Letters to the Editor 1148 single-agent treatments (Figure 2d and Supplementary Figures 2Department of Pediatric Oncology, Dana Farber Cancer Institute, 4a–d). Importantly, the combination treatment also led to a Boston, MA, USA; significant increase in apoptosis measured by Annexin V and PI, as 3Department of Haematology, University College London Cancer compared with the result with either drug alone (Figure 2e and Institute, University College London, London, UK; Supplementary Figures 5a–c). 4Cancer Science Institute of Singapore, National University of In conclusion, our study shows that ABT-199 in submicromolar Singapore, Singapore, Singapore; doses is not active in the killing of most cell lines representing 5Department of Medical Oncology, Dana Farber Cancer Institute, more-differentiated T-ALL cells. Intriguingly, the LOUCY Boston, MA, USA and cell line, the only T-ALL cell line with the expression signature 6Department of Computer Science, Metropolitan College, Boston of early immature T-ALL, showed exquisite sensitivity to ABT-199. University, Boston, MA, USA In addition, ABT-199 showed striking synergy with cytarabine in E-mail: [email protected] or [email protected] the LOUCY cell line. Although our results are based on a single 7These authors contributed equally to this work. early immature T-ALL cell line, they are potentially important because primary cases of early immature T-ALL are highly resistant to current regimens of combination chemotherapy. As other early REFERENCES immature T-ALL cell lines are not available, the possibility that 1 Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet 2008; 371: ABT-199 may show clinically significant activity in early immature 1030–1043. T-ALL cases will need to be pursued in ‘primagraft models’ of this 2 Goldberg JM, Silverman LB, Levy DE, Dalton VK, Gelber RD, Lehmann L et al. disease (established by engrafting early immature T-ALL samples Childhood T-cell acute lymphoblastic leukemia: the Dana-Farber Cancer Institute directly into immunodeprived mice). Alternatively, as ABT-199 acute lymphoblastic leukemia consortium experience. J Clin Oncol 2003; 21: 3616–3622. is now in clinical trials for chronic lymphocytic leukemia 3 Coustan-Smith E, Mullighan CG, Onciu M, Behm FG, Raimondi SC, Pei D et al. and as primagraft models of early immature T-ALL cells Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic have not been reported, this BH3 mimetic could be tested leukaemia. Lancet Oncol 2009; 10: 147–156. directly in phase I/II clinical trials, either alone or in combination 4 Van Vlierberghe P, Ambesi-Impiombato A, Perez-Garcia A, Haydu JE, Rigo I, with cytarabine, for relapsed/refractory early immature T-ALL Hadler M et al. ETV6 mutations in early immature human T cell leukemias. patients. J Exp Med 2011; 208: 2571–2579. 5 Rothenberg EV, Moore JE, Yui MA. Launching the T-cell-lineage developmental programme. Nature reviews. Immunology 2008; 8: 9–21. CONFLICT OF INTEREST 6 Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012; 481: 157–163. The authors declare no conflict of interest. 7 Davids MS, Letai A. Targeting the B-cell lymphoma/leukemia 2 family in cancer. J Clin Oncol 2012; 30: 3127–3135. 8 Zhang L, Ming L, Yu J. BH3 mimetics to improve cancer therapy; mechanisms and ACKNOWLEDGEMENTS examples. Drug Resist Updat 2007; 10: 207–217. This study was supported by 4R00CA134743 and ACS Grant IRG-72-001-36 to HF; and 9 Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S et al. ABT-263: 1R01 CA176746 and P01 CA109901 to ATL. The authors thank J Etchin, A Kentsis and R a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 2008; 68: Segal for reagents. IH acknowledges training support through NHLB1 T32 HL7501. 3421–3428. 10 Gandhi L, Camidge DR, Ribeiro de Oliveira M, Bonomi P, Gandara D, Khaira D et al. NM Anderson1,7, I Harrold1,7, MR Mansour2,3, T Sanda4, Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients M McKeown5, N Nagykary1, JE Bradner5, G Lan Zhang6, with small-cell lung cancer and other solid tumors. J Clin Oncol 2011; 29: 909–916. AT Look2 and H Feng1 11 Vandenberg CJ, Cory S. ABT-199, a new Bcl-2-specific BH3 mimetic, has in vivo 1 efficacy against aggressive Myc-driven mouse lymphomas without provoking Department of Pharmacology & Experimental Therapeutics, thrombocytopenia. Blood 2013; 121: 2285–2288. The Center for Cancer Research, Section of Hematology 12 Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al. ABT-199, and Medical Oncology, Boston University School a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing of Medicine, Boston, MA, USA; platelets. Nat Med 2013; 19: 202–208.

