Letters to the Editor 1422 ACKNOWLEDGEMENTS factors in 494 patients from the grupo Espanol de Linfomas/Transplante Autologo This work was undertaken at University College London Hospitals/University College de Medula Osea Spanish cooperative group. J Clin Oncol 2001; 19: 1395–1404. London, which received a proportion of funding from the Department of Health’s 6 Jabbour E, Hosing C, Ayers G, Nunez R, Anderlini P, Pro B et al. Pretransplant NIHR Biomedical Research Centres funding Scheme. The funding sources had no positive positron emission tomography/gallium scans predict poor outcome involvement in study design; in the collection, analysis and interpretation of data; in in patients with recurrent/refractory Hodgkin . 2007; 109: the writing of the report; or in the decision to submit the paper for publication. 2481–2489. 7 Moskowitz AJ, Yahalom J, Kewalramani T, Maragulia JC, Vanak JM, Zelenetz AD et KJ Thomson1,2, I Kayani2, K Ardeshna2, EC Morris2, al. Pretransplantation functional imaging predicts outcome following autologous R Hough1,2, A Virchis2, AH Goldstone2, stem transplantation for relapsed and refractory Hodgkin lymphoma. Blood 2010; 116: 4934–4937. DC Linch1,2 and KS Peggs1,2 1 8 Peggs KS, Hunter A, Chopra R, Parker A, Mahendra P, Milligan D et al. Clinical University College London Cancer Institute, London, UK and evidence of a graft-versus-Hodgkin’s-lymphoma effect after reduced-intensity 2 Department of Haematology, University College London, allogeneic transplantation. Lancet 2005; 365: 1934–1941. University College London Hospitals NHS Trust, 9 Sureda A, Robinson S, Canals C, Carella AM, Boogaerts MA, Caballero D et al. London, UK Reduced-intensity conditioning compared with conventional allogeneic stem-cell E-mail: [email protected] 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. 10 Peggs KS, Sureda A, Qian W, Caballero D, Hunter A, Urbano-Ispizua A et al. REFERENCES Reduced-intensity conditioning for allogeneic haematopoietic stem cell trans- 1 Viviani S, Zinzani PL, Rambaldi A, Brusamolino E, Levis A, Bonfante V et al. ABVD plantation in relapsed and refractory Hodgkin lymphoma: impact of alemtuzu- versus BEACOPP for Hodgkin’s lymphoma when high-dose salvage is planned. N mab and donor lymphocyte infusions on long-term outcomes. Br J Haematol Engl J Med 2011; 365: 203–212. 2007; 139:70–80. 2 Linch DC, Winfield D, Goldstone AH, Moir D, Hancock B, McMillan A et al. Dose 11 Peggs KS, Kayani I, Edwards N, Kottaridis P, Goldstone AH, Linch DC et al. Donor intensification with autologous bone-marrow transplantation in relapsed and resistant lymphocyte infusions modulate relapse risk in mixed chimeras and induce dur- Hodgkin’s disease: results of a BNLI randomised trial. Lancet 1993; 341: 1051–1054. able salvage in relapsed patients after T-cell-depleted allogeneic transplantation 3 Schmitz N, Pfistner B, Sextro M, Sieber M, Carella AM, Haenel M et al. Aggressive for Hodgkin’s lymphoma. J Clin Oncol 2011; 29: 971–978. conventional chemotherapy compared with high-dose chemotherapy with 12 Meignan M, Gallamini A, Haioun C. Report on the first international workshop on autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Interim-PET-Scan in lymphoma. Leuk Lymphoma 2009; 50: 1257–1260. Hodgkin’s disease: a randomised trial. Lancet 2002; 359: 2065–2071. 