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.