Letters to the Editor 1908 Haematopoietic and Lymphoid Tissue, 4th edn, vol. 2. World 13 Zenz T, Vollmer D, Trbusek M, Smardova J, Benner A, Soussi T Health Organization: Lyon, France, 2008, pp 229–232. et al. TP53 mutation profile in chronic lymphocytic leukemia: 7 Zainuddin N, Murray F, Kanduri M, Gunnarsson R, Smedby KE, evidence for a disease specific profile from a comprehensive Enblad G et al. TP53 Mutations are infrequent in newly diagnosed chronic lymphocytic leukemia. Leuk Res 2011; 35: 272–274. analysis of 268 mutations. Leukemia 2010; 24: 2072–2079. 8 Flordal Thelander E, Ichimura K, Collins VP, Walsh SH, Barbany G, 14 Bea S, Ribas M, Hernandez JM, Bosch F, Pinyol M, Hernandez L Hagberg A et al. Detailed assessment of copy number alterations et al. Increased number of chromosomal imbalances and high- revealing homozygous deletions in 1p and 13q in mantle cell level DNA amplifications in mantle cell lymphoma are associated lymphoma. Leuk Res 2007; 31: 1219–1230. with blastoid variants. Blood 1999; 93: 4365–4374. 9 Hartmann EM, Campo E, Wright G, Lenz G, Salaverria I, Jares P 15 Salaverria I, Zettl A, Bea S, Moreno V, Valls J, Hartmann E et al. et al. Pathway discovery in mantle cell lymphoma by integrated Specific secondary genetic alterations in mantle cell lymphoma analysis of high-resolution gene expression and copy number provide prognostic information independent of the gene expression- profiling. Blood 2010; 116: 953–961. 10 Greiner TC, Moynihan MJ, Chan WC, Lytle DM, Pedersen A, based proliferation signature. JClinOncol2007; 25: 1216–1222. Anderson JR et al. p53 mutations in mantle cell lymphoma are 16 Zenz T, Krober A, Scherer K, Habe S, Buhler A, Benner A et al. associated with variant cytology and predict a poor prognosis. Monoallelic TP53 inactivation is associated with poor prognosis in Blood 1996; 87: 4302–4310. chronic lymphocytic leukemia: results from a detailed genetic 11 Hernandez L, Fest T, Cazorla M, Teruya-Feldstein J, Bosch F, characterization with long-term follow-up. Blood 2008; 112: Peinado MA et al. p53 gene mutations and protein overexpression 3322–3329. are associated with aggressive variants of mantle cell lymphomas. 17 Young KH, Leroy K, Moller MB, Colleoni GW, Sanchez-Beato M, Blood 1996; 87: 3351–3359. 12 Olivier M, Hollstein M, Hainaut P. TP53 mutations in human Kerbauy FR et al. Structural profiles of TP53 gene mutations predict cancers: origins, consequences, and clinical use. Cold Spring Harb clinical outcome in diffuse large B-cell lymphoma: an interna- Perspect Biol 2010; 2: a001008. tional collaborative study. Blood 2008; 112: 3088–3098.

