Letters to the Editor 1419 Program, The Japanese Cord Blood Bank Network and The Japanese Society of myeloid leukemia or myelodysplastic syndrome who have 5 Pediatric Hematology. We also thank Ms Takako Sakai, data manager of the Japan and/or 7 abnormalities. Haematologica 2005; 90: 1339 -- 1345. Society for Hematopoietic Cell Transplantation Data Registry, for their excellent 2 Slovak ML, Kopecky KJ, Cassileth PA, Harrington DH, Theil KS, Mohamed A assistance. et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern K Ishiyama1,2, A Takami1,13, Y Kanda3,13, S Nakao1, M Hidaka4, Cooperative Oncology Group Study. Blood 2000; 96: 4075 -- 4083. T Maeda5, T Naoe6, S Taniguchi7, K Kawa8, T Nagamura9, 3 Kurosawa S, Yamaguchi T, Miyawaki S, Uchida N, Kanamori H, Usuki K et al. K Tabuchi10, Y Atsuta11 and H Sakamaki12 A Markov decision analysis of allogeneic hematopoietic cell transplantation 1Department of Cellular Transplantation Biology, versus chemotherapy in patients with acute myeloid leukemia in first remission. Kanazawa University Graduate School of Medical Sciences, Blood 2011; 117: 2113 -- 2120. 4 Slovak ML, Gundacker H, Bloomfield CD, Dewald G, Appelbaum FR, Larson RA Kanazawa, Ishikawa, Japan; 2 et al. A retrospective study of 69 patients with t(6;9)(p23;q34) AML emphasizes Department of Hematology, Tokyo Metropolitan the need for a prospective, multicenter initiative for rare ‘poor prognosis’ myeloid Ohtsuka Hospital, Toshima, Tokyo, Japan; malignancies. Leukemia 2006; 20: 1295 -- 1297. 3 Division of Hematology, Saitama Medical Center, 5 Ishiyama K, Takami A, Kanda Y, Nakao S, Hidaka M, Maeda T et al. Allogeneic Jichi Medical University, Saitama, Saitama Prefecture, Japan; hematopoietic stem cell transplantation for acute myeloid leukemia with 4Department of Hematology, National Hospital Organization t(6;9)(p23;q34) dramatically improves the patient prognosis: a matched-pair Kumamoto Medical Center, Kumamoto, analysis. Leukemia 2011; e-pub ahead of print 26 August 2011; doi:10.1038/ Kumamoto Prefecture, Japan; leu.2011.229. 5Department of Hematology and Oncology, Osaka University 6 Scrucca L, Santucci A, Aversa F. Competing risk analysis using R: an easy guide for clinicians. Bone Marrow Transplant 2007; 40: 381 -- 387. Hospital, Suita, Osaka, Japan; 6 7 Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities Department of Hematology and Oncology, Nagoya University in the presence of competing risks: new representations of old estimators. Graduate School of Medicine, Nagoya, Aichi, Japan; Stat Med 1999; 18: 695 -- 706. 7 Department of Hematology, Toranomon Hospital, 8 Garcon L, Libura M, Delabesse E, Valensi F, Asnafi V, Berger C et al. DEK-CAN Minato, Tokyo, Japan; molecular monitoring of myeloid malignancies could aid therapeutic stratifica- 8Department of Hematology/Oncology, Osaka Medical Center tion. Leukemia 2005; 19: 1338 -- 1344. and Research Institute for Maternal and Child Health, 9 Frohling S, Schlenk RF, Breitruck J, Benner A, Kreitmeier S, Tobis K et al. Prognostic Izumi, Osaka, Japan; significance of activating FLT3 mutations in younger adults (16 to 60 years) with 9Department of Cell Processing and Transfusion, Institute of acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm. Blood 2002; 100: 4372 -- 4380. Medical Science, University of Tokyo, Minato, Tokyo, Japan; 10 10 Oyarzo MP, Lin P, Glassman A, Bueso-Ramos CE, Luthra R, Medeiros LJ. Acute Department of Pediatrics, Hematology/Oncology, myeloid leukemia with t(6;9)(p23;q34) is associated with dysplasia and a high Kanagawa Children’s Medical Center, frequency of flt3 mutations. Am J Clin Pathol 2004; 122: 348 -- 358. Yokohama, Kanagawa, Japan; 11 Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U et al. Analysis of 11Department of Hematopoietic Stem Cell Transplantation FLT3-activating mutations in 979 patients with acute myelogenous leukemia: Data Management/Biostatistics, Nagoya University association with FAB subtypes and identification of subgroups with poor School of Medicine, Nagoya, Aichi, Japan and prognosis. Blood 2002; 99: 4326 -- 4335. 12Department of Hematology, Tokyo Metropolitan Cancer 12 Kuchenbauer F, Kern W, Schoch C, Kohlmann A, Hiddemann W, Haferlach T et al. and Infectious Diseases Center Komagome Hospital, Detailed analysis of FLT3 expression levels in acute myeloid leukemia. Haematologica 2005; 90: 1617 -- 1625. Bunkyo, Tokyo, Japan 13 Koh Y, Park J, Ahn KS, Kim I, Bang SM, Lee JH et al. Different clinical importance of E-mail: [email protected] FLT3 internal tandem duplications in AML according to FAB classification: possible 13 These authors contributed equally to this work. existence of distinct leukemogenesis involving monocyte differentiation pathway. Ann Hematol 2009; 88: 1089 -- 1097. 14 Small D. Targeting FLT3 for the treatment of leukemia. Semin Hematol 2008; 45(3 Suppl 2): S17 -- S21. REFERENCES 15 Breitenbuecher F, Markova B, Kasper S, Carius B, Stauder T, Bohmer FD et al. 1 van der Straaten HM, van Biezen A, Brand R, Schattenberg AV, Egeler RM, A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in Barge RM et al. Allogeneic stem cell transplantation for patients with acute AML. Blood 2009; 113: 4063 -- 4073.

