REFERENCES1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM GLOBOCAN 2008,

Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 (Internet).2010 (cited; available from: http://globocan.iarc.fr).

2 Landgren O, Weiss BM. Patterns of monoclonal gammopathy ofundetermined significance and multiple myeloma in various ethnic/racialgroups: support for genetic factors in pathogenesis. Leukemia 2009; 23:1691 -- 1697.

3 Hayden PJ, Tewari P, Morris DW, Staines A, Crowley D, Nieters A et al. Variation inDNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma. Hum MolGenet 2007; 16: 3117 -- 3127.

4 Zintzaras E, Giannouli S, Rodopoulou P, Voulgarelis M. The role of MTHFR gene inmultiple myeloma. J Hum Genet 2008; 53: 499 -- 507.

5 Singh MS, Michael M. Role of xenobiotic metabolic enzymes in cancerepidemiology. Methods Mol Biol 2009; 472: 243 -- 264.

6 Tang L, Bergevoet SM, Gilissen C, de Witte T, Jansen JH, van der Reijden BA et al.Hematopoietic stem cells exhibit a specific ABC transporter geneexpression profile clearly distinct from other stem cells. BMC Pharmacol 2010;10: 12.

7 Landgren O, Kyle RA, Hoppin JA, Beane Freeman LE, Cerhan JR, Katzmann JA et al.Pesticide exposure and risk of monoclonal gammopathy of undeter-mined significance in the Agricultural Health Study. Blood 2009; 113:6386 -- 6391.

8 Lope V, Perez-Gomez B, Aragones N, Lopez-Abente G, Gustavsson P, Plato N et al.Occupation, exposure to chemicals, sensitizing agents, and risk of

multiple myeloma in Sweden. Cancer Epidemiol Biomarkers Prev 2008; 17:3123 -- 3127.

9 Leal-Ugarte E, Gutierrez-Angulo M, Macias-Gomez NM, Peralta-Leal V, Duran-Gonzalez J, De La Luz Ayala-Madrigal M et al. MDR1 C3435T polymorphism inMexican children with acute lymphoblastic leukemia and in healthy individuals.Hum Biol 2008; 80: 449 -- 455.

10 Urayama KY, Wiencke JK, Buffler PA, Chokkalingam AP, Metayer C, Wiemels JL.MDR1 gene variants, indoor insecticide exposure, and the risk of childhoodacute lymphoblastic leukemia. Cancer Epidemiol Biomarkers Prev 2007; 16:1172 -- 1177.

11 Jamroziak K, Balcerczak E, Calka K, Piaskowski S, Urbanska-Rys H, Salagacka A et al.Polymorphisms and haplotypes in the multidrug resistance 1 gene (MDR1/ABCB1)and risk of multiple myeloma. Leuk Res 2009; 33: 332 -- 335.

12 Krupoves A, Seidman EG, Mack D, Israel D, Morgan K, Lambrette P et al.Associations between ABCB1/MDR1 gene polymorphisms and Crohn0sdisease: a gene-wide study in a pediatric population. Inflamm Bowel Dis 2009;15: 900 -- 908.

13 Yuan HY, Chiou JJ, Tseng WH, Liu CH, Liu CK, Lin YJ et al. FASTSNP: an alwaysup-to-date and extendable service for SNP function analysis and prioritization.Nucleic Acids Res 2006; 34(Web Server issue): W635 -- 641.

14 Lecuyer E, Hoang T. SCL: from the origin of hematopoiesis to stem cells andleukemia. Exp Hematol 2004; 32: 11 -- 24.

15 Palii CG, Perez-Iratxeta C, Yao Z, Cao Y, Dai F, Davison J et al. Differential genomictargeting of the transcription factor TAL1 in alternate haematopoietic lineages.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 theIgE mast-cell receptor b chain and KIT V654ALeukemia (2012) 26, 1422 -- 1425; doi:10.1038/leu.2011.354;published online 16 December 2011

