Letters to the Editor 1606 leukemia mouse model. This effect was proposed to be due, at REFERENCES least in part, to inhibition of FBXW7, a negative regulator of Notch 1 Ferrando AA, Neuberg DS, Staunton J, Loh ML, Huard C, Raimondi SC et al. Gene 8 signaling. However, FBXW7 targets the degradation of other expression signatures define novel oncogenic pathways in T cell acute lympho- oncogenic proteins, such as c-Myc and mTOR, and the expression blastic leukemia. Cancer Cell 2002; 1: 75–87. of miR-223 is significantly elevated in TAL1-positive T-ALL cases 2 Cardoso BA, de Almeida SF, Laranjeira AB, Carmo-Fonseca M, Yunes JA, (Supplementary Figure 2), suggesting that the oncogenic function Coffer PJ et al. TAL1/SCL is downregulated upon histone deacetylase of this microRNA may extend beyond mere collaboration in Notch- inhibition in T-cell acute lymphoblastic leukemia cells. Leukemia 2011; 25: induced leukemia. Moreover, the proleukemic role of mir-223 may 1578–1586. 3 O’Neil J, Shank J, Cusson N, Murre C, Kelliher M. TAL1/SCL induces leukemia be also achieved by downregulating targets such as E2F1, FOXO1, by inhibiting the transcriptional activity of E47/HEB. Cancer Cell 2004; 5: RHOB or EPB41L3, which have been associated with induction of 587–596. apoptosis and/or have tumor-suppressive roles (Supplementary 4 Sanda T, Lawton LN, Barrasa MI, Fan ZP, Kohlhammer H, Gutierrez A et al. Table 5). Interestingly, the intriguing possibility that miR-223 may Core transcriptional regulatory circuit controlled by the TAL1 complex potentially act downstream of TAL1 to negatively regulate MEF2C, in human T cell acute lymphoblastic leukemia. Cancer Cell 2012; 22: recently identified as an oncogene in T-ALL,15 would be in line with 209–221. the observations that TAL1 and MEF2C tend to segregate, defining 5 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; two discrete T-ALL subsets.15 116: 281–297. In summary, our studies identify and validate for the first time 6 Schotte D, Pieters R, Den Boer ML. MicroRNAs in acute leukemia: from biological players to clinical contributors. Leukemia 2012; 26: 1–12. a small set of TAL1-regulated microRNA genes whose role may 7 Fabbri M, Croce CM, Calin GA. MicroRNAs in the ontogeny of leukemias and be important in the context of hematopoiesis and T-cell lymphomas. Leuk Lymphoma 2009; 50: 160–170. leukemogenesis. 8 Mavrakis KJ, Van Der Meulen J, Wolfe AL, Liu X, Mets E, Taghon T et al. A coop- erative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet 2011; 43: 673–678. 9 Fukao T, Fukuda Y, Kiga K, Sharif J, Hino K, Enomoto Y et al. An evolutionarily CONFLICT OF INTEREST conserved mechanism for microRNA-223 expression revealed by microRNA gene The authors declare no conflict of interest. profiling. Cell 2007; 129: 617–631. 10 Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen MM, Brugman MH et al. Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human ACKNOWLEDGEMENTS T-cell development from CD34 þ cells: implications for leukemogenesis in gene This study was supported by grants from Fundac¸a˜o para a Cieˆncia e a Tecnologia therapy. Leukemia 2007; 21: 754–763. (FCT; PTDC/BIM-ONC/1548/2012) and Liga Portuguesa Contra o Cancro (Terry Fox 11 Ghisi M, Corradin A, Basso K, Frasson C, Serafin V, Mukherjee S et al. Modulation of Award) to JTB; and FWO Vlaanderen (G056413N) and UGent (GOA 01G01910W) to FS. microRNA expression in human T-cell development: targeting of NOTCH3 by miR- NCC and KD have PhD fellowships from FCT and IWT Vlaanderen, respectively. PR has 150. Blood 2011; 117: 7053–7062. a post-doctoral grant from FWO Vlaanderen. We would like to thank Steve Lefever for 12 Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O et al. primer design. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature 2008; 451: 1125–1129. 13 Pulikkan JA, Dengler V, Peramangalam PS, Peer Zada AA, Muller-Tidow C, Bohlander SK et al. Cell-cycle regulator E2F1 and microRNA-223 comprise an 1 2 1 2 2 autoregulatory negative feedback loop in acute myeloid leukemia. Blood 2010; NC Correia , K Durinck , AP Leite , M Ongenaert , P Rondou , 115: 1768–1778. 2 1 1 F Speleman , FJ Enguita and JT Barata 14 Agatheeswaran S, Singh S, Biswas S, Biswas G, Pattnayak NC, Chakraborty S. 1 Instituto de Medicina Molecular, Faculdade de Medicina da BCR-ABL mediated repression of miR-223 results in the activation of MEF2C and Universidade de Lisboa, Lisbon, Portugal and PTBP2 in chronic myeloid leukemia. Leukemia 2012; e-pub ahead of print 21 2Center for Medical Genetics, Department of Pediatrics and Genetics, December 2012. doi:10.1038/leu.2012.339. Ghent University, Ghent, Belgium 15 Homminga I, Pieters R, Langerak AW, de Rooi JJ, Stubbs A, Verstegen M et al. E-mail: [email protected] Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia. Cancer Cell 2011; 19: 484–497.

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

All-trans retinoic acid and arsenic trioxide resistance of acute promyelocytic leukemia with the variant STAT5B-RARA fusion gene

Leukemia (2013) 27, 1606–1610; doi:10.1038/leu.2012.371 to NPM1, NUMA1, PRKAR1A, FIP1L1, BCOR and STAT5B have been described.2–5 Prolonged disease-free survival of APL patients is achieved by combining all-trans retinoic acid (ATRA) and chemotherapy.6 The genetic hallmark of acute promyelocytic leukemia (APL) is In addition, treatment with arsenic trioxide (ATO) is of proven the t(15;17)(q22;q21)/PML-RARA rearrangement, which is detect- value in relapsed disease and also effective during induction able by fluorescence in situ hybridization (FISH) or reverse or consolidation therapy or both.5–7 However, the RARA transcription-polymerase chain reaction (RT-PCR) in 495% of fusion partner has an important impact on the biology of the morphologically defined APL.1,2 Moreover, seven variant fusion disease, in particular with regard to retinoic acid (RA) sensitivity. genes, which fuse RARA at low frequency to ZBTB16 or very rarely Therefore, APL can be divided in two disease subtypes: a RA-

Accepted article preview online 28 December 2012; advance online publication, 22 January 2013

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited Letters to the Editor 1607 RARA fusions.1–4 The sensitivity of variant RARA fusions to ATO has not been well documented, but ZBTB16–RARA-positive APL appears to be resistant.2,5 In this respect, it is noteworthy that the direct interaction of ATO with the PML moiety of the PML- RARA fusion protein leading to its degradation may provide an explanation for the specificity of ATO treatment in PML-RARA- positive APL.8 Herein, we describe a new case of APL with a STAT5B-RARA fusion in an adolescent—the first pediatric patient—which brings the number of patients with this rare APL variant to eight.9–15 Furthermore, we summarize the therapeutic approaches and clinical data of the cases described so far, demonstrating that patients with STAT5B-RARA-positive APL are unresponsive to both ATRA and ATO. This emphasizes the clinical importance of identifying this specific fusion gene in PML-RARA- negative APL. A 17-year-old boy underwent a routine checkup for a several day history of fatigue without any other clinical evidence of leukemia. However, 10% and 80% blast cells with AML M3 morphology and prominent Auer rods were detected in the peripheral blood and the bone marrow (BM), respectively. Immunophenotyping showed MPO þ , CD13 þ , CD33 þ , CD34 À , HLA-D À myeloid blast cells and, although clinically not apparent, coagulation was abnormal: fibrinogen 84 mg/dl, D-dimers 19.6 mg/ml. Cytogenetics revealed a 46,XY,?der(13), ?der(17q) karyotype without the typical t(15;17) and RT-PCR for PML-RARA was negative. Whereas FISH using the Vysis LSI PML/ RARA dual color dual fusion translocation probe resulted in an ambiguous FISH scoring with only 7% of interphase cells showing a split signal of the RARA probe (Figure 1a), hybridization with the Vysis LSI PML-RARA single fusion FISH probe kit suggested a deletion of RARA (Figure 1b). FISH employing the RARA-specific cosmid cos1241 and a STAT5B-specific BAC clone showed one co-localization of the probes (Figure 1c). Moreover, by applying additional FISH probes loss of genetic material at both loci as well as the presence of an inversion event were observed, indicating a complex