Polymorphisms in xenobiotic transporters ABCB1, ABCG2, ABCC2, ABCC1, ABCC3 and multiple myeloma risk: a case--control study in the context of the International Multiple Myeloma rESEarch (IMMEnSE) consortium

Leukemia (2012) 26, 1419--1422; doi:10.1038/leu.2011.352; case--control studies supports the idea that genetic factors are 2 published online 20 December 2011 involved in MM pathogenesis. Therefore, several studies focusing on various and pathways have tried to identify single- nucleotide polymorphisms (SNPs) associated with the suscept- 3,4 Multiple myeloma (MM) is a hematological neoplasm that arises ibility to the disease. from a single clone of malignant plasma cells in the bone marrow. The detoxification and elimination of xenobiotic compounds is In Europe, 4.6/100 000 males and 3.2/100 000 females every year one of the most investigated processes in cancer susceptibility, with 5 develop MM, with a median age at diagnosis around 60 years.1 several evidences of its association with cancer risk. ATP-binding The observation of a higher risk to develop MM among first- cassette (ABC) subfamily B, member 1 (ABCB1 or MDR1); subfamily degree relatives of MM patients in several population-based G, member 2 (ABCG2 or BCRP); subfamily C, member 2 (ABCC2 or

& 2012 Macmillan Publishers Limited Leukemia (2012) 1402 -- 1448 Letters to the Editor 1420 Table 1. Demographical characteristics of cases and controls

Cases Controls Center Gender M/F Mean age Median age Gender M/F Mean age Median age (total) (±s.d.) (min-max) (total) (±s.d.) (min-max)

First Study Population (IMMEnSE consortium) Pisa, Italya 115/108 (223) 62.60 (±9.90) 63 (35-87) 129/105 (234) 58.77 (±10.89) 58.5 (35-89) Lodz, Polandb 50/47 (97) 61.80 (±10.55) 62 (39-86) 66/80 (146) 69.53 (±6.71) 69 (55-98) Salamanca, Spainc 66/58 (124) 62.70 (±11.60) 62 (31-93) 137/125 (262) 65.56 (±12.85) 66 (24-92) Granada, Spaind 23/36 (59) 63.25 (±10.30) 63 (39-86) Lyon, Francee 43/32 (74) 55.60 (±8.97) 57 (34-75) 47/47 (94) 33.31 (±14.80) 30 (18-60) Braga, Portugalf 26/29 (55) 66.78 (±10.49) 68 (43-86) 54/45 (99) 60.76 (±7.72) 58 (51-85) Total 323/310 (633) 62.10 (±10.64) 62 (31-93) 433/402 (835) 60.15 (±15.18) 62 (18-98)