Mast cells exit the bone marrow as immature multilineage cellsthat circulate in the blood and subsequently undergo fulldifferentiation upon reaching a target organ (reviewed in Gilfillanand Tkaczyk1). Unlike other hematopoietic cells, differentiatedmast cells continue to express KIT, a transmembrane receptor witha bipartite intracellular kinase domain.1 The main ligand of KIT isstem cell factor (also known as KIT ligand), which stimulates mast-cell survival, development, maturation and activation.1 Mast cellsalso express a high-affinity cell-surface IgE receptor, FceRI, whichrecognizes the Fc portion of IgE and is required for mast-cellsurvival via signaling through LYN and SYK kinases.1

Mast-cell leukemia (MCL) is an aggressive form of systemicmastocytosis characterized by the overproliferation of atypicalmast cells, promastocytes and blasts that produce, amongother substances, tryptase.2 MCL is often linked to somaticallyacquired activating mutations in the KIT receptor that result inuncontrolled ligand-independent signaling by KIT and hyper-proliferation of mast cells (reviewed in Ustun et al.3). The KITD816V mutation causes resistance to imatinib therapy.3 Mutationsin TET2 and NRAS have also been described in mastocytosispatients and each of them segregates with the KIT D816Vmutation,4,5 suggesting that more than one lesion is required todrive leukemogenesis. In order to identify novel MCL determi-nants, we utilized two approaches to undertake the firstcomprehensive study of the DNA changes in an MCL patient.This study was approved by the Institutional Review Boards of theNorth Shore-LIJ Health System and the Cold Spring HarborLaboratory. The patient gave written informed consent inaccordance with the Declaration of Helsinki.

The patient, a 42-year-old female, presented with epigastric pain,fever, weight loss and urticaria. She was found to have splenome-

galy, anemia and elevated levels of tryptase and histamine. Marrowfindings were consistent with MCL (Supplementary Figures S1 andS2). The karyotype was 46, XX [20] and negative for KIT D816Vmutation by PCR. The pertinent treatment details are: inductiontherapy with cladribine, cytarabine and filgrastim plus dailydasatinib; day 21 re-induction with high-dose cytarabine plusidarubicin; and day 41 imatinib 400 mg daily for 14 days. Bonemarrow biopsies on treatment days 21, 41 and 78 revealedpersistent MCL. The patient expired 96 days after diagnosis (seeSupplementary Information for a detailed summary).

Array comparative genomic hybridization was performed inorder to identify chromosomal copy number changes. Tumor andgermline DNA were hybridized to Affymetrix 6.0 SNP arrays(Affymetrix; Santa Clara, CA, USA) and data were analyzed andviewed with Nexus Copy Number V6 software (BioDiscovery, ElSegundo, CA, USA). Array comparative genomic hybridizationrevealed two somatic copy number changes in the tumor: copynumber neutral loss of heterozygosity on chr1p36.33-p31.1(81.3 Mb) and a focal hemizygous deletion (2.6 Mb) on chr10q21.1containing two genes, PCDH15 (a protocadherin) and hsa-mir-548f-1(Figures 1a and b). Members of the hsa-mir-548 microRNA genefamily are differentially expressed in cancer cells6 and have beenimplicated in tumorigenesis.7

In order to identify tumor-specific single-nucleotide variants,tumor and germline DNA were enriched for exonic regions(SeqCap EZ Human Exome Library v2.0; Roche NimbleGen,Madison, WI, USA) and 76 bp paired-end-sequenced on a GenomeAnalyzer IIX platform (Illumina, San Diego, CA, USA). Paired-endreads were aligned and single-nucleotide variants in each genomewere identified (Supplementary Information).

Thirty-eight tumor-specific single-nucleotide variants wereidentified in the tumor genome: 5 variants outside a codingregion (intron, untranslated region or intergenic), 12 synonymouscoding changes and 21 non-synonymous coding changes, thelatter of which were further evaluated. The amino acid changes

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

Figure 1. Hemizygous deletion of chromosome 10q21.1 detected by high-resolution SNP array in leukemic cells and independentsequence confirmation for two non-synonymous somatic tumor variants. (a) The majority of chromosome 10 has 2N copy number (CN) andheterozygous B allele frequency (BAF), except for a hemizygous deletion (b) of chromosome 10q21.1; 2.6Mb (chr10:54,305,820 --56,908,099).This region encompasses one protein coding gene, PCDH15, and one microRNA gene, hsa-mir-548f-1. (c and d) Germline (saliva DNA) andtumor (leukemic) DNA were PCR amplified for (c) KIT (top two panels) and (d) MS4A2 (bottom two panels), and capillary sequenced.The germline genotype for KIT is homozygous T/T; GTG encodes for Valine 654. The tumor is heterozygous T/C; GCG encodes for Alanine.The germline genotype for MS4A2 is homozygous C/C; CTC encodes for Leucine 188. The tumor is heterozygous C/T; TTC encodes forPhenylalanine.