intrachromosomal rearrangement. A more detailed description of the FISH data and all probes used are provided in the Supplementary Information. STAT5B-RARA-specific RT-PCR using primers STAT5Bex14-F1 50-GGCAATGGTTTGACGGTGTG-30 and RARAex3-R1 50-AGGGAGGGCTGGGCACTATC-30 and sequen- cing of the amplification product confirmed that, like in all cases described so far,9–15 STAT5B exon 15 was fused to RARA exon 3 Figure 1. FISH, RT-PCR and flow cytometric MRD analysis of STAT5B- (Figures 1d and e). RARA-positive APL. (a) Interphase FISH with the Vysis LSI PML/RARA dual color dual fusion translocation probe resulted in a splitting of Based on epidemiological data, 495% of APL harbor a PML-RARA rearrangement, and ZBTB16-RARA and NPM1-RARA the RARA probe (green signals) in only 7% of the cells, indicating a 1 possible RARA rearrangement. (b) Hybridization of a metaphase with account for o1% and 0.5% of the cases, respectively. Including the Vysis LSI PML/RARA single fusion FISH probe kit showing a the patient described herein, now eight STAT5B-RARA-positive APL complete deletion of the RARA probe (green signals). (c) FISH using cases have been identified;9–15 however, due to the lack of the RARA-specific cosmid cos124 (green signals) combined with the epidemiological data the incidence of this APL variant remains to STAT5B-spanning BAC clone (RP11–358B23; red signals) showing be determined. The median age of the patients was 36.5 years one co-localization (indicated by arrows) of the two probes, (range 17–67 years), and remarkably, seven of the eight patients suggesting the presence of a STAT5B-RARA fusion. Of note, RP11– were male (Table 1). A similar biased sex ratio has also been 358B23 spans the entire STAT5B gene but shows only two signals 0 observed in ZBTB16-RARA-positive APL in that amongst 11 adult indicating a deletion of the STAT5 3 -end. (d) RT-PCR analysis 1 for STAT5B-RARA: lane 1, patient sample; lane 2, negative control. patients 10 were male. In contrast, PML-RARA-positive APL shows (e) Sequence chromatogram showing the fusion between STAT5B an almost equal sex distribution in both childhood and adult 7,16 exon 15 and RARA exon 3. (f) Flow cytometric analysis of BM cells APL. taken at diagnosis (day 0) and after the second induction course After informed consent was obtained, the patient was treated (day 56). Based on diagnostic immunophenotyping, which showed according to the AML-BFM 2004 (ClinicalTrials.gov: NCT00111345) hyperexpression of CD99 and CD33, samples were stained using a protocol,7 and during intensification and maintenance therapy patient-specific four-color combination (CD99/CD45RA/CD33/CD34). seven courses of ATRA (each for 14 days) were administered. Represented dot plots show blasts in red color (background cells in Morphological remission was achieved after induction gray). SSC, side light scattering characteristics. chemotherapy; however, despite treatment with ATRA, coagulation tests remained abnormal during the first two responsive subtype, which includes APL with RARA fusions to PML, courses of chemotherapy. Minimal residual disease (MRD) levels NPM1 and NuMA, and possibly also to BCOR, PRKAR1A and FIP1L1; determined by flow cytometry, tracking the aberrant CD33 þþ, and a RA-unresponsiveness subtype characterized by the CD45RA þ , CD99 þþ and CD34 À blast cell-specific phenotype presence of the ZBTB16-RARA and most probably the STAT5B- (Figure 1f), declined slowly: 10% at day 15, 0.85% after the second

& 2013 Macmillan Publishers Limited Leukemia (2013) 1567 – 1614 1608 ekma(03 57–1614 – 1567 (2013) Leukemia

Table 1. Clinical and biologic data of STAT5B-RARA-positive APL patients

No Sex/age Karyotype FAB WBC DIC Therapy Outcomea Ref (years) ( Â 109/l)

1 M/67 45,X,-Y,add(17)(q?)[40] M1/ 6.7 þ NA; no response to ATRA; blast cells insensitive to ATRA in vitro NA 9 M3v 2 M/57 46,XY,t(10;11)(q22;q25), M3 23.8 þþ ATRA (no granulocytic differentiation; discontinued) 10 i(17)(q10) Induction therapy: cytarabine, daunorubicin First CR First relapse BM at 9 months Gemtuzumab ozogamicin Second CR Non-myeloablative alloSCT BMT-related complications Dead at 18 months 3 M/42 46,X, À Y, þ 11[9]/46,XY[11] M3 3.6 þþ ATRA (improvement of DIC without granulocytic differentiation) 11 Induction therapy: cytarabine, daunorubicin, mitoxantrone, etoposide First CRm Post-remission therapy: cytarabine Secondary MDS (RCMD) at 41 months First relapse BM at 75 months ATO (no response) Induction therapy: cytarabine, idarubicin Pulmonary bleeding

Dead at 76 months Editor the to Letters 4 M/41 47,XY,del(9)(q?),add(17)(q12), M3 77.8 þ ATRA (no granulocytic differentiation) 12 þ mar1[3]/ Induction therapy: cytarabine, idarubicin First CRm 48,XY,idem, þ mar1[17] Consolidation therapy: cytarabine, idarubicin First relapse meningeal cytarabine, intrathecal chemotherapy, whole-brain irradiation Second CRm Second relapse BM Dead at 17 months 5 M/32 46,XY M3 3.8 þ Induction therapy: ATRA, ATO, mitoxantrone (no granulocytic differentiation) 13 FLAG (fludarabine, cytarabine, G-CSF) First CR Consolidation therapy: FLAG alloPBSCT (HLA-matched sister) CRm at 28 months 6 F/29 46,XX,t(3;17)(q26;q21) M3 5.6 þ Induction therapy: AIDA (ATRA, idarubicin); cytarabine CR, but no CRm 14 Molecular relapse FLA (fludarabine, cytarabine) No CRm Myeloablative alloBMT (sibling) Clinical CR, BMT-related complications Dead at 15 months 7 M/26 46,XY NA 6.6 þ ATRA (no granulocytic differentiation) 15 Induction therapy: idarubicin, cytarabine ATRA, ATO No CR CAG (cytarabine, aclarubicin, G-CSF) No CR Intracranial haemorrhage Dead at 6 months 8 M/17 46,XY,?der(13),?der[17q][14]/ M3 2.8 þ ATRA This 46,XY[6] Induction therapy: AIE (cytarabine, idarubicin, etoposide) First CR; but no CRf work

& Consolidation/intensification therapy:

03McilnPbihr Limited Publishers Macmillan 2013 AI (ATRA, cytarabine, idarubicin) HAE (etoposide, high-dose cytarabine, ATRA) No CRf haM (cytarabine, mitoxantrone, ATRA) First relapse testis at 24 months ATO (no response), DNX-FLA (fludarabine, cytarabine, daunoxome), FLA, Second CR treosulfan, fludarabine, thiotepa; autoSCT Second relapse BM at 40 months

FLA (fludarabine, cytarabine) Refractory disease, infectious complications FLAMSA (fludarabine, amsacrine, cytarabine) Dead at 53 months Abbreviations: ATRA, all-trans retinoic acid; alloPBSCT, allogeneic peripheral blood stem cell transplantation; alloSCT, allogeneic stem cell transplantation; ATO, arsenic trioxide; autoSCT, autologous stem cell transplantation; BM, bone marrow; BMT bone marrow transplantation; CR, complete morphological remission; CRf, flow remission; CRm, molecular remission; DIC, disseminated intravascular coagulation: À none, þ moderate, þþ severe; FAB, French–American–British; GCS-F, granulocyte colony-stimulating factor; MDS, myelodysplastic syndrome; NA, not available; RCMD, refractory cytopenia with multilineage dysplasia; WBC, white blood cell. aIf not otherwise specified, follow-up time from diagnosis Letters to the Editor 1609 and 0.06% after the third course of chemotherapy. Two years after died of transplantation-related complications or progressive diagnosis, the patient had an isolated extramedullary relapse in disease. Notably, the only patient (case 5) who remains the right testis that was treated with ATO (50 days) without any relapse-free for more than 2 years received an allogeneic response. A decrease of testicular size was achieved with transplant in first molecular CR. Although in APL the fludarabine, cytarabine and daunoxome; and the testis was occurrence of extramedullary disease both at presentation and removed. Another block of fludarabine and cytarabine was relapse is a rare event,2,5,6 one case each had an isolated CNS administered, followed by high-dose therapy with treosulfan, and testis relapse. Even at molecular relapse, CNS involve- fludarabine and thiotepa, as well as autologous stem cell rescue. ment is not uncommon, which therefore, should be Three and a half years after initial diagnosis, he had a second excluded in all APL patients at diagnosis and at molecular or isolated BM relapse with blast cells treatment-resistant to frank relapse.6 fludarabine, cytarabine and amsacrine. Without having achieved In summary, STAT5B-RARA-positive APL is unresponsive to both a third CR, stem cell transplantation was pursued; however, during ATRA and ATO, belongs to a highly relapse-prone APL subgroup, conditioning therapy, he died of fulminant pulmonary and and its potential refractoriness to chemotherapy at relapse cerebral aspergillosis. suggests that this subgroup of APL patients might benefit from Regarding the clinical outcome of STAT5B-RARA-positive APL BM transplantation in first remission. Therefore, the accurate patients on the whole, we aimed to collect the therapy identification of this rare subtype of APL is essential to guide regimens administered and the most recent follow-up data therapeutic decisions. of all patients described to date. As summarized in Table 1, all of them were treated with ATRA, and four also with ATO (two each at diagnosis and relapse) combined with different poly- CONFLICT OF INTEREST chemotherapy regimens. Importantly, although administration of The authors declare no conflict of interest. ATRA resulted in an improvement of disseminated intravascular coagulation, no differentiation of the blast cells was achieved, emphasizing that STAT5B-RARA-positive APL is unresponsive to ACKNOWLEDGEMENTS ATRA (Table 1). This work was supported by the St. Anna Kinderkrebsforschung e.V. The authors More specifically, in patient 2, due to the lack of granulocytic would like to thank Robert E Gallagher for critically reviewing the manuscript and differentiation ATRA therapy was discontinued and only after Angela Schumich for conducting the FACS analysis. induction chemotherapy a CR was achieved. A first relapse occurred 9 months after initial presentation, and a second brief CR was obtained with gemtuzumab ozogamicin, followed by non-myeloa- AUTHOR CONTRIBUTIONS blative allogeneic stem cell transplantation. However, the patient SS initiated the study, collected all clinical data and drafted the manuscript. MK succumbed to transplantation-related complications. In case 3, conducted FISH and molecular experiments. MND is responsible for the flow polychemotherapy resulted in a CR, however 41 months from cytometric data. HB, BWC, AA and MND contributed to the writing of the diagnosis this patient developed a secondary myelodysplastic manuscript. HB, BWC, KS, S-JZ, H-YC, AA and MND provided, and are responsible syndrome, and after more than 6 years the STAT5B-RARA-positive for the clinical data. All authors gave their final approval of the manuscript. APL recurred. Patient 4 experienced two relapses, a first extra- medullary relapse, and after a short CR, a second relapse in the BM S Strehl1,MKo¨nig1, H Boztug2, BW Cooper3, K Suzukawa4, occurred and the patient died of progressive disease. S-J Zhang5, H-Y Chen6, A Attarbaschi2 and MN Dworzak1,2 Furthermore, the patient described herein (case 8), who at first 1CCRI, Children’s Cancer Research Institute, extramedullary relapse in the testis received ATO without any St. Anna Kinderkrebsforschung e.V, Vienna, Austria; evidence of responsiveness, suggests that STAT5B-RARA-positive 2St. Anna Children’s Hospital, Department of Pediatrics, APL is not only unresponsive to ATRA but also to ATO. Supporting Medical University of Vienna, Vienna, Austria; this notion, in case 3 at relapse ATO was not effective, in case 5 3University Hospitals of Cleveland, Case Medical Center, despite induction therapy with ATRA and ATO no granulocytic Cleveland, OH, USA; differentiation was observed, and in case 7 neither by the 4Department of Hematology, Division of Clinical Medicine, administration of ATRA nor by ATO and polychemotherapy a CR University of Tsukuba, Ibaraki, Japan; was achieved. 5First Affiliated Hospital of Nanjing Medical University, Of particular interest, in case 6 monitoring of MRD by RT-qPCR Nanjing, China and showed that the patient, although in morphological CR, never 6Jingjiang People’s Hospital, Jiangsu Province, China obtained a molecular remission. Post-treatment MRD analysis E-mail: [email protected] indicated an impending relapse and further therapy followed by allogeneic BMT was given; however, the patient died of transplanta- tion-related complications. In line with the persistent MRD in case 6, REFERENCES flow MRD levels in the patient described herein indicate that no molecular remission might have been obtained. Unfortunately, lack of 1 Grimwade D, Biondi A, Mozziconacci MJ, Hagemeijer A, Berger R, Neat M et al. Characterization of acute promyelocytic leukemia cases lacking the classic appropriate material precluded analysis of STAT5B-RARA fusion t(15;17): results of the European Working Party. Groupe Francais de Cytogene- transcript levels. In view of the clinical importance of detecting tique Hematologique, Groupe de Francais d’Hematologie Cellulaire, UK Cancer persistent molecular MRD at the end of consolidation therapy or Cytogenetics Group and BIOMED 1 European Community-Concerted Action 6 recurrent molecular disease in PML-RARA-positive APL patients, ‘Molecular Cytogenetic Diagnosis in Haematological Malignancies’. Blood 2000; assessment of STAT5B-RARA transcript levels by RT-qPCR,14 which 96: 1297–1308. provides a higher sensitivity as compared to flow MRD, will facilitate 2 Mistry AR, Pedersen EW, Solomon E, Grimwade D. The molecular pathogenesis of clinical decision-making regarding allogeneic transplant as well as early acute promyelocytic leukaemia: implications for the clinical management of the treatment intervention at any signs of an impending relapse also in disease. Blood Rev 2003; 17: 71–97. this APL subtype. 