Replication Study population (Heidelberg Myeloma Group) Heidelberg, Germanyg 323/241 (564) 54.58 (±7.82) 56 (25-66) 789/682 (1471) 56.31 (±9.89) 59 (18-68)

Overall population Total 646/551 (1197) 58.43 (±10.10) 59 (25-93) 1222/1084 (2306) 56.78 (±12.73) 59 (18-98)

aDeparment of Oncology, Transplants and Advanced Technologies, Section of Haematology, Pisa University Hospital, Pisa, Italy. bDeparment of Hematology, Medical University of Lodz, Lodz, Poland. cHematology Division, University Hospital of Salamanca, Salamanca, Spain. dHematology and Hemotherapy Department, University Hospital Virgen de las Nieves, Granada, Spain. eHospices Civils de Lyon, Lyon, France. fLife and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. gMedizinische Klinik V, Universitaetsklinikum Heidelberg, Heidelberg, Germany.

Table 2. Significant associations of ABCB1 SNPs rs10264990 and rs17327442 with MM risk in the overall population

SNP (rs) Cases N (%) Controls N (%) ORa 95%CI P-value P-trend P-perm

ABCB1 rs10264990 C/C 549 (47.1) 958 (41.7) 1 Ref. --- 0.023 C/T 475 (40.7) 1059 (46.0) 0.77 0.66--0.90 0.001 T/T 142 (12.2) 283 (12.3) 0.85 0.68--1.07 0.171 0.869* C/T + T/T 617 (52.9) 1342 (58.3) 0.79 0.68--0.91 0.001 0.015**

ABCB1 rs17327442 A/A 824 (69.7) 1600 (69.9) 1 Ref. --- 0.191 A/T 309 (26.1) 643 (28.1) 0.92 0.78--1.08 0.309 T/T 50 (4.2) 46 (2.0) 1.99 1.32--3.02 0.001 0.009* A/T + T/T 359 (30.3) 689 (30.1) 0.99 0.85--1.16 0.928 1** Genotype distribution among MM cases and controls in the overall population of the ABCB1 SNPs rs10264990 and rs17327442. aOdds Ratios (OR) are adjusted for age, gender and region of origin. Differences in sample numbers are due to failures in genotyping. Results in bold show Po0.05. *P-value obtained after 100 000 permutations following a co-dominant model. **P-value obtained after 100 000 permutations following a dominant model.

MRP2); subfamily C, member 1 (ABCC1 or MRP1) and subfamily C, ABCC1 and ABCC3 to test their impact on MM susceptibility member 3 (ABCC3 or MRP3) are efflux pumps that have a key role in (a complete list of the selected SNPs is available in Supplementary determining the intracellular levels of xenobiotic and toxic Table I). In the context of an International Consortium named compounds, thus protecting cells. ABC transporters are expressed IMMEnSE (International Multiple Myeloma rESEarch), we collected in many tissues, including the blood, and they have been recently more than 700 MM cases and 900 controls from Italy, Spain, showed to be characteristically expressed in hematopoietic stem Poland, Portugal and France. A subset of 633 MM patients and 835 cells (HSCs).6 Beside, the exposure to pesticides and toxic healthy controls with a similar distribution between gender and compounds, most of which are substrates of ABC transporters, age (w2 P ¼ 0.753, Kruskal--Wallis P ¼ 0.307) was available for this has been shown to increase MM and MGUS (monoclonal study and was used for the initial screening of the 58 genetic gammopathy of undetermined significance) risk.7,8 Therefore, it is variants selected. Baseline characteristics of the population are a reasonable hypothesis that genetic variation within these genes reported in Table 1. The IMMEnSE biobank is set up at the German can affect the exposure of hematopoietic cells to toxic compounds Cancer Research Center (DKFZ) in Heidelberg, where genotyping resulting in an increased/decreased risk of MM. has been conducted using both TaqMan (ABI, Applied Biosystems, Despite the large number of studies investigating the role of Foster City, CA, USA) and KASPar (KBioscence, Hoddesdon, UK) variants in ABC transporters in cancer susceptibility, including technologies and adequate quality control procedures (as hematological malignancies,9,10 to date only one investigated the described in the Supplementary methods). All the SNPs were in role of three variants in ABCB1 gene and MM susceptibility.11 Hardy--Weinberg Equilibrium (HWE) among controls (P40.001) Therefore, we performed a two-phase candidate gene association with the exception of the ABCC2 SNP rs3740073 (Po0.001) that study to comprehensively evaluate the role of genetic variants of was therefore excluded from further analysis. ABCs transporter genes in relation to MM risk. We selected 54 Logistic regression was used to assess the main effects of the SNPs in ABCB1, ABCG2 and ABCC2 genes using a tagging approach genetic polymorphisms on MM risk using a co-dominant and a to take into account for all the common genetic variability within dominant inheritance model adjusted for age, gender and region these genes and four additional functional polymorphisms in of origin. For each SNP, the more common allele in the controls