13 Barrington SF, Qian W, Somer EJ, Franceschetto A, Bagni B, Brun E et al. Con- 4 Sweetenham JW, Carella AM, Taghipour G, Cunningham D, Marcus R, Della VA et cordance between four European centres of PET reporting criteria designed for al. High-dose therapy and autologous stem-cell transplantation for adult patients use in multicentre trials in Hodgkin lymphoma. Eur J Nucl Med Mol Imaging 2010; with Hodgkin’s disease who do not enter remission after induction chemother- 37: 1824–1833. apy: results in 175 patients reported to the European group for blood and marrow 14 Moskowitz CH, Matasar MJ, Zelenetz AD, Nimer SD, Gerecitano J, Hamlin P et al. transplantation. Lymphoma working party. J Clin Oncol 1999; 17: 3101–3109. Normalization of pre-ASCT, FDG-PET imaging with second-line, non-cross resis- 5 Sureda A, Arranz R, Iriondo A, Carreras E, Lahuerta JJ, Garcia-Conde J et al. Auto- tant, chemotherapy programs improves event-free survival in patients with logous stem-cell transplantation for Hodgkin’s disease: results and prognostic 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 profile from BCR–ABL1 fusion

Leukemia (2013) 27, 1422–1424; doi:10.1038/leu.2012.332 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 De Braekeleer et al.1 published the first case of acute (Pts 1, 3 and 5) were still alive, survival ranging from 12 to 97 lymphoblastic leukemia (ALL) associated with a t(1;9)(q24;q34) months. One patient, Pt 1, received bone marrow transplantation and showed colocalization of bacterial artificial (BAC) 31 months following diagnosis (after a first relapse) and was alive clones containing the RCSD1 and ABL1 genes, suggesting the 66 months later. Another patient, Pt 3, had allogeneic hemato- presence of a yet undescribed fusion gene. Mustjoki et al.2 poietic stem cell transplantation 4 months following diagnosis but identified a second patient. Sequencing of the PCR products relapsed 12 months later. A third patient, Pt 2, had three bone showed that the first three exons of RCSD1 were fused to ABL1, marrow transplantations, each time following relapse, but died 84 starting at exon 4. months following diagnosis. We obtained biological samples from patients diagnosed in Conventional on bone marrow cells using R- and/ Finland, United Kingdom, Kuwait and Ecuador. Five patients (three or G- and/or Q-banding revealed a clone containing a males and two females) were then included in this study (Table 1). t(1;9)(q24;q34) along with a normal clone in four patients while Their age ranged from 11 to 40 years. All had B-cell ALL. additional material of unknown origin was identified at chromo- Immunophenotyping showed the blasts to be CD10 þ , CD19 þ , some 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 Letters to the Editor 1423 Table 1. Clinical and hematological characteristics of five patients with t(1;9)(q24;q34)

Patient Country Sex Age Hb WBC Platelets PB BM Complete Event-free Survival Reference no. (years) (g/ ( 109/ ( 109/l) blasts blasts remission survival (months) dl) l) (%) (%) (months)

1 France M 11 11.5a 6.4a 104a 47a 92a Yes 11 97 þ b De Braekeleer et al.1 2 England F 15 5.8 348 25 NA NA Yes 1c 84 De Braekeleer et al.5 3 Finland M 40 11.1 24.3 291 34 80 Yes 16d 66 þ Mustjoki et al.2 4 Kuwait F 18 8.0 110 10 87 92 LTF LTF LTF Zamecnikova17 5 Ecuador M 18 9.1 470 56 52 58 —e 12 þ 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 Furthermore, the retained N-terminal part of the partner BAC clones RP11-232M22 (containing the RCSD1 gene) and RP11- proteins contain a coiled-coil domain or a helix-loop-helix domain 83J21 (containing ABL1) was attempted in four patients (P1, P2, P4 that is necessary for oligomerization of the fusion ABL1 protein and P5) and showed two fusion signals, suggesting the presence and required for its tyrosine kinase activation.5 However, although of a RCSD1–ABL1 chimeric gene. its structure is not well known, the RCSD1 protein does not have a Primers designed by Mustjoki et al.2 covering the kinase domain coiled-coil domain or a helix-loop-helix domain.7 Therefore, it is of ABL1 