis. A complete remission was achieved in three patients (Pts 1, 2 and 3) while one patient, Pt 5, was not compliant to treatment and did not achieve complete remission. Three of the four patients for whom data were available (Pts 1, 3 and 5) were still alive, survival ranging from 12 to 97 months. One patient, Pt 1, received bone marrow transplantation 31 months following diagnosis (after a first relapse) and was alive 66 months later. Another patient, Pt 3, had allogeneic hematopoietic stem cell transplantation 4 months following diagnosis but relapsed 12 months later. A third patient, Pt 2, had three bone marrow transplantations, each time following relapse, but died 84 months following diagnosis. Conventional cytogenetics on bone marrow cells using R- and/ or G- and/or Q-banding revealed a clone containing a t(1;9)(q24;q34) along with a normal clone in four patients while additional material of unknown origin was identified at chromosome Xp22 in the t(1;9)-containing clone in the fifth patient (P1)

Accepted article preview online 20 November 2012; advance online publication, 7 December 2012

Leukemia (2013) 1394 – 1440

& 2013 Macmillan Publishers Limited


1423 Table 1.

Clinical and hematological characteristics of five patients with t(1;9)(q24;q34)

Patient no.

Country

Sex

Age (years)

Hb (g/ dl)

1

France

M

11

11.5a

2

England

F

15

5.8

3 4 5

Finland Kuwait Ecuador

M F M

40 18 18

11.1 8.0 9.1

WBC ( 109/ l) 6.4a 348 24.3 110 470

Platelets ( 109/l)

PB blasts (%)

BM blasts (%)

Complete remission

Event-free survival (months)

Survival (months)

104a

47a

92a

Yes

11

97 þ b

25

NA

NA

Yes

1c

84

291 10 56

34 87 52

80 92 58

Yes LTF —e

16d LTF

66 þ LTF 12 þ

Reference

De Braekeleer et al.1 De Braekeleer et al.5 Mustjoki et al.2 Zamecnikova17

Abbreviations : BM, bone marrow; F, female; Hb hemoglobin; M, male; NA, not available; LTF, lost to follow-up (moved to another country); PB, Peripheral blood; WBC, white blood cells. aAt relapse. bBM transplantation 31 months following diagnosis. cBM transplantations performed at 4, 35, 84 months after initial diagnosis (relapses at 3, 33, 75 months after initial diagnosis). dAllogeneic hematopoietic stem cell transplantation 4 months following diagnosis. Relapse 12 months after allotransplant. Treated with dasatinib, switched to imatinib because of immunological side effects. eNo compliance to treatment.

(Supplementary Table 1). Fluorescent in situ hybridization with BAC clones RP11-232M22 (containing the RCSD1 gene) and RP1183J21 (containing ABL1) was attempted in four patients (P1, P2, P4 and P5) and showed two fusion signals, suggesting the presence of a RCSD1–ABL1 chimeric gene. Primers designed by Mustjoki et al.2 covering the kinase domain of ABL1 and most of the exons of RCSD1 were used on complementary DNA (cDNA) obtained from patients P1, P2, P4 and P5 and extracted from the cytogenetic cell pellet. PCR analysis from leukemic cells showed a sole RCSD1–ABL1-positive product, whereas two RCSD1–ABL1-positive products of slightly different molecular weights were found by Mustjoki et al. in patient P3 (ref. 2) (Supplementary Table 1). Sequencing of PCR products showed that the first three exons of RCSD1 were fused to exons 4– 11 of ABL1 in all five patients (Supplementary Figure 1). The shorter PCR product identified in P3 consisted of the first two exons of RCSD1 fused to exon 4 of ABL1, presumably resulting from alternative splicing of the fusion gene. All fusion genes were inframe and encoded the tyrosine kinase domain of ABL1. Two other cases of B-cell ALL associated with a RCSD1–ABL1 fusion gene were reported in the literature. Inokuchi et al.3 described a 31-year-old man with t(1;9)(q24;q34) in whom imatinib and dasatinib, combined with dexamethasone, achieved transient clinical effects. Leukemic cells rapidly became refractory to the treatment following the subsequent development of more cytogenetic abnormalities and the patient deceased 6 months following diagnosis.3 Roberts et al.4 reported a 15-year-old male in whom RNA-seq analysis showed a RCSD1– ABL1 fusion. No cytogenetic and clinical data were available.4 In both cases, a chimeric transcript consisting of the first three exons of RCSD1 fused to the ABL1 gene starting from exon 4 was observed, as in the cases described here. Seven genes are known to fuse to the ABL1 gene.5 All the fusion genes result from the joining of 50 sequences of the partner gene with the 30 sequences of the ABL1 gene. The breakpoint occurs in intron 1 or 2 of ABL1 in five different types of fusion gene. Breakpoint in intron 3 of the ABL1 gene was found in the sole case of ALL associated with a SFPQ–ABL1 fusion and in all cases with RCSD1–ABL1 thus far studied. As a consequence, the fusion protein contains part of the SH2 domain of ABL1, the SH1 domain (that has tyrosine kinase function), the three nuclear localization signal domains, the three DNA-binding regions and the F-actin-binding domain.5 Roumiantsev et al.6 demonstrated that SH2 domain of BCR–ABL1 was required for efficient induction of CML-like disease in mice. However, it was not required for transformation of primary bone marrow B-lymphoid progenitors in vitro or for induction of B-lymphoid leukemia in mice.6 This could explain why all the patients thus far reported to carry the RCSD1–ABL1 gene were diagnosed with B-cell ALL. & 2013 Macmillan Publishers Limited