Leukemia (2012) 1402 -- 1448 & 2012 Macmillan Publishers Limited Letters to the Editor 1421 was assigned as the reference category (for detailed material and regulation, one can hypothesize that the introduction of a binding methods see Supplementary Information). Of the 58 variants site for the transcription factor by the rs17327442_T allele could screened, 13 SNPs resulted associated with MM risk in the determine its interaction with TAL1. This could therefore result in IMMEnSE population at the conventional P-value level of Po0.05 an aberrant regulation of ABCB1 expression during normal HSC (Supplementary Table II). Haplotype analysis (Supplementary development. Owing to the broad spectrum of substances Table III) and gene--gene interaction analysis did not add any transported by this pump, including regulators and mediators of significant information to the single SNP analysis. With the aim to B-cell growth and survival, an alteration of its normal expression further investigate the associations found in the first phase, an could enhance the exposure of HSCs to mutagens or proliferating independent case--control set of German origin was collected signals that could therefore justify the increased risk to develop through the collaboration with the Heidelberg Myeloma Group. MM associated with the rs17327442 T/T genotype. Using the same methodology described above, we genotyped In conclusion, our results suggest that genetic variants within the 13 SNPs associated in the first phase in 564 MM cases and ABCB1 gene can have a role on the risk to develop MM. 1471 healthy controls from Germany (Table 1) with a similar Nevertheless the association of the ABCB1 SNP rs17327442 T/T distribution between genders (w2 P ¼ 0.141), although age was homozygotes with a twofold higher risk to develop MM has to be significantly higher for the controls (Kruskal--Wallis P ¼ 0.0001). All replicated on independent populations to clarify its real impact. the SNPs resulted in HWE among controls (P40.001). The two ABCB1 SNPs rs2235013 and rs10256836 were replicated in the CONFLICT OF INTEREST Heidelberg population (Supplementary Table IV). The authors declare no conflict of interest. We analyzed the results for the whole population of 1197 MM cases and 2306 controls (Supplementary Tables V, VI). Interestingly, ACKNOWLEDGEMENTS the ABCB1 SNPs rs10264990 and rs17327442 resulted associated with MM risk reaching the P-value threshold adjusted for multiple We acknowledge support by the recruiting hospitals and physicians of the study testing (P 0.0014; Table 2). To evaluate heterogeneity of the regions as well as their collaborating nurses and technicians. We thank Dr Fabienne o Lesueur (IARC, Lyon, France) for the collection on French controls and Dr Victor findings between populations, we performed a meta-analysis across Moreno (IDBELL, Barcelona, Spain) for the recruitment of Spanish controls. We thank all the different regions in the IMMEnSE and Heidelberg popula- Prof. Bugert at the Institute of Transfusion Medicine and Immunology, German Red tions. Results showed the absence of statistically significant Cross Blood Service Baden-Wu¨rttemberg-Hessen for providing German control DNA heterogeneity among different case--control sets and confirmed samples. This work was partially supported by the grants P08-CVI-4116 from the the associations (Supplementary Figure 1). To further assess the Consejerı´a de Salud de la Junta de Andalucia (Sevilla, Spain), PI081051 from the robustness of our findings, we performed 100 000 permutations to Fondo de Investigaciones Sanitarias (Madrid, Spain) and NN402178334 from the compare P-values from randomly generated empirical distributions Polish Ministry of Science and Higher Education (Lodz, Poland). for the SNPs rs10264990 and rs17327442 with the observed ones. We confirmed in this way the statistically significant associations of A Martino1, D Campa1, G Buda2, J Sainz3,4, R Garcı´a-Sanz5, the T carriers for the rs10264990 with a decreased risk of MM K Jamroziak6, RM Reis7,8, N Weinhold9, M Jurado3,4,RRı´os3,4, (P ¼ 0.015) and of the T/T homozygous for the rs17327442 with an Z Szemraj-Rogucka6, H Marques7, J Szemraj10, A Stein1, R Kumar11, increased risk of MM (P ¼ 0.009; Table 2). E Orciuolo2, F Gemignani12, S Landi12, H Goldschmidt9, M Petrini2, At the best of our knowledge, this is the largest study on genetic C Dumontet13, F Canzian1 and AM Rossi12 susceptibility of MM and has a sufficient statistical power (over 0.80 1Genomic Epidemiology Group, German Cancer for a co-dominant model) to detect an odds ratio ¼ 1.57 at Research Center DKFZ, Heidelberg, Germany; a ¼ 0.0014 (study-wise significance P-threshold) for a SNP with a 2Oncology, Transplants and Advanced Technologies, minor allele frequency (MAF) ¼ 0.05 in the overall population. Section of Hematology, Pisa University, Pisa, Italy; Besides, this is the first comprehensive study that captures all the 3Genomic Oncology Area, Genyo - Pfizer-University of common genetic variation within ABCB1, ABCG2 and ABCC2 genes Granada-Andalusian Government Centre for Genomics and and investigates variants in ABCC1 and ABCC3 in relation to MM risk. Oncological Research, Granada, Spain; Our results confirm the findings of the only study already published 4Department of Hematology, Virgen de las Nieves in the literature showing no associations of the same three ABCB1 University Hospital, Granada, Spain; variants previously tested (or their tag SNP) with MM risk.11 5University Hospital of Salamanca, Universidad de Our results suggest a possible low-penetrance role for the two Salamanca-Consejo Superior de Investigaciones genetic variants rs10264990 and rs17327442 within ABCB1 gene in Cientı´ficas, Hematology Division, Salamanca, Spain; modulating individual susceptibility to MM. 6Department of Hematology, Medical University The SNP rs17327442 has been already found associated with of Lodz, Lodz, Poland; genetic susceptibility to Crohn’s disease,12 whereas the SNP 7Life and Health Sciences Research Institute (ICVS), rs10264990 has not been associated with any disease risk. Both School of Health Sciences, University of these SNPs are located in intronic regions of the ABCB1 gene and Minho, Braga, Portugal; no functional data have been reported to date. To explain our 8Molecular Oncology Research Center, Barretos Cancer Hospital, observation, we investigated the putative function of the two Barretos, Sao Paulo, Brazil; SNPs with the FastSNP tool.13 Although the ABCB1 SNP 9Medizinische Klinik V, Universitaetsklinikum Heidelberg, rs10264990 has no known or predicted function, the genetic Department of Internal Medicine, Heidelberg, Germany; ABCB1 variant rs17327442 is predicted to be an ‘intronic 10Department of Medical Biochemistry, enhancer’. In particular, the rs17327442_T allele is predicted to Medical University of Lodz, Lodz, Poland; introduce a binding site for the transcription factors T-cell acute 11Division of Molecular Genetic Epidemiology, lymphocytic leukemia 1 (TAL1). This latter finding is particularly German Cancer Research Center DKFZ, intriguing. It has been shown that TAL1 is a crucial factor for HSC Heidelberg, Germany; differentiation and its deregulation can lead to oncogenesis in 12Department of Biology, Section of Genetics, T cells by altering several downstream genes. Several ABC family University of Pisa, Pisa, Italy and members are regulated by TAL1, and in particular ABCG2, ABCE1 13INSERM U590, Laboratoire de Cytologie Analytique, and ABCB10 are activated in erythroid cell lines after TAL1 Faculte de Medecine Rockefeller, Universite Claude knocking down, while ABCC1 expression is repressed.14,15 Bernard Lyon I, Lyon, France Although ABCB1 seems not to be one of the targets for TAL1 E-mail: [email protected]

& 2012 Macmillan Publishers Limited Leukemia (2012) 1402 -- 1448 Letters to the Editor 1422 REFERENCES multiple myeloma in Sweden. Cancer Epidemiol Biomarkers Prev 2008; 17: 1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM GLOBOCAN 2008, 3123 -- 3127. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 (Internet). 9 Leal-Ugarte E, Gutierrez-Angulo M, Macias-Gomez NM, Peralta-Leal V, Duran- 2010 (cited; available from: http://globocan.iarc.fr). Gonzalez J, De La Luz Ayala-Madrigal M et al. MDR1 C3435T polymorphism in 2 Landgren O, Weiss BM. Patterns of monoclonal gammopathy of Mexican children with acute lymphoblastic leukemia and in healthy individuals. undetermined significance and multiple myeloma in various ethnic/racial Hum Biol 2008; 80: 449 -- 455. groups: support for genetic factors in pathogenesis. Leukemia 2009; 23: 10 Urayama KY, Wiencke JK, Buffler PA, Chokkalingam AP, Metayer C, Wiemels JL. 1691 -- 1697. MDR1 gene variants, indoor insecticide exposure, and the risk of childhood 3 Hayden PJ, Tewari P, Morris DW, Staines A, Crowley D, Nieters A et al. Variation in acute lymphoblastic leukemia. Cancer Epidemiol Biomarkers Prev 2007; 16: DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma. Hum Mol 1172 -- 1177. Genet 2007; 16: 3117 -- 3127. 11 Jamroziak K, Balcerczak E, Calka K, Piaskowski S, Urbanska-Rys H, Salagacka A et al. 4 Zintzaras E, Giannouli S, Rodopoulou P, Voulgarelis M. The role of MTHFR gene in Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1) multiple myeloma. J Hum Genet 2008; 53: 499 -- 507. and risk of multiple myeloma. Leuk Res 2009; 33: 332 -- 335. 5 Singh MS, Michael M. Role of xenobiotic metabolic in cancer 12 Krupoves A, Seidman EG, Mack D, Israel D, Morgan K, Lambrette P et al. epidemiology. Methods Mol Biol 2009; 472: 243 -- 264. Associations between ABCB1/MDR1 gene polymorphisms and Crohn0s 6 Tang L, Bergevoet SM, Gilissen C, de Witte T, Jansen JH, van der Reijden BA et al. disease: a gene-wide study in a pediatric population. Inflamm Bowel Dis 2009; Hematopoietic stem cells exhibit a specific ABC transporter gene 15: 900 -- 908. expression profile clearly distinct from other stem cells. BMC Pharmacol 2010; 13 Yuan HY, Chiou JJ, Tseng WH, Liu CH, Liu CK, Lin YJ et al. FASTSNP: an always 10:12. up-to-date and extendable service for SNP function analysis and prioritization. 7 Landgren O, Kyle RA, Hoppin JA, Beane Freeman LE, Cerhan JR, Katzmann JA et al. Nucleic Acids Res 2006; 34(Web Server issue): W635 -- 641. Pesticide exposure and risk of monoclonal gammopathy of undeter- 14 Lecuyer E, Hoang T. SCL: from the origin of hematopoiesis to stem cells and mined significance in the Agricultural Health Study. Blood 2009; 113: leukemia. Exp Hematol 2004; 32: 11 -- 24. 6386 -- 6391. 15 Palii CG, Perez-Iratxeta C, Yao Z, Cao Y, Dai F, Davison J et al. Differential genomic 8 Lope V, Perez-Gomez B, Aragones N, Lopez-Abente G, Gustavsson P, Plato N et al. targeting of the transcription factor TAL1 in alternate haematopoietic lineages. Occupation, exposure to chemicals, sensitizing agents, and risk of EMBO J 2010; 30: 494 -- 509.