and most of the exons of RCSD1 were used on likely that the RCSD1–ABL1 protein exerts its transforming effects complementary DNA (cDNA) obtained from patients P1, P2, P4 through different mechanisms. Treatment with tyrosine kinase and P5 and extracted from the cytogenetic cell pellet. PCR inhibitors, such as imatinib and dasatinib, was only applied in two analysis from leukemic cells showed a sole RCSD1–ABL1-positive patients, with variable results.2,3 It is therefore difficult to assess product, whereas two RCSD1–ABL1-positive products of slightly the effectiveness of tyrosine kinase inhibitors in patients with different molecular weights were found by Mustjoki et al. in patient RCSD1–ABL1 fusion. P3 (ref. 2) (Supplementary Table 1). Sequencing of PCR products Total RNA was extracted from frozen leukemic cells obtained showed that the first three exons of RCSD1 were fused to exons 4– for patients P1 and P3, reverse transcribed to obtain cDNA that 11 of ABL1 in all five patients (Supplementary Figure 1). The shorter was used as a template to synthesize biotinylated cRNA. PCR product identified in P3 consisted of the first two exons of Unfortunately, no high-quality RNA suitable for gene expression RCSD1 fused to exon 4 of ABL1, presumably resulting from profiling was available from the other three patients. Labeled alternative splicing of the fusion gene. All fusion genes were in- cRNA was then fragmented and hybridized to Human HT-12 v3 frame and encoded the tyrosine kinase domain of ABL1. Expression BeadChips (Illumina, San Diego, CA, USA) according to Two other cases of B-cell ALL associated with a RCSD1–ABL1 the manufacturer’s instructions. The ArrayMiner 5.3 software fusion gene were reported in the literature. Inokuchi et al.3 (Optimal Design, Brussels, Belgium) was used to compare described a 31-year-old man with t(1;9)(q24;q34) in whom the gene expression profiles, first, between ALL patients imatinib and dasatinib, combined with dexamethasone, with an ABL1 chimeric gene (RCSD1–ABL1 or BCR–ABL1) and achieved transient clinical effects. Leukemic cells rapidly became nine patients with a MLL rearrangement; second, between refractory to the treatment following the subsequent two patients with a RCSD1–ABL1 gene (studied in duplicate) and development of more cytogenetic abnormalities and the patient four Philadelphia-positive B-ALL patients carrying the BCR–ABL1 deceased 6 months following diagnosis.3 Roberts et al.4 reported a gene. 15-year-old male in whom RNA-seq analysis showed a RCSD1– Analysis of patients with an ABL1 fusion gene using unsuper- ABL1 fusion. No cytogenetic and clinical data were available.4 In vised two-dimensional hierarchic clustering algorithm revealed both cases, a chimeric transcript consisting of the first three exons that this group had a distinct signature from the MLL-rearranged of RCSD1 fused to the ABL1 gene starting from exon 4 was group (Supplementary Figure 2). Among the genes that showed observed, as in the cases described here. the highest overexpression among the ABL1 group, several of Seven genes are known to fuse to the ABL1 gene.5 All the fusion them, such as JAK1, CD27, PTP4A3, TP53INP1, FOXO1 and NLRC5, genes result from the joining of 50 sequences of the partner gene are involved in cell proliferation, differentiation and apoptosis with the 30 sequences of the ABL1 gene. The breakpoint occurs in control (Supplementary Figure 3). intron 1 or 2 of ABL1 in five different types of fusion gene. Gene expression signature of both patients with RCSD1–ABL1 Breakpoint in intron 3 of the ABL1 gene was found in the sole case fusion gene was distinct from that of ALL patients with BCR–ABL1 of ALL associated with