Furthermore, the retained N-terminal part of the partner proteins contain a coiled-coil domain or a helix-loop-helix domain that is necessary for oligomerization of the fusion ABL1 protein and required for its tyrosine kinase activation.5 However, although its structure is not well known, the RCSD1 protein does not have a coiled-coil domain or a helix-loop-helix domain.7 Therefore, it is likely that the RCSD1–ABL1 protein exerts its transforming effects through different mechanisms. Treatment with tyrosine kinase inhibitors, such as imatinib and dasatinib, was only applied in two patients, with variable results.2,3 It is therefore difficult to assess the effectiveness of tyrosine kinase inhibitors in patients with RCSD1–ABL1 fusion. Total RNA was extracted from frozen leukemic cells obtained for patients P1 and P3, reverse transcribed to obtain cDNA that was used as a template to synthesize biotinylated cRNA. Unfortunately, no high-quality RNA suitable for gene expression profiling was available from the other three patients. Labeled cRNA was then fragmented and hybridized to Human HT-12 v3 Expression BeadChips (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The ArrayMiner 5.3 software (Optimal Design, Brussels, Belgium) was used to compare the gene expression profiles, first, between ALL patients with an ABL1 chimeric gene (RCSD1–ABL1 or BCR–ABL1) and nine patients with a MLL rearrangement; second, between two patients with a RCSD1–ABL1 gene (studied in duplicate) and four Philadelphia-positive B-ALL patients carrying the BCR–ABL1 gene. Analysis of patients with an ABL1 fusion gene using unsupervised two-dimensional hierarchic clustering algorithm revealed that this group had a distinct signature from the MLL-rearranged group (Supplementary Figure 2). Among the genes that showed the highest overexpression among the ABL1 group, several of them, such as JAK1, CD27, PTP4A3, TP53INP1, FOXO1 and NLRC5, are involved in cell proliferation, differentiation and apoptosis control (Supplementary Figure 3). Gene expression signature of both patients with RCSD1–ABL1 fusion gene was distinct from that of ALL patients with BCR–ABL1 gene (Supplementary Figure 2). Several genes overexpressed in the RCSD1–ABL1 group compared with the BCR–ABL1 group code transcription factors such as AFF1 and PBX4 (Supplementary Figure 4). AFF1 (alias AF4–AF4/FMR2 family, member 1) is part of a higher order complex, known as the AEP complex, also containing other AFF1 and ENL family proteins and PTEF-b (positive transcription elongation factor b).8 This complex promotes CD133 transcription, a gene encoding a pentaspan transmembrane glycoprotein thought to function in maintaining stem cell properties by suppressing differentiation. Overexpression of AFF1 would lead to higher CD133 transcript levels, inducing ALL cell growth and survival.9 Leukemia (2013) 1394 – 1440