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Whole-exome sequencing in del(5q) myelodysplastic syndromes in transformation to acute myeloid leukemia

Leukemia (2014) 28, 1148–1151; doi:10.1038/leu.2013.381 (WES) has been used to identify novel gene mutations in myeloid malignancies, including MDS.2 The value of this approach in the study of MDS transforming to AML has recently been illustrated by Walter et al.3 To date, however, only a small number of cases of Myelodysplastic syndromes (MDSs) are clonal disorders of the MDS have been thus studied. hematopoietic stem cell.1 MDS represents an excellent model of Interstitial deletion within the long arm of 5 leukemic transformation with B30–40% of MDS patients (del(5q)) is one of the most frequent cytogenetic abnormalities 4 developing acute myeloid leukemia (AML).1 The molecular basis observed in MDS, occurring in B10–20% of patients. In order to of leukemic transformation in MDS remains poorly understood investigate the molecular events associated with disease and there is a compelling need to identify the specific molecular progression in MDS with del(5q), we performed WES on paired events driving this process. Recently, whole-exome sequencing samples from two MDS cases with del(5q) (patient 1 and patient 2)

Accepted article preview online 24 December 2013; advance online publiction, 21 January 2014

Leukemia (2014) 1129 – 1174 & 2014 Macmillan Publishers Limited Letters to the Editor 1149 Table 1. List of mutations identified by WES in two MDS del(5q) patients (patients 1 and 2) and one MDS patient without del(5q) (patient 3) in samples obtained before and after transformation to AML

Patient 1 (with del(5q))

MDS (at diagnosis) 46,XX,del(5)(q13:q32)[12]/46,XX[18] AML (31 months from diagnosis) 46,XX,del(5)(q13:q32)[11]/46,XX,del(5)(q13:q32), þ 8,add(12)(p13),add(16)(q24), À 18[39]

Gene Mutation Mutant allele frequency Depth of coverage (x) Mutant allele frequency Depth of coverage (x) (variant/total reads) (variant/total reads)

Mutations present at both MDS and AML stages PCNX S670Rfs*21 15.8 304 45.3 285 SMC1B R790P 16.5 79 47.1 87

Mutations present at the AML stage only RLF I988R — 173 57.9 285 FAM188A R75L — 71 40.9 66 RBM23 Q97H — 203 35.8 173 TP53 K132E — 51 55.0 40 GPATCH8 D1246A — 136 36.1 144 FBXL12 R174C — 122 31.5 108 RYR1 R1140C — 43 45.2 84 HUWE1 V3033L — 106 33.3 93 ERCC6L K1230N — 212 46.4 196 CES2 H571Q — 62 53.1 32

Patient 2 (with del(5q))

MDS (at diagnosis) 44,XX,del(5)(q13:q22), À 17, À 18, À 22, AML (4 months from diagnosis) NA þ mar[15]/46,XX[15]

Gene Mutation Mutant allele frequency Depth of coverage (x) Mutant allele frequency Depth of coverage (x) (variant/total reads) (variant/total reads)

Mutations present at both MDS and AML stages RYR1 W545Cfs*52 5.9 118 23.0 135

Mutations present at the AML stage only PTPRE R291M — 69 23.7 76 UBAC2 S223C — 251 22.1 217 TP53 F270S — 226 24.3 185 CLSTN2 K397N — 56 25.0 60 DNAH14 L899V — 107 21.7 115 WRN L211P — 207 22.4 196

Patient 3 (without del(5q))

MDS (at diagnosis) 46,XY[30] AML (25 months from diagnosis) 47,XY, þ 8[30]

Gene Mutation Mutant allele frequency Depth of coverage (x) Mutant allele frequency Depth of coverage (x) (variant/total reads) (variant/total reads)

Mutations present at both MDS and AML stages GSTM2 P2A 21.1 38 51.4 37 COL6A3 A3050P 27.8 90 41.7 60 MFSD7 V93I 32.2 59 45.5 44 TET2 Q769* 24.1 83 45.1 71 SYNE1 P3948A 37.2 78 38.5 52 C7orf60 M288I 24.5 53 48.0 50 NOC2L Y619C 30.4 207 42.4 118 DNHD1 Q4123H 38.1 63 52.4 42 ANKS1B R938C 31.3 99 42.6 61 CECR2 Q195P 33.8 133 41.6 101 PCSK6 Q923K 33.6 128 43.8 80 PLCD3 P654L 31.8 148 47.3 93

& 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174 Letters to the Editor 1150 Table 1. (Continued )

Patient 3 (without del(5q))

MDS (at diagnosis) 46,XY[30] AML (25 months from diagnosis) 47,XY, þ 8[30]

Gene Mutation Mutant allele frequency Depth of coverage (x) Mutant allele frequency Depth of coverage (x) (variant/total reads) (variant/total reads)

Mutations present at the AML stage only ZNF835 A340T — 87 39.6 53

Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; WES, whole-exome sequencing. WES data were aligned to the hg19/GRCh37 reference genome build using Stampy (http://www.well.ox.ac.uk/project-stampy). Identification of variant sites and alleles was performed using Platypus (http://www.well.ox.ac.uk/platypus). ANNOVAR (http://www.openbioinformatics.org/annovar) was then used to functionally annotate the variants. Variants for Sanger sequencing validation were selected based on the following criteria: (a) not reported as a known SNP in dbSNP unless reported in COSMIC; (b) leading to a non-synonymous amino-acid change, frameshift, stop codon creation or in-frame insertion/deletion; (c) high-quality score (among 10% best score values); (d) present in o7% reads in the constitutional sample; (e) present in 45% reads in the MDS sample; and (f) present in sequencing reads from both directions. Sanger sequencing was used for validation of the mutations identified by WES and for the screening of a large MDS cohort for mutations in selected (the exons containing the mutations were sequenced). Genomic DNA was isolated from patient bone marrow or peripheral blood samples. The selected regions were PCR-amplified and bidirectionally sequenced using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 Genetic Analyzer. Sequence data were analyzed using Mutation Surveyor V3.25 (Softgenetics, State College, PA, USA).

CECR2 genes have been previously reported in MDS and AML.3,5–8 The allele frequency of all the mutations identified was higher in the AML stage samples compared with the MDS stage samples (Figure 1; Supplementary Figure 1). This is consistent with previous observations made by Walter et al.3 showing that nearly all clones harboring mutations underwent expansion with disease progression from MDS to AML. Interestingly, we found that most mutations identified in the two del(5q) cases in our study were present at the AML stage only. In contrast, most mutations identified in the case without del(5q) were present at both the MDS and the AML stage (Figure 1; Supplementary Figure 1). The mutation pattern where most of the mutations found after AML transformation are already Figure 1. Scatter plot showing the changes in the mutant allele present in the paired MDS sample, with a subset of AML stage- frequencies from MDS to AML in the three patients studied. specific mutations, has been observed previously.3,9 We identified recurrent mutations of TP53 and RYR1 at the AML stage of both patients 1 and 2 (with del(5q)), but no mutations of these two genes were found in patient 3 (without del(5q)), or in and one MDS case without del(5q) (patient 3) before and after another MDS case with del(7q) also investigated using WES, both progression to AML. Genomic DNA was isolated from bone before and following leukemic transformation, by our group.9 marrow cells collected at the time of diagnosis and at leukemic Both TP53 mutations identified are inactivating and located in the transformation (following informed consent) and subjected to DNA-binding domain of the . p53, the guardian of the WES. T cells were used as a source of constitutional DNA (CD3 þ genome, is also known to have a role in the regulation of cells, purity 497%). Tumor and constitutional DNA were used for hematopoietic stem cell quiescence and self-renewal. It is well exome capture and high-throughput sequence analysis using recognized that mutation of TP53, resulting in loss of the tumor- Illumina technology (San Diego, CA, USA). suppressor activities of the p53 protein, as well as in the In patient 1 (with del(5q)), we identified 12 somatic non- acquisition of new oncogenic activities, can lead to an increase synonymous variations (SNVs), of which two were present at both in genomic instability.6 We suggest that the complex karyotype the MDS and AML stages and 10 at the AML stage only (Table 1). observed in the AML stage of patient 1 (with del(5q)) is likely In patient 2 (with del(5q)), we identified seven SNVs, of which one secondary to the presence of this TP53 mutation. Recent data was present at both the MDS and AML stages and six only after AML indicate that mutation of TP53 may be one of the molecular transformation (Table 1). In patient 3 (without del(5q)), we identified events necessary for clonal progression of some del(5q) MDS 13 SNVs, of which 12 were present at both the MDS and AML stages, patients to AML.4–6 The fact that both cases with the del(5q) whereas one was only present at the AML stage (Table 1). showed mutation of TP53 following leukemic transformation to The functional effects of the missense mutations identified were AML in our WES study further supports this suggestion. evaluated using PolyPhen2 and SIFT (Supplementary Table 1). None The RYR1 gene encodes a calcium release channel and of the genes found to be mutated in the two del(5q) cases lie within mutations of RYR1 have been reported in several other cancers the deleted region at 5q. All mutations identified were validated in (COSMIC database), but not previously in MDS or AML. The RYR1 the AML stage samples using Sanger sequencing. In all three cases, mutation in patient 1 was a missense mutation, whereas the RYR1 WES analysis and Sanger sequencing of constitutional DNA mutation in patient 2 was a frameshift variant (producing a confirmed the somatic nature of the variants. truncated protein) located in the N-terminal domain within a Thevastmajorityofthemutatedgenesidentifiedinthisstudy hotspot region for mutations associated with malignant have not been previously reported in myeloid malignancies, hyperthermia. Mutations in this region of RYR1 are thought to although all are known to be mutated in other cancers (as reported cause aberrations in RYR1 Ca2 þ channel activity.10 Deep RNA in the COSMIC database). Mutations in the TET2, TP53, RLF and sequencing data show that RYR1 is expressed at low levels in bone