Sequence analysis of 515 kinase genes in chronic lymphocytic leukemia

Leukemia (2011) 25, 1908–1910; doi:10.1038/leu.2011.163; sequenced unidirectionally. All mutations were confirmed in published online 24 June 2011 independently generated amplicons. A total of 9003 amplicons were considered to be of high enough quality to be scored for The pathogenesis of chronic lymphocytic leukemia (CLL) mutations. To be considered eligible for scoring at least 50% of remains incompletely understood.1 Although acquired chromo- the bases in 50% of the samples for a given amplicon had to somal aberrations have been demonstrated to influence CLL have a Phred score of 20 and it further had to be judged to be of biology and clinical behavior, it remains unclear what single good quality by visual inspection. A total of 8798 (97.7%) gene defects other than p53 or ATM mutations cause or amplicons of the 9003 reported in this study had 20 or more contribute to the CLL phenotype.2 In particular, recurrent gene samples that were scored for mutations. Mutations were scored mutations that are increasingly found in other hematological in all 24 samples for 6763 (75%) of the amplicons, and only malignancies have not yet been identified in CLL. One recent 12 amplicons had as few as 12 samples that were scored. The CLL gene re-sequencing study reported the analysis of selected average Phred score for all of the bases in all of samples in all of exons of 70 tyrosine kinase genes in 95 CLL patients and amplicons reported in this study was 54.3. reported no somatically acquired mutations.3 Given the frequent Six somatically acquired mutations were identified, each identification of stereotypical immunoglobulin receptor genes in occurring once in the kinases WEE1, NEK1, BRAF, KDR, CLL, it has been suggested that antigen engagement of the B-cell MAP4K3 and TRPM6 (Table 1). Because clinically approved receptor on CLL cells serves a critical role in CLL cell survival therapeutics that target BRAF are available, we subsequently and CLL disease etiology. Further, the reduced expression of analyzed all BRAF coding exons in 120 CLL cases and exons del(13q)(14)-resident microRNAs has been implicated in early 11 and 15 selectively in an additional 130 cases (the sites for the CLL pathogenesis in a subset of cases.4 It is unknown whether vast majority of BRAF mutations affect amino acid residue 6008). CLL is driven by high-frequency recurrent gene mutations in one Primers to amplify and sequence all coding exons of BRAF and or a few genes. adjacent intronic sequences, including splice junctions, were To address this question for phosphokinases, we sequenced designed using the primer 3 program (http://frodo.wi.mit. the coding regions of 515 kinases (for a listing of kinase genes edu/primer3/) and sequence information was generated as sequenced and kinase family classification see Supplementary described.6 Somatic mutations were confirmed using paired Table S1) in DNA from CD19 þ sorted cells from 23 CLL cases.5 patient CD3 þ /buccal DNA as templates. In total, four BRAF This research was approved by the University of Michigan mutations were found, none involving BRAF amino acid residue Institutional Review Board (IRBMED #2004-0962), and written 600 (Table 1 and Supplementary Table 2). informed consent was obtained from all patients before Amino acid substitutions in WEE1, NEK1, BRAF, KDR, enrollment. CD19 þ and CD3 þ cells were purified from CLL MAP4K3 and TRPM6 were also analyzed using the CHASM samples using FACS as described.6 Clinical and molecular algorithm9,10 to estimate the probability that they impact protein characteristics of the CLL cases studied are summarized in activity in a manner relevant to oncogenicity. We trained an Supplementary Table S2. Primers used for sequence analysis of ensemble of decision trees11,12 (Random Forest) with 3285 8308 distinct coding exons from 515 kinase genes were derived likely oncogenic somatic missense mutations from the COSMIC from prior sequencing projects.7 A summary of kinase gene database13 and 3300 ‘passenger’ mutations synthetically gener- reference sequences, primer sequences and exon coverage can ated by a computer algorithm to mimic the cancer mutation be found in Supplementary Table S3. Amplicons were spectrum. To ensure an unbiased score, 13 unique BRAF amino

Leukemia Letters to the Editor 1909 Table 1 Listing of kinase gene names, transcript accession ID and mutations for the six mutated kinase genes in CLL

Gene Transcript accession Coding Tumor Nucleotide (genomic) Nucleotide (cDNA) Amino acid ID exon (protein)

BRAF CCDS5863.1 15 CLL-19 g.chr7: 139906318A4AG c.1801A4AG p.K601KE BRAF CCDS5863.1 15 CLL-32 g.chr7: 139906318A4AG c.1801A4AG p.K601KE BRAF CCDS5863.1 15 CLL-50 g.chr7: 139906333G4GC c.1786G4GC p.G596GR BRAF CCDS5863.1 11 CLL-192 g.chr7: 139934586G4GC c.1406G4GC p.G469GA WEE1 CCDS7800.1 11 CLL-10 g.chr11: 9566645A4AG c.1861A4AG p.R621RG NEK1 NM_012224 9 CLL-29 g.chr4: 170876731C4CA c.860C4CA p.P287PH KDR CCDS3497.1 30 CLL-52 g.chr4: 55787080C4CG c.4027C4CG p.L1343LV MAP4K3 CCDS1803.1 30 CLL-58 g.chr2: 39397320T4TA c.2368T4TA p.C790CS TRPM6 CCDS6647.1 22 CLL-80 g.chr9: 74627268G4GA c.2975G4GA p.G992GE Abbreviations: cDNA, complementary DNA; CLL, chronic lymphocytic leukemia. acid residue substitution mutations were removed from the CLL32 (BRAF mutants K601E) compared with the majority of training set because they occurred at the same position as cases with wild-type BRAF,18 see Supplementary Figure 2. Of mutations of interest. For each mutation, the CHASM score is note, BRAF mutant K601E has previously been demonstrated to the fraction of trees that assign it to the passenger class; the have increased catalytic activity ex vivo, and it therefore P-value measures the statistical significance of the score and is remains unsettled from this data what effects BRAF K601E corrected for multiple testing (false discovery rate). For CLL, we mutants may have on CLL cells. did not have sufficient data to estimate its spectrum, and we thus In summary, our data provide practical information about the used the better-characterized spectrum of colorectal cancer. mutational state of the CLL kinome with implications for CLL Using this algorithm, mutations in BRAF and a mutation in biology/pathogenesis. Given the substantial interest in the TRPM6 were found to be statistically significant as likely CLL research community to identify drivers and modifiers of driver mutations (Supplementary Table S4). Only the BRAF CLL pathogenesis, these data provide important albeit largely mutations occurred within the catalytic kinase domain and in negative information about the mutational state of the kinome in codons previously reported as sites of recurrent mutations in CLL, extending prior negative findings in 70 tyrosine kinase solid tumors and lymphomas (COSMIC database cite genes in CLL.3 We also identify a small subset of CLL that PMID:20952405); they may have biological roles in the affected harbors an activated RAS–BRAF pathway that could be targeted CLL cells. Mutations in the remaining kinases did not receive therapeutically. This data should motivate future genome-wide statistically significant driver scores but mutations in all these pathogenetic CLL gene discovery efforts to determine whether genes except NEK1 have previously been identified in other other, potentially targetable genetic alterations can be found in tumors (breast, colorectum, pancreas and glioblastoma multi- this disease. forme), and WEE1,14 BRAF15 and KDR16 have established roles in cancer biology. Amino acid substitutions were also analyzed using the Conflict of interest program SIFT (Sorting Intolerant from Tolerant; SIFT analysis was performed using the instructions found at http://sift.jcvi.org/). The authors declare no conflict of interest. Using this algorithm, mutations in BRAF and TRPM6 scored as ‘Affecting Protein Function’ and thus may have biological roles in Acknowledgements the affected CLL cells; mutations in the remaining four kinases scored as ‘Tolerated’. This study was supported by the Virginia and DK Ludwig Fund for Our large validation screen had identified four CLL cases with Cancer Research, National Institutes of Health Grants CA136537 heterozygous BRAF mutations (CLL19 and CLL32, both with (SNM), CA135877 (RK), CA 43460 (BV), the Translational K601K/E; CLL50 with G596G/R; and CLL192 with G469G/A). Research Program of the Leukemia and Lymphoma Society of We mapped the three BRAF mutations to an X-ray crystal America (SM), NSF Grant DBI 0845275 (RK), DOD NDSEG structure of inactive BRAF (engineered mutant V600E) in fellowship 32 CFR 168a (HC) and the University of Michigan0s complex with the RAF inhibitor BAY43-9006 (PDB ID: 1UWJ). Cancer Center Support Grant (5 P30 CA46592). We are grateful Interestingly, the mutations are close to each other and to the for services provided by the microarray core of the University of inhibitor in three-dimensional space. All three mutations occur Michigan Comprehensive Cancer Center. in important functional elements of the protein that are critical 2 þ to coordinating ligand (ATP, Mg ) interactions and catalytic 1,5 2,5 3 3 3 activity and could potentially impact inhibitor binding (Supple- X Zhang , M Reis , R Khoriaty ,YLi, P Ouillette , J Samayoa4, H Carter4, R Karchin4,MLi2, LA Diaz Jr2, mentary Figure 1). No CLL case displayed mutations of the 2 2 2 amino acid residue 600, which is the predominant BRAF VE Velculescu , N Papadopoulos , KW Kinzler , B Vogelstein2 and SN Malek3 mutation found across human tumors. Next, N-Ras and K-Ras 1 exons 2 and 3 were sequenced in 234 CLL cases and N-RAS Department of Medicine, Massachusetts General Hospital, Boston, MA, USA; codon 61 mutations were found in 2 cases (CLL155 with Q61Q/R 2The Ludwig Center for Cancer Genetics and Therapeutics and and CLL172 with Q61Q/L). Howard Hughes Medical Institute at Johns Hopkins, Using p-ERK immunoblotting of lysates from unstimulated Baltimore, MD, USA; CLL cases (N ¼ 13), we discovered substantially increased basal 3Departments of Internal Medicine, Division of p-ERK levels in CLL27 (BRAF wild type) and CLL50 and 192 Hematology and Oncology University of Michigan, (BRAF mutants G596R17 and G469A18) but not in CLL19 or Ann Arbor, MI, USA and

Leukemia Letters to the Editor 1910 4 Department of Biomedical Engineering, Institute for 9 Carter H, Chen S, Isik L, Tyekucheva S, Velculescu VE, Kinzler KW Computational Medicine, Johns Hopkins University, et al. Cancer-specific high-throughput annotation of somatic Baltimore, MD, USA mutations: computational prediction of driver missense mutations. E-mail: [email protected] Cancer Res 2009; 69: 6660–6667. 5These authors contributed equally to this work. 10 Carter H, Samayoa J, Hruban RH, Karchin R. Prioritization of driver mutations in pancreatic cancer using cancer-specific high-throughput annotation of somatic mutations (CHASM). References Cancer biol ther 2010; 10: 582–587. 11 Amit YaG D. Shape quantization and recognition with random 1 Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. trees. Neural Comput 1997; 9: 1545–1588. N Engl J Med 2005; 352: 804–815. 12 Breiman L. Random Forest. Machine Learning 2001; 45: 5–32. 2 Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger 13 Forbes SA, Tang G, Bindal N, Bamford S, Dawson E, Cole C et al. L et al. Genomic aberrations and survival in chronic lymphocytic COSMIC (the Catalogue of Somatic Mutations in Cancer): a leukemia. N Engl J Med 2000; 343: 1910–1916. resource to investigate acquired mutations in human cancer. 3 Brown JR, Levine RL, Thompson C, Basile G, Gilliland DG, Nucleic Acids Res 2010; 38 (Database issue): D652–D657. Freedman AS. Systematic genomic screen for tyrosine kinase 14 Backert S, Gelos M, Kobalz U, Hanski ML, Bohm C, Mann B et al. mutations in CLL. Leukemia 2008; 22: 1966–1969. Differential gene expression in colon carcinoma cells and 4 Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al. tissues detected with a cDNA array. Int J Cancer 1999; 82: Frequent deletions and down-regulation of micro- RNA genes 868–874. miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc 15 Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Natl Acad Sci USA 2002; 99: 15524–15529. Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and 5 Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. mismatch-repair status. Nature 2002; 418: 934. The protein kinase complement of the human genome. Science 16 Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis (New York, NY) 2002; 298: 1912–1934. K et al. Somatic mutations affect key pathways in lung 6 Kujawski L, Ouillette P, Erba H, Saddler C, Jakubowiak A, Kaminski adenocarcinoma. Nature 2008; 455: 1069–1075. M et al. Genomic complexity identifies patients with aggressive 17 Garnett MJ, Rana S, Paterson H, Barford D, Marais R. chronic lymphocytic leukemia. Blood 2008; 112: 1993–2003. Wild-type and mutant B-RAF activate C-RAF through distinct 7 Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ et al. mechanisms involving heterodimerization. Mol cell 2005; 20: The genomic landscapes of human breast and colorectal cancers. 963–969. Science (New York, NY) 2007; 318: 1108–1113. 18 Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, 8 Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Good VM et al. Mechanism of activation of the RAF-ERK signaling Mutations of the BRAF gene in human cancer. Nature 2002; 417: pathway by oncogenic mutations of B-RAF. Cell 2004; 116: 949–954. 855–867.

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

Role of BCL2L10 methylation and TET2 mutations in higher risk myelodysplastic syndromes treated with 5-Azacytidine

Leukemia (2011) 25, 1910–1913; doi:10.1038/leu.2011.170; according to the Gimema multicenter clinical trial MDS0205 published online 15 July 2011 (EudraCT number 2005-004811-31). Therapy consisted of valproic acid given orally to reach a plasma concentration Epigenetic gene regulation has a critical role during normal above 50 mg/ml and 5-azacytidine (5-AZA) at a standard dose of development and neoplastic transformation. Several tumor 75 mg/sqm daily, subcutaneously, for 7 days every 4 weeks. suppressor genes are found to be abnormally methylated and Response rate and survival for the whole patient group have silenced in hematological malignancies, and the distribution of been previously reported.5 The validation group was composed DNA methylation follows specific and distinct patterns in acute of a retrospective series of 27 patients treated at the Universita’ myeloid leukemia (AML) and myelodysplastic syndromes Cattolica Sacro Cuore (Rome, Italy) between September 2007 (MDS). However, the mechanisms mediating aberrant methyl- and June 2010. Inclusion criteria were: diagnosis of higher-risk cytosine patterns in MDS have not been defined. MDS and treatment with 5-AZA at 75 mg/sqm daily, subcuta- TET2 is a close relative of TET1 and TET3, a family of enzymes neously, 7 days for a median of 4 cycles (range 2–30 cycles). sharing two highly conserved domains, which convert DNA 5- Clinical characteristics of these patients are described in Table 1. methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The Both patient groups had not received any specific treatment gene encoding for TET2 resides at chromosome 4q24 and is before starting 5-AZA, except for supportive therapy. DNA was frequently mutated in myeloid malignancies, including about extracted from bone marrow mononuclear cells obtained from 25% of MDS, 40–50% chronic myelomonocytic leukemia, 15% all patients before 5-AZA exposure. All patients signed informed myeloproliferative neoplasms, 10–20% of AML, in particular in consent in accordance with the Declaration of Helsinki, cases secondary to MDS.1–3 Most recently, Ko et al.4 showed following institutional guidelines. that TET2 mutations compromise the hydroxymethyl-catalytic We found that TET2 was mutated in 12/38 (32%) patients with activity of the protein, with lower levels of 5-hydroxymethylcy- Int-2/high risk MDS included in the Gimema multicenter study tosine in genomic DNA from TET2-mutated samples compared MDS0205.5 Mutational analysis of TET2 coding exons 3–11 with controls. performed by PCR-based denaturing high pressure liquid We studied the prognostic role of TET2 mutations and chromatography using a WAVE-MDTMSystem (Transgenomic, methylation profiling in 38 patients treated with 5-azacytidine Omaha, NE, USA) equipped with a DNASep Cartridge, detected (Vidaza, Celgene Corp., Summit, NJ, USA) and valproic acid, five frameshift, three nonsense, six missense (two recurrent and

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