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

Mast-cell leukemia exome sequencing reveals a mutation in the IgE mast-cell receptor b chain and KIT V654A

Leukemia (2012) 26, 1422--1425; doi:10.1038/leu.2011.354; galy, anemia and elevated levels of tryptase and histamine. Marrow published online 16 December 2011 findings were consistent with MCL (Supplementary Figures S1 and S2). The karyotype was 46, XX [20] and negative for KIT D816V mutation by PCR. The pertinent treatment details are: induction Mast cells exit the bone marrow as immature multilineage cells therapy with cladribine, cytarabine and filgrastim plus daily that circulate in the blood and subsequently undergo full dasatinib; day 21 re-induction with high-dose cytarabine plus differentiation upon reaching a target organ (reviewed in Gilfillan idarubicin; and day 41 imatinib 400 mg daily for 14 days. Bone and Tkaczyk1). Unlike other hematopoietic cells, differentiated marrow biopsies on treatment days 21, 41 and 78 revealed mast cells continue to express KIT, a transmembrane receptor with persistent MCL. The patient expired 96 days after diagnosis (see a bipartite intracellular kinase domain.1 The main ligand of KIT is Supplementary Information for a detailed summary). stem cell factor (also known as KIT ligand), which stimulates mast- Array comparative genomic hybridization was performed in cell survival, development, maturation and activation.1 Mast cells order to identify chromosomal copy number changes. Tumor and also express a high-affinity cell-surface IgE receptor, FceRI, which germline DNA were hybridized to Affymetrix 6.0 SNP arrays recognizes the Fc portion of IgE and is required for mast-cell (Affymetrix; Santa Clara, CA, USA) and data were analyzed and survival via signaling through LYN and SYK kinases.1 viewed with Nexus Copy Number V6 software (BioDiscovery, El Mast-cell leukemia (MCL) is an aggressive form of systemic Segundo, CA, USA). Array comparative genomic hybridization mastocytosis characterized by the overproliferation of atypical revealed two somatic copy number changes in the tumor: copy mast cells, promastocytes and blasts that produce, among number neutral loss of heterozygosity on chr1p36.33-p31.1 other substances, tryptase.2 MCL is often linked to somatically (81.3 Mb) and a focal hemizygous deletion (2.6 Mb) on chr10q21.1 acquired activating mutations in the KIT receptor that result in containing two genes, PCDH15 (a protocadherin) and hsa-mir-548f-1 uncontrolled ligand-independent signaling by KIT and hyper- (Figures 1a and b). Members of the hsa-mir-548 microRNA gene proliferation of mast cells (reviewed in Ustun et al.3). The KIT family are differentially expressed in cancer cells6 and have been D816V mutation causes resistance to imatinib therapy.3 Mutations implicated in tumorigenesis.7 in TET2 and NRAS have also been described in mastocytosis In order to identify tumor-specific single-nucleotide variants, patients and each of them segregates with the KIT D816V tumor and germline DNA were enriched for exonic regions mutation,4,5 suggesting that more than one lesion is required to (SeqCap EZ Human Exome Library v2.0; Roche NimbleGen, drive leukemogenesis. In order to identify novel MCL determi- Madison, WI, USA) and 76 bp paired-end-sequenced on a Genome nants, we utilized two approaches to undertake the first Analyzer IIX platform (Illumina, San Diego, CA, USA). Paired-end comprehensive study of the DNA changes in an MCL patient. reads were aligned and single-nucleotide variants in each genome This study was approved by the Institutional Review Boards of the were identified (Supplementary Information). North Shore-LIJ Health System and the Cold Spring Harbor Thirty-eight tumor-specific single-nucleotide variants were Laboratory. The patient gave written informed consent in identified in the tumor genome: 5 variants outside a coding accordance with the Declaration of Helsinki. region (intron, untranslated region or intergenic), 12 synonymous The patient, a 42-year-old female, presented with epigastric pain, coding changes and 21 non-synonymous coding changes, the fever, weight loss and urticaria. She was found to have splenome- latter of which were further evaluated. The amino acid changes

Leukemia (2012) 1402 -- 1448 & 2012 Macmillan Publishers Limited