Leukemia (2014) 1129 – 1174 & 2014 Macmillan Publishers Limited Letters to the Editor 1151 marrow CD34 þ cells of MDS patients (data not shown). RYR1 may Research Centre Programme. Whole-exome sequencing was performed at the Oxford represent a new recurrently mutated gene associated with MDS Genomics Centre, Wellcome Trust Centre for Human Genetics, Oxford, UK. del(5q) transformation to AML. A mutation in the SMC1B gene in patient 1 (with del(5q)) was identified at both the MDS and AML stages. The SMC1B gene DISCLAIMER encodes a cohesin component and mutations involving other components of the cohesin complex have been recently reported The views expressed are those of the author(s) and not necessarily those of the in MDS.11 SMC1B may therefore represent a new mutated cohesin NHS, the NIHR or the Department of Health. complex gene. In addition, we identified mutations of the RLF and 1,5 1,5 1,5 RBM23 genes in patient 1 (with del(5q)) following transformation A Pellagatti , M Fernandez-Mercado , C Di Genua , 2 3 1 4 2 to AML. Interestingly, mutation of RLF has previously been MJ Larrayoz , S Killick , H Dolatshad , A Burns , MJ Calasanz , 4 1 reported in one MDS case transforming to AML3 and we A Schuh and J Boultwood 1 therefore conclude that it may represent a new recurrent LLR Molecular Haematology Unit, Nuffield Division of Clinical mutation in AML secondary to MDS, illustrating the value of Laboratory Sciences, Radcliffe Department of Medicine, University of comparing WES data sets in MDS. RBM23 is a RNA-binding Oxford, Oxford, UK; 2 SR-related factor. SR have a role in pre-messenger RNA Department of Genetics, University of Navarra, Pamplona, Spain; 3 splicing and are important regulators of alternative splicing.12 Department of Haematology, Royal Bournemouth Hospital, Recurrent mutations in splicing factor genes have been recently Bournemouth, UK and 4 identified by us and others in MDS and have revealed a new NIHR Biomedical Research Centre, University of Oxford, Oxford, UK leukemogenic pathway involving spliceosomal dysfunction in E-mail: [email protected] 5 MDS.2 We also identified mutation of the CECR2 gene in patient 3 These authors contributed equally to this work. (without del(5q)) at both the MDS and AML stages. CECR2 was recently identified as a novel DNA damage response protein, important for g-H2AX formation and DNA double-strand break REFERENCES repair.13 Mutation of CECR2 has been previously reported in one 1 Heaney ML, Golde DW. Myelodysplasia. N Engl J Med 1999; 340: 1649–1660. AML case8 and therefore CECR2 may also be a new recurrently 2 Boultwood J, Dolatshad H, Varanasi SS, Yip BH, Pellagatti A. The role of splicing mutated gene in MDS and AML. factor mutations in the pathogenesis of the myelodysplastic syndromes. Adv Biol A single mutation of DNAH14 was found in patient 2 (with Regul 2013; e-pub ahead of print 15 September 2013; doi:10.1016/j.jbior. del(5q)), and a mutation of DNHD1 in patient 3 (without del(5q)). 2013.09.005. In addition, we and others have previously reported mutation of 3 Walter MJ, Shen D, Ding L, Shao J, Koboldt DC, Chen K et al. Clonal architecture 3,9,14,15 DNAH11 in MDS and AML. These genes encode dyneins and of secondary acute myeloid leukemia. NEnglJMed2012; 366: 1090–1098. may represent a new group of mutated genes in myeloid 4 Boultwood J, Pellagatti A, McKenzie AN, Wainscoat JS. Advances in the disorders. Dyneins are involved in spindle assembly checkpoint, 5q- syndrome. Blood 2010; 116: 5803–5811. a process that is crucial for cell division. 