a SFPQ–ABL1 fusion and in all cases with gene (Supplementary Figure 2). Several genes overexpressed in RCSD1–ABL1 thus far studied. As a consequence, the the RCSD1–ABL1 group compared with the BCR–ABL1 group code contains part of the SH2 domain of ABL1, the SH1 domain (that transcription factors such as AFF1 and PBX4 (Supplementary has tyrosine kinase function), the three nuclear localization signal Figure 4). domains, the three DNA-binding regions and the F-actin-binding AFF1 (alias AF4–AF4/FMR2 family, member 1) is part of a higher domain.5 Roumiantsev et al.6 demonstrated that SH2 domain of order complex, known as the AEP complex, also containing other BCR–ABL1 was required for efficient induction of CML-like disease AFF1 and ENL family proteins and PTEF-b (positive transcription in mice. However, it was not required for transformation of elongation factor b).8 This complex promotes CD133 transcription, a primary bone marrow B-lymphoid progenitors in vitro or for gene encoding a pentaspan transmembrane glycoprotein thought induction of B-lymphoid leukemia in mice.6 This could explain why to function in maintaining stem cell properties by suppressing all the patients thus far reported to carry the RCSD1–ABL1 gene differentiation. Overexpression of AFF1 would lead to higher CD133 were diagnosed with B-cell ALL. transcript levels, inducing ALL cell growth and survival.9

& 2013 Macmillan Publishers Limited Leukemia (2013) 1394 – 1440 Letters to the Editor 1424 PBX4 (pre-B-cell leukemia homeobox 4) encodes a member of 3Service de Cytoge´ne´tique, Cytologie et Biologie de la Reproduction, the pre-B-cell leukemia transcription factor family.10 Pbx functions Hoˆpital Morvan, CHRU Brest, Brest, France; are in part mediated by the interaction of Pbx proteins with 4Service des Maladies du Sang, CHU Angers, Angers, France; members of the Hox and Meis/Prep families. They are not only 5Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) Hox cofactors, but also at the crossroads of several signaling U892, Angers, France; pathways. Through pbx-containing complexes, they participate in 6Northern Service, Newcastle upon Tyne, UK; the recruitment of chromatin-remodeling enzymes such as HAT, 7Hematology Research Unit Helsinki, Department of Medicine, HDAC, CBP or the whole SWI/SNF complex.11 University of Helsinki and Helsinki University Central Hospital, A major difference between both groups was the deregulation Helsinki, Finland; of the BCL2 signaling pathway. Indeed, three genes, BCL2, CREB1 8Department of Hematology, Kuwait Cancer Control Center, and CREB5, were overexpressed while BAX was under-expressed in Kuwait City, Kuwait; the RCSD1–ABL1 group. Overexpression of BCL2 (B-cell CLL/ 9Instituto de Investigaciones Biome´dicas, Universidad de las lymphoma 2), being localized in mitochondria, blocks the Ame´ricas, Quito, Ecuador; apoptotic death of pro-B-lymphocytes.12 CREB1 (cyclic AMP 10Service d’He´matologie Clinique, Institut de Cance´rologie et response element-binding protein) is a known BCL2 d’He´matologie, Hoˆpital Morvan, CHRU Brest, Brest, France and transcription factor; it has a critical role in regulation of 11Instituto del Cancer SOLCA, Cuenca, Ecuador apoptosis of B cells.13 CREB5 protein, which is highly E-mail: [email protected] homologous with CREB1, binds to CRE (cyclic AMP response element) as a heterodimer with CREB1 and functions as a CRE- dependent trans-activator.14 Following phosphorylation, CREB1 translocates to the nucleus, where it binds to the BCL2 , REFERENCES 13 which in turn, activates BCL2 gene expression. Both CREB1 and 1 De Braekeleer E, Douet-Guilbert N, Le Bris MJ, Berthou C, Morel F, De Braekeleer CREB5 were overexpressed