1424 PBX4 (pre-B-cell leukemia homeobox 4) encodes a member of the pre-B-cell leukemia transcription factor family.10 Pbx functions are in part mediated by the interaction of Pbx proteins with members of the Hox and Meis/Prep families. They are not only Hox cofactors, but also at the crossroads of several signaling pathways. Through pbx-containing complexes, they participate in the recruitment of chromatin-remodeling enzymes such as HAT, HDAC, CBP or the whole SWI/SNF complex.11 A major difference between both groups was the deregulation of the BCL2 signaling pathway. Indeed, three genes, BCL2, CREB1 and CREB5, were overexpressed while BAX was under-expressed in the RCSD1–ABL1 group. Overexpression of BCL2 (B-cell CLL/ lymphoma 2), being localized in mitochondria, blocks the apoptotic death of pro-B-lymphocytes.12 CREB1 (cyclic AMP response element-binding protein) is a known BCL2 transcription factor; it has a critical role in regulation of apoptosis of B cells.13 CREB5 protein, which is highly homologous with CREB1, binds to CRE (cyclic AMP response element) as a heterodimer with CREB1 and functions as a CREdependent trans-activator.14 Following phosphorylation, CREB1 translocates to the nucleus, where it binds to the BCL2 promoter, which in turn, activates BCL2 gene expression.13 Both CREB1 and CREB5 were overexpressed in patients with RCSD1–ABL1 fusion. The BAX (BCL2-associated X protein) protein encoded by the BAX gene belongs to the BCL2 protein family. It forms a heterodimer with BCL2, functions as an apoptotic activator and induces cell death by acting on mitochondria.15 This gene was underexpressed in patients with RCSD1–ABL1 fusion. Modifications in the expression of these four genes may have led to an antiapoptotic effect of the fusion gene. Roumantsev et al.16 showed that the ZNF198–FGFR1 fusion kinase associated with the 8p11 myeloproliferative syndrome used a different pathway than the BCR–FGFR1 fusion kinase inducing chronic myeloid leukemia. They concluded that different signaling pathways originating from both the kinase domain and the NH2-terminal fusion partner were implicated.16 It is likely that their conclusions also apply to our results and that the RCSD1– ABL1 and BCR–ABL1 fusion proteins use different signaling pathways to generate distinct forms of leukemia. In conclusion, the t(1;9)(q24;q34) is a recurrent cytogenetic abnormality present in B-cell ALL associated with RCSD1–ABL1 fusion gene. This rearrangement appears to be rare, as only one case was identified in a cohort of 231 children with B-cell ALL.4 More cases and functional studies are necessary to determine its pathogenic consequences, which may involve the BCL2 signaling pathway.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work has been funded in part by the ‘Ligue contre le Cancer-Comite´ du Finiste`re’.

E De Braekeleer1,2, N Douet-Guilbert1,2,3, P Guardiola4,5, D Rowe6, S Mustjoki7, A Zamecnikova8, S Al Bahar8, G Jaramillo9, C Berthou10, N Bown6, K Porkka7, C Ochoa11 and M De Braekeleer1,2,3 1 Laboratoire d’Histologie, Embryologie et Cytogeńe´tique, Faculte´ de Me´decine et des Sciences de la Sante´, Universite´ de Bretagne Occidentale, Brest, France; 2 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U1078, Brest, France;

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Service de Cytogeńe´tique, Cytologie et Biologie de la Reproduction, Hoˆpital Morvan, CHRU Brest, Brest, France; 4 Service des Maladies du Sang, CHU Angers, Angers, France; 5 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U892, Angers, France; 6 Northern Genetics Service, Newcastle upon Tyne, UK; 7 Hematology Research Unit Helsinki, Department of Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; 8 Department of Hematology, Kuwait Cancer Control Center, Kuwait City, Kuwait; 9 Instituto de Investigaciones Biome´dicas, Universidad de las Ame´ricas, Quito, Ecuador; 10 Service d’He´matologie Clinique, Institut de Cance´rologie et d’He´matologie, Hoˆpital Morvan, CHRU Brest, Brest, France and 11 Instituto del Cancer SOLCA, Cuenca, Ecuador E-mail: [email protected]

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia (2013) 1394 – 1440

& 2013 Macmillan Publishers Limited