3 Catalano A, Dawson MA, Somana K, Opat S, Schwarer A, Campbell LJ et al. The PRKAR1A gene is fused to RARA in a new variant acute promyelocytic leukemia. In contrast to the overall relapse rate of about 10–15% in 2,5,6 Blood 2007; 110: 4073–4076. APL, 86% (6/7) of patients with STAT5B-RARA-positive 4 Yamamoto Y, Tsuzuki S, Tsuzuki M, Handa K, Inaguma Y, Emi N. BCOR as a novel APL—including one case with a molecular relapse—experienced fusion partner of retinoic acid receptor alpha in a t(X;17)(p11;q12) variant of acute single or multiple relapses (Table 1); and all of them promyelocytic leukemia. Blood 2010; 116: 4274–4283.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1567 – 1614 Letters to the Editor 1610 5 Lengfelder E, Hofmann WK, Nowak D. Impact of arsenic trioxide in the treatment 11 Kusakabe M, Suzukawa K, Nanmoku T, Obara N, Okoshi Y, Mukai HY et al. Detection of acute promyelocytic leukemia. Leukemia 2012; 26: 433–442. of the STAT5B-RARA fusion transcript in acute promyelocytic leukemia with the 6 Sanz MA, Grimwade D, Tallman MS, Lowenberg B, Fenaux P, Estey EH et al. normal chromosome 17 on G-banding. Eur J Haematol 2008; 80: 444–447. Management of acute promyelocytic leukemia: recommendations from an 12 Iwanaga E, Nakamura M, Nanri T, Kawakita T, Horikawa K, Mitsuya H et al. Acute expert panel on behalf of the European LeukemiaNet. Blood 2009; 113: promyelocytic leukemia harboring a STAT5B-RARA fusion gene and a G596V 1875–1891. missense mutation in the STAT5B SH2 domain of the STAT5B-RARA. Eur J Hae- 7 Creutzig U, Zimmermann M, Dworzak M, Urban C, Henze G, Kremens B et al. matol 2009; 83: 499–501. Favourable outcome of patients with childhood acute promyelocytic leukaemia 13 Qiao C, Zhang SJ, Chen LJ, Miao KR, Zhang JF, Wu YJ et al. Identification of the after treatment with reduced cumulative anthracycline doses. Br J Haematol 2010; STAT5B-RARalpha fusion transcript in an acute promyelocytic leukemia patient 149: 399–409. without FLT3, NPM1, c-Kit and C/EBPalpha mutation. Eur J Haematol 2011; 86: 8 Zhang XW, Yan XJ, Zhou ZR, Yang FF, Wu ZY, Sun HB et al. Arsenic trioxide 442–446. controls the fate of the PML-RARalpha oncoprotein by directly binding PML. 14 Jovanovic JV, Rennie K, Culligan D, Peniket A, Lennard A, Harrison J et al. Science 2010; 328: 240–243. Development of real-time quantitative polymerase chain reaction assays to track 9 Arnould C, Philippe C, Bourdon V, Gregoire MJ, Berger R, Jonveaux P. The signal treatment response in retinoid resistant acute promyelocytic leukemia. Front transducer and activator of transcription STAT5b gene is a new partner of retinoic Oncol 2011; 1: 35. acid receptor alpha in acute promyelocytic-like leukaemia. Hum Mol Genet 1999; 15 Chen H, Pan J, Yao L, Wu L, Zhu J, Wang W et al. Acute promyelocytic leukemia 8: 1741–1749. with a STAT5b-RARalpha fusion transcript defined by array-CGH, FISH, and RT- 10 Gallagher RE, Mak S, Paietta E, Cooper B, Ehmann WC, Tallman MS. Identification PCR. Cancer Genet 2012; 205: 327–331. of a second acute promyelocytic leukemia (APL) patient with the STAT5b-RAR 16 Schnittger S, Bacher U, Haferlach C, Kern W, Alpermann T, Haferlach T. Clinical fusion gene among PML-RAR -negative Eastern Cooperative Oncology Group impact of FLT3 mutation load in acute promyelocytic leukemia with t(15;17)/PML- (ECOG) APL Protocol Registrants. Blood 2004; 104: 821a. RARA. Haematologica 2011; 96: 1799–1807.