in patients with RCSD1–ABL1 fusion. M. A new partner gene fused to abl1 in a t(1;9)(q24;q34)-associated B-cell acute The BAX (BCL2-associated X protein) protein encoded by the BAX lymphoblastic leukemia. Leukemia 2007; 21: 2220–2221. gene belongs to the BCL2 protein family. It forms a heterodimer 2 Mustjoki S, Hernesniemi S, Rauhala A, Kahkonen M, Almqvist A, Lundan T et al. A with BCL2, functions as an apoptotic activator and induces cell novel dasatinib-sensitive RCSD1-ABL1 fusion transcript in chemotherapy-refrac- death by acting on mitochondria.15 This gene was under- tory adult pre-B lymphoblastic leukemia with t(1;9)(q24;q34). Haematologica 2009; expressed in patients with RCSD1–ABL1 fusion. Modifications in 94: 1469–1471. the expression of these four genes may have led to an anti- 3 Inokuchi K, Wakita S, Hirakawa T, Tamai H, Yokose N, Yamaguchi H et al. RCSD1- apoptotic effect of the fusion gene. ABL1-positive B lymphoblastic leukemia is sensitive to dexamethasone and 16 tyrosine kinase inhibitors and rapidly evolves clonally by chromosomal translo- Roumantsev et al. showed that the ZNF198–FGFR1 fusion cations. Int J Hematol 2011; 94: 255–260. kinase associated with the 8p11 myeloproliferative syndrome 4 Roberts KG, Morin RD, Zhang J, Hirst M, Zhao Y, Su X et al. Genetic alterations used a different pathway than the BCR–FGFR1 fusion kinase activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic inducing chronic myeloid leukemia. They concluded that different leukemia. Cancer Cell 2012; 22: 153–166. signaling pathways originating from both the kinase domain and 5 De Braekeleer E, Douet-Guilbert N, Rowe D, Bown N, Morel F, Berthou C et al. ABL1 the NH2-terminal fusion partner were implicated.16 It is likely that fusion genes in hematological malignancies: a review. Eur J Haematol 2011; 86:361–371. their conclusions also apply to our results and that the RCSD1– 6 Roumiantsev S, de Aos IE, Varticovski L, Ilaria RL, Van Etten RA. The src homology 2 ABL1 and BCR–ABL1 fusion proteins use different signaling domain of Bcr/Abl is required for efficient induction of chronic myeloid leukemia- pathways to generate distinct forms of leukemia. like disease in mice but not for lymphoid leukemogenesis or activation of phosphatidylinositol 3-kinase. Blood 2001; 97: 4–13. In conclusion, the t(1;9)(q24;q34) is a recurrent cytogenetic 7 Eyers CE, McNeill H, Knebel A, Morrice N, Arthur SJC, Cuenda A et al. The phos- abnormality present in B-cell ALL associated with RCSD1–ABL1 phorylation of CapZ-interacting protein (CapZIP) by stress-activated protein fusion gene. This rearrangement appears to be rare, as only one kinases triggers its dissociation from CapZ. Biochem J 2005; 389: 127–135. 4 case was identified in a cohort of 231 children with B-cell ALL. 8 Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML. A higher-order complex More cases and functional studies are necessary to determine its containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and pathogenic consequences, which may involve the BCL2 signaling physiologic MLL-dependent transcription. Cancer Cell 2010; 17: 198–212. pathway. 9 Mak AB, Nixon AM, Moffat J. The mixed lineage leukemia (MLL) fusion-associated gene AF4 promotes CD133 transcription. Cancer Res 2012; 72: 1929–1934. 10 Wagner K, Mincheva A, Korn B, Lichter P, Popperl H. Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis. Mech CONFLICT OF INTEREST Dev 2001; 103: 127–131. The authors declare no conflict of interest. 11 Laurent A, Bihan R, Omilli F, Deschamps S, Pellerin I. PBX proteins: much more than Hox cofactors. 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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