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were filtered through PolyPhen-2 and annotated for whether theamino acid change is predicted to be benign or damaging to thefunction of the protein (Table 1). Nine of the non-synonymousvariants occurred in genes within the chr1p uniparental disomy(UPD) region and underwent loss of heterozygosity. However,eight of these were not evaluated further because they eitherretained the reference allele in the tumor or were present indbSNP130 at high allele frequencies and are predicted byPolyPhen-2 to be benign. The remaining 13 candidate disease-relevant missense mutations that caused protein-coding changeswere evaluated further (Table 1). Of these, 11 single-nucleotidevariants were validated independently using Sanger sequencingof PCR products generated from the original patient DNA (two ofthese are shown in Figures 1c and d). The majority of these genesare annotated as targets of cancer-associated somatic mutations(COSMIC database http://www.sanger.ac.uk), although not all ofthem in hematologic cancers; some have been linked to cancer viachanges in copy number or expression levels (Table 1 andSupplementary Information). Of greatest interest are the variantswe found in two genes, KIT and MS4A2.

As part of the clinical evaluation, this patient was screened forthe KIT D816V mutation and was found to be negative. However,exome sequencing revealed a mutation in KIT, V654A, previouslyreported in gastrointestinal stromal tumor (GIST),8 but not inleukemia. This mutation may confer resistance to imatinibtreatment as the V654A substitution directly affects the bindingof imatinib to the receptor.9 Prior knowledge of this mutationwould have altered the decision to administer imatinib, to whichthe patient showed no response. Our results suggest thatsequencing of larger regions of the KIT gene may be informative.

We also identified a missense mutation in MS4A2, the gene thatencodes the b chain of the high-affinity tetrameric cell-surface IgE(FceRI) mast-cell receptor. To date, a mutation in this gene hasonly been reported in ovarian carcinoma (COSMIC). The mutationfound here, L188F, is within the last of the four transmembranedomains and is predicted to be damaging to protein function. Inaddition to its role in immunologic responses, FceRI has antigen-independent effects, in particular enhancing mast-cell survival inthe absence of allergen (reviewed in Gilfillan and Tkaczyk1, andKraft and Kinet10). Cell-surface expression of the tetramericreceptor (a, b and two g chains) is regulated at several levelsand, once engaged, sets off a complex intracellular signalingcascade via the interaction of the b chain with LYN and SYKkinases.10 The full-length b chain acts as an amplifier of surface-tetramer expression as it promotes the maturation and transportof the tetramer subunits to the cell surface.10 In addition,tetrameric cell-surface expression is regulated by cell-type specificalternative splicing of the b chain that produces two truncatedisoforms of the protein.11,12 Each truncated isoform antagonizesthe function of the full-length protein resulting in less surfaceexpression and less downstream signaling, which limits cellproliferation/survival. This mutation identified in the FceRI b chainis an intriguing finding as the mast cell tetrameric receptorcomplex is involved in enhancing mast-cell survival.10 In principle,increasing the levels of mast-cell receptor signaling could lead toprolonged or aberrant mast-cell survival, increasing the likelihoodthat the dividing cells acquire additional mutations, includingthose that block differentiation and promote leukemogenesis. Thisis particularly relevant for mast cells as they spend a majority oftheir lives not fully differentiated.

Although we cannot rule out the possibility that the mutantb chain of the FceRI receptor identified in this patient isinactivating or inert, we hypothesize that this mutation results inincreased signaling/survival in one of the following ways: (1) themutant protein may have a higher affinity for the immaturea chain and as such can more efficiently transport it to the Golgifor processing, which would result in increased surface expression,(2) the mutant protein is mislocalized in the cell, yet can functionTa

ble

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andintracellular

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7Dam

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multiple

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7369

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OPhosphatase;

underexpressed

inlungcancer

199

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3225

GG/G

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785/18

27Dam

aging

Yes

Lu,O,B,N,

Tran

scription

2410

5575

5491

bC

C/T

T--LO

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PC

DH

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929/19

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aging

Yes

O,P

Protocadherin

1711

5986

1461

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MS4

A2

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aging

Yes

1in

Obch

ainofIgEmastcellreceptorthat

isrequired

formastcellsurvival

1281

2420

49C

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Dam

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Yes

1in

OEstablish/m

aintain

receptordistribution

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6203

4632

GG/G

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37Dam

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Yes

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nel;ove

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1944

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14Dam

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Mitogen

-activated

protein

kinase

18

Abbreviations:B,b

reast;Chr,ch

romosome;

H+L,

hem

ato+lymph;K,kidney

;L,

liver;Lu

,lung;N,central

nervo

ussystem

;ND,notdone;

NP,notpossible

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le;O,ovary;P,

pan

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UPD

regionofch

r1.

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3572

0.bHem

izyg

ousregionofch

r10

.Po

lyPhen

-2prediction:dam

agingincludes

possibly

andprobab

ly.

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

as an aberrant signal transducer in a different cellular compart-ment, similar to what has been shown for mutant FLT3 and otherreceptor tyrosine kinases13 or (3) the mutation causes the surface-bound tetramer to be locked in a constitutively active form thatcan very efficiently signal to downstream effectors---either withoutrequiring ligand or by more efficiently interacting with LYN/SYK.Future functional studies are required to determine the role of thismutant receptor in leukemogenesis. If SYK is activated, wepropose that the recently described SYK inhibitors, whichshowed efficacy in lymphoma14 and promoted differentiation ofAML cells,15 should be evaluated for their potential therapeuticvalue in MCLs harboring mutations of the components of theIgE mast-cell receptor.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSThis research was funded by The Don Monti Memorial Research Foundation (SLA andSWL) and by The Ryan Gibson Foundation (MSS). SWL is a Howard Hughes MedicalInstitute Investigator. We gratefully acknowledge Dr W Richard McCombie forguidance with sequencing, Dr Shiroo Parshad for assistance with patient medicalcare, Dr Judith Brody for bone marrow interpretation, Dr Saul Teichberg for electronmicroscopy, Dr Piers Patten for scientific discussion, Candace Schiffer and Erin Boylefor patient sample collection and the members of the Laboratory of ExperimentalImmunology of the Feinstein Institute for Medical Research for their assistance incryopreserving cells. Drs Chris Vakoc, Iris Applemann and Zhen Zhao are thanked fortheir critical reading of the manuscript.

MS Spector1, I Iossifov1, A Kritharis2,3, C He1, JE Kolitz2,3,SW Lowe1,4 and SL Allen2,3

1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA;2Department of Medicine, North Shore University Hospital-Long

Island Jewish Medical Center, Lake Success, NY, USA and3Department of Medicine, Hofstra North Shore-LIJ School of

Medicine, Hempstead, NY, USAE-mail: lowes@mskcc.org

4Current address: Cancer Biology and Genetics Program, MemorialSloan-Kettering Cancer Center, New York, NY, USA

REFERENCES1 Gilfillan AM, Tkaczyk C. Integrated signalling pathways for mast-cell activation.

Nat Rev Immunol 2006; 6: 218 -- 230.2 Valent P, Horny HP, Escribano L, Longley BJ, Li CY, Schwartz LB et al. Diagnostic

criteria and classification of mastocytosis: a consensus proposal. Leuk Res 2001;25: 603 -- 625.

3 Ustun C, Deremer DL, Akin C. Tyrosine kinase inhibitors in the treatment ofsystemic mastocytosis. Leuk Res 2011; 35: 1143 -- 1152.

4 Tefferi A, Levine RL, Lim KH, Abdel-Wahab O, Lasho TL, Patel J et al. Frequent TET2mutations in systemic mastocytosis: clinical, KITD816V and FIP1L1-PDGFRAcorrelates. Leukemia 2009; 23: 900 -- 904.

5 Wilson TM, Maric I, Simakova O, Bai Y, Chan EC, Olivares N et al. Clonal analysis ofNRAS activating mutations in KIT-D816V systemic mastocytosis. Haematologica2011; 96: 459 -- 463.