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

A G polymorphism in the CRBN gene acts as a biomarker of response to treatment with lenalidomide in low/int-1 risk MDS without del(5q)

Leukemia (2013) 27, 1610–1613; doi:10.1038/leu.2013.59 Sixty-six MDS patients from five hematological centers with low/ int-1 risk MDS without del5q were included in this study between January 2010 and December 2012. All patients were anemic and E3 ubiquitin ligase protein Cereblon (CRBN) has been identified as resistant to erythropoiesis-stimulating agents either epoetin a direct molecular target for the teratogenicity of thalidomide and alpha/beta (60 000 U/week) or darbopoetin (300 mg/week) during cytotoxicity of immunomodulatory drugs (IMiDs) such as lenali- 12 weeks, and remained transfusion dependent (X4U of Red Blood domide.1,2 CRBN gene is highly conserved and its related protein Cells during the previous 8 weeks). They received a dose of 10 mg/ of 442 AA is ubiquitously expressed in humans. CRBN is part of a day of lenalidomide for 21 days every 28 days, and the erythroid functional E3 ubiquitin ligase complex together with the DNA response rate, hematological improvement-erythroid was evaluated damage-binding protein-1 (DDB1), Cullin 4A and Roc1, and acts as according to the IWG 2006 criteria after at least 3 months of a substrate receptor for client proteins to be ubiquitinated and treatment. Fifteen patients were not evaluable due to treatment degraded by proteasome.1 CRBN itself undergoes auto- toxicity or early death. The characteristics of the 51 remaining ubiquitination, and thalidomide is described as an inhibitory patients who completed 3 months of treatment (26 RARS, 8 RCMD- molecule for CRBN auto-ubiquitination activity. RS, 7 RCMD, 4 RA, 5 RAEB-1 and 1 CMML) are described in Table 1. Thalidomide or lenalidomide have demonstrated their efficacy Forty-one (80%) patients had a normal karyotype, five presented in the treatment of various hematological disorders including with trisomy 8, two presented del11q and one had del20q. myeloma3 and myelodysplastic syndromes (MDS), either with or Karyotype was unavailable for one patient. Comparisons using without deletion of 5q chromosome.4–6 It has been recently Pearson w2 or Fischer’s exact test for qualitative data, and Wilcoxon shown that changes in gene expression patterns induced by test for quantitative data were performed on Stata MP 12.1 software lenalidomide are abrogated by the depletion of CRBN gene in (Ritme, Paris, France). All tests were two-sided and a Po0.05 were myeloma cell lines.7 Furthermore, 485% patients with low considered to have statistical significance. expression of CRBN are resistant to treatment with thalidomide, Fourteen (27%) patients were responders to lenalidomide, and, conversely, high CRBN gene levels correlate with better according to IWG 2006 with 8 (57%) major responses and 6 (43%) survival in myeloma patients treated with maintenance thalido- minor responses according to IWG 2000, and 37 (73%) were mide.7,8 In low/int-1 risk MDS, the erythroid response rate to nonresponders. No difference of age (P ¼ 0.901), sex (P ¼ 0.334) or thalidomide is quite low (16–30%) and responses are usually International Prognostic Scoring System (IPSS; P ¼ 0.324) at short,9 whereas patients with low/int-1 risk MDS with del5q have a treatment onset was observed between the two groups. The high response rate (50–67%) to lenalidomide.5,10 By contrast, overall response rate was equivalent to that reported in the 430% of low/int-1 MDS patients without del(5q) respond to this literature.6,11 treatment.6,11 Altogether, these data provided us with a rationale The CRBN gene located in 3p26.2 contains 11 predicted exons. to investigate the role of CRBN gene in the response to To address the putative role of CRBN in the response to lenalidomide in low/int-1 MDS patients without del(5q). lenalidomide in MDS patients, we conducted genetic studies

Accepted article preview online 26 February 2013; advance online publication, 26 April 2013

Leukemia (2013) 1567 – 1614 & 2013 Macmillan Publishers Limited