5 Fernandez-Mercado M, Burns A, Pellagatti A, Giagounidis A, Germing U, Agirre X et al. In order to determine whether some of the mutations identified Targeted resequencing analysis of 25 genes commonly mutated in myeloid disorders by WES are recurrent in MDS, we next screened a large series of 96 in del(5q) myelodysplastic syndromes. Haematologica 2013; 98: 1856–1864. MDS cases for mutations in selected genes that we found to be 6 Jadersten M, Saft L, Smith A, Kulasekararaj A, Pomplun S, Gohring G et al. TP53 mutated, using Sanger sequencing. We selected the SMC1B, mutations in low-risk myelodysplastic syndromes with del(5q) predict disease RBM23, CECR2 and PCNX genes. In addition, we screened 14 MDS progression. J Clin Oncol 2011; 29: 1971–1979. del(5q) cases with advanced disease (that is, excess blasts) for 7 Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P et al. Clinical and biological implications of driver mutations in myelodysplastic mutations in the RYR1 gene. We did not find any additional syndromes. Blood 2013; 122: 3616–3627. mutations of these genes in the patient cohorts analyzed; 8 Welch JS, Ley TJ, Link DC, Miller CA, Larson DE, Koboldt DC et al. however, our analysis represented only a partial screen, as it The origin and evolution of mutations in acute myeloid leukemia. Cell 2012; 150: was limited to the exons containing the mutations identified. 264–278. In summary, WES performed in a cohort of three MDS patients 9 Fernandez-Mercado M, Pellagatti A, Di Genua C, Larrayoz MJ, Winkelmann N, both with and without the del(5q), at diagnosis and following Aranaz P et al. Mutations in SETBP1 are recurrent in myelodysplastic syndromes progression to AML, identified many somatic gene mutations, and often coexist with cytogenetic markers associated with disease progression. most of which have not been previously reported in MDS or AML Br J Haematol 2013; 163: 235–239. and some of which are recurrent. Several mutations were 10 Kobayashi S, Yamamoto T, Parness J, Ikemoto N. Antibody probe study of Ca2 þ identified in the AML stage only and thus may have a role in channel regulation by interdomain interaction within the ryanodine receptor. disease progression of MDS to AML. In addition, our data further Biochem J 2004; 380: 561–569. support a role for TP53 mutation in the progression of del(5q) MDS 11 Kon A, Shih LY, Minamino M, Sanada M, Shiraishi Y, Nagata Y et al. Recurrent to AML. These data further our knowledge of the molecular mutations in multiple components of the cohesin complex in myeloid neoplasms. genetic events that occur during evolution from MDS to AML. The Nat Genet 2013; 45: 1232–1237. analysis of larger cohorts of MDS del(5q) cases transforming to 12 Long JC, Caceres JF. The SR protein family of splicing factors: master regulators of gene expression. Biochem J 2009; 417: 15–27. AML is warranted in order to determine the prevalence of the 13 Lee SK, Park EJ, Lee HS, Lee YS, Kwon J. Genome-wide screen of human mutated genes identified by our study. bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein. Mol Cells 2012; 34: 85–91. CONFLICT OF INTEREST 14 Kim Y, Lee I, Kim D, Won Jung C, Jang J, Kim H et al. Prognostic impact of NPM1, The authors declare no conflict of interest. IDH1/2 and DNAH11 gene mutations on normal karyotype acute myeloid leukemia patients not harboring FLT3/ITD mutation. Haematologica 2013; 98(Suppl 1): 280 (abstract P633). ACKNOWLEDGEMENTS 15 Walter MJ, Shen D, Shao J, Ding L, White BS, Kandoth C et al. Clonal diversity of This work was supported by Leukaemia and Lymphoma Research of the United recurrently mutated genes in myelodysplastic syndromes. Leukemia 2013; 27: Kingdom and by the National Institute for Health Research (NIHR) Oxford Biomedical 1275–1282.

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

& 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174