6 Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz Jr LA, Sjoblom T et al. The colorectalmicroRNAome. Proc Natl Acad Sci USA 2006; 103: 3687 -- 3692.

7 Piriyapongsa J, Jordan IK. A family of human microRNA genes from miniatureinverted-repeat transposable elements. PLoS One 2007; 2: e203.

8 Hartmann K, Wardelmann E, Ma Y, Merkelbach-Bruse S, Preussner LM,Woolery C et al. Novel germline mutation of KIT associated with familialgastrointestinal stromal tumors and mastocytosis. Gastroenterology 2005; 129:1042 -- 1046.

9 Roberts KG, Odell AF, Byrnes EM, Baleato RM, Griffith R, Lyons AB et al. Resistanceto c-KIT kinase inhibitors conferred by V654A mutation. Mol Cancer Ther 2007; 6:1159 -- 1166.

10 Kraft S, Kinet JP. New developments in FceRI regulation, function and inhibition.Nat Rev Immunol 2007; 7: 365 -- 378.

11 Donnadieu E, Jouvin MH, Rana S, Moffatt MF, Mockford EH, Cookson WO et al.Competing functions encoded in the allergy-associated FceRIbeta gene. Immunity2003; 18: 665 -- 674.

12 Cruse G, Kaur D, Leyland M, Bradding P. A novel FceRIbeta-chain truncationregulates human mast cell proliferation and survival. FASEB J 2010; 24:4047 -- 4057.

13 Choudhary C, Olsen JV, Brandts C, Cox J, Reddy PN, Bohmer FD et al. Mislocalizedactivation of oncogenic RTKs switches downstream signaling outcomes. Mol Cell2009; 36: 326 -- 339.

14 Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, LaCasce A et al.Inhibition of Syk with fostamatinib disodium has significant clinical activity innon-Hodgkin lymphoma and chronic lymphocytic leukemia. Blood 2010; 115:2578 -- 2585.

15 Hahn CK, Berchuck JE, Ross KN, Kakoza RM, Clauser K, Schinzel AC et al. Proteomicand genetic approaches identify Syk as an AML target. Cancer Cell 2009; 16:281 -- 294.

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

Reversal of T-cell tolerance in myelodysplastic syndromethrough lenalidomide immune modulationLeukemia (2012) 26, 1425 -- 1429; doi:10.1038/leu.2011.359;published online 23 December 2011

Myelodysplastic syndromes (MDS) represent a spectrum ofsenescence-dependent, hematopoietic stem cell disorders1 withdysplastic cytological features, ineffective hematopoiesis, and apropensity for transformation into acute myeloid leukemia.2

Response biomarkers to inform delegation of FDA-approvedtherapies such as the thalidomide analog lenalidomide (Revlimid,Celgene Inc., Warren, NJ, USA) are needed to improve outcomes.High rates of erythroid response to lenalidomide occur in del(5q)-MDS patients because of suppression of haplodeficient phospha-tases encoded within the proximal commonly deleted region.3 Aprevious report showing that bone marrow lymphoid aggregatesappear in association with hematological response implicatesimmune modulation in this process.4 Thalidomide, lenalidomideand other structural analogs of this drug class induce potentimmune modulation independent of del(5q), with documented

activation of T-cells and NK-cells both in vitro and in vivo inmultiple myeloma and chronic lymphocytic leukemia.5 -- 7

In an effort to understand how lenalidomide’s immunomodu-latory activity may be linked to hematological response in MDS,we evaluated T-cell activity before and after lenalidomidetreatment in vitro, and examined in vivo immune correlationrelated to hematological response based on InternationalWorking Group 2000 criteria. For this analysis, 100 patients withpathologically defined MDS were consented at Moffitt CancerCenter to evaluate immune responses. A total of 13 of these werelow-risk, treated with lenalidomide, and had samples collectedbefore and after treatment. Blood samples from an additional fivepatients with only lenalidomide pretreatment samples availablewere used for in vitro experiments, but did not contribute tohematological response analysis. Clinical characteristics andlenalidomide responses are shown in Supplementary Table 1.There was no difference between responders (R) and nonrespon-ders (NR) with regard to international prognostic score, WorldHealth Organization classification or age (P¼ 0.224).

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