Letters to the Editor 1124 most of them the study population does not reach 200 cases. S Pigullo1, R Haupt2, C Dufour1, P Di Michele1, MG Valsecchi3, G Basso4, C Rizzari5, A Biondi5, M Lanciotti1 Interestingly, studies with larger cohorts give results similar to 1 ours. In a study of 197 patients (163 Caucasians and 34 blacks) Hematology Unit, Department of Pediatric Hemato- Chen et al5 found a higher prevalence of the double null GSTT1 Oncology, G Gasline Children’s Hospital, Genova, Italy; 2Epidemiology and Biostatistics Section, Scientific Directorate, and GSTM1 genotype among black children. In a large series of 8 G Gaslini Children’s Hospital, Genova, Italy; 651 (616 Caucasians and 35 black) ALL patients Davies et al 3Section of Medical Statistics, University of Milano-Bicocca, found GSTT1 and GSTM1 gene frequencies similar to those of Monza, Italy; controls, but they do not confirm the data on black children. In 4Pediatric Onco-Hematology Clinic, University of Padova, contrast a Canadian study on 177 cases showed an increased Padova, Italy; frequency of GSTM1 null genotype in children with ALL.9 5Centro Ricerca Tettamanti, Pediatric Clinic, University of A smaller number of studies have been reported on the Milano-Bicocca, Monza, Italy possible influence of GSTP1 genotype on childhood ALL. All of E-mail: [email protected] them had negative results; only one on 278 cases reported a role of GSTP1 in the susceptibility to childhood ALL in individuals carrying the variants Val105/Ala114 but did not shown clear results on the role of each variant.10 In these studies, positive References results were obtained in a French-Canadian population, suggesting that the influence of GSTs on the risk of leukemia 1 Salinas AE, Wong MG. Glutathione transferases: a review. Curr Med could differ among populations, or a different exposure could be Chem 1999; 6: 279–309. involved in leukemogenesis. 2 Rebbeck TR. Molecular epidemiology of the human Glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Several factors must be considered in the design of a reliable Cancer Epid Biomark Prevent 1997; 6: 733–743. case–control study. A large sample size with an adequate power 3 Zimniak P, Nanduri B, Pikula S, Bandorowicz-Pikula J, Singhal SS, is one of the most important factors. The choice of the control Srivastava SK et al. Naturally occurring human Glutathione population is also crucial because the possible different S-transferase GSTP1 isoforms with isoleucin and valin in position exposure to environmental toxicants should be considered. All 105 differ in enzymatic properties. Eur J Biochem 1994; 224: these factors were taken in account in our study and, in order to 893–899. 4 Ye Z, Song H. Glutathione s-transferase polymorphisms rule out geographical differences in genetic backgrounds and in (GSTM1, GSTP1 and GSTT1) and the risk of acute leukaemia: a environmental exposure to carcinogens, the normal controls systematic review and meta-analysis. Eur J Cancer 2005; 41: were matched not only for gender and age, but also for 980–989. geographical area of residence. This is the first study that takes 5 Chen CL, Liu Q, Pui CH, Rivera GK, Sandlund JT, Ribeiro R et al. into consideration also the residence area as a criterion for the Higher frequency of Glutathione S-transferase deletions in black selection of controls. children with acute lymphoblastic leukemia. Blood 1997; 89: 8 1701–1707. In addition this is the second study after that of Davies et al 6 Harries LW, Stubbins MJ, Forman D, Howard GC, Wolf CR. that considered the possible influence of genotype on the Identification of genetic polymorphisms at the Glutathione patients’ clinical characteristics, confirming the lack of associa- S-transferase Pi locus and association with susceptibility to tion between ALL subtype and GST polymorphism. bladder, testicular and prostate cancer. Carcinogenesis 1997; 18: In conclusion, this study represents one of the largest studies 641–644. on GSTT1 and GSTM1 genotypes, and the largest study on 7 Sala S, Lanciotti M, Valsecchi MG, Di Michele P, Dufour C, Haupt R et al. Genotypes of the glutathione S-transferase super- GSTP1 genotypes in children with ALL. Our data do not support family do not correlate with outcome of childhood acute a role of GST genotypes in genetic susceptibility to ALL. Our lymphoblastic leukaemia. Leukemia 2003; 17: 981–983. findings, although negative, contribute to give a definitive 8 Davies SM, Bhatia S, Ross JA, Kiffmeyer WR, Gaynon PS, Radloff answer to the question on the role of GST genotype in childhood GA et al. Glutathione S-transferase genotype, genetic susceptibility, ALL etiology at least in the Caucasian population. and outcome of therapy in childhood acute lymphoblastic leukemia. Blood 2002; 100: 67–71. 9 Krajinovic M, Labuda D, Richer C, Karimi S, Sinnett D. Suscept- Acknowledgements ibility to childhood acute lymphoblastic leukemia: influence of CYP1A1, CYP2D6, GSTM1 and GSTT1 genetic polymorphisms. Blood 1999; 93: 1496–1501. This work was supported by Compagnia di S. Paolo; Fondazione 10 Krajinovic M, Labuda D, Sinnett D. Glutathione S-transferase P1 CARIGE, ERG S.p.A. We thank Dr Anna Capurro for critical genetic polymorphisms and susceptibility to childhood acute reading of the manuscript. lymphoblastic leukaemia. Pharmacogenetics 2002; 12: 655–658. Expression and mutation status of candidate kinases in multiple myeloma Leukemia (2007) 21, 1124–1127. doi:10.1038/sj.leu.2404612; chain locus is found in 15% of patients. FGFR3 mutations, published online 8 March 2007 activating the receptor, are found in both primary patient samples and cell lines, supporting the role of FGFR3 signaling in the pathogenesis of the disease. In recent years, several other Kinase hyperactivity often results in deregulation of cellular kinases have also been reported to be somatically mutated in pathways involved in proliferation and survival. In multiple hematologic and solid organ malignancy. Furthermore, mutated myeloma (MM), for example, the translocation of FGFR3, a kinases are ideal targets for therapeutics. These observations receptor tyrosine kinase (RTK), to the immunoglobulin heavy have led us (and others) to attempt kinome-wide sequencing in Leukemia Letters to the Editor 1125 the hope of identifying other, as yet unidentified, somatic IDs on Affymetrix U133 Plus 2 corresponding to known kinases. mutations in MM by focusing on RTKs. Of note, major Analysis of these kinases in healthy controls, MGUS, smoulder- sequencing efforts are currently being launched that may in ing MM (SMM), MM and MM cell lines revealed significant part be duplicative. Subsequently, we report here our data to differential expression in 185 unique kinases (Supplementary date derived from the sequencing of the kinase domains of 31 Information). Supervised hierarchical clustering indicated by the candidate genes and one kinase-regulatory gene CKS1B. heatmap in Figure 2b grouped the normals, cell lines and patient Although we did not find recurrent mutation in genes other samples as separate clusters. When normal plasma cells were than FGFR3, we have noted non-synonymous sequence compared with the malignant counterparts of MGUS, SMM or differences in ROR2, EGFR, GUCY2F and EPHB4 that are not MM, 64% (65/102) of kinases showed at least 2.84-fold higher present in dbSNP or pooled DNA from ethnically diverse expression in the disease state whereas 36% of the kinases (37/ populations that may now be further studied in larger cohorts of 102) showed diminished expression. From this list of upregu- MM patients for any pharmacogenetic significance. lated RTKs, seven RTKs were identified for sequencing: JAK3, Our general approach is detailed in Figure 1. Genomic DNA MET, KIT, CSK, ABL1, FGFR3 and ROR2. from 32 MM cell lines, and from CD138 þ purified bone marrow ROR2 expression showed a pattern reminiscent of the gene plasma cells from 24 MM patients (including 12 hyperdiploid), ‘spikes’ observed in translocated FGFR3, albeit at a level 10-fold eight monoclonal gammopathy of undetermined significance less than FGFR3 (Figure 3). Like FGFR3 mutations, ROR2 allelic (MGUS) and five plasma cell leukemia (total of 37 samples) mutations cause skeletal malformations in that inactivating were used in the study. Patient samples were obtained from mutations result in autosomal dominant brachydactyly type B3 Mayo Clinic (Scottsdale, AR, USA) and Princess Margaret and autosomal recessive Robinow syndrome.4 ROR2 is a Hospital (Toronto, ON, Canada) after informed consent. When negative regulator of Wnt signaling5 and its expression is linked necessary, a whole genome amplification method (GenomiPhi, in patients to the absence of bone disease (not shown). The GE Health Care, Piscata-way, NJ, USA Pharmacia) was used to parallel skeletal phenotype of germline mutations in both FGFR3 amplify a limited amount of patients’ genomic DNA. and ROR2, together with the spiked and differential pattern of PCR amplification of genomic DNA was performed in 96-well gene expression noted above, led us to consider ROR2 as strong plates using primers that have been previously described1,2 or candidate for kinase mutation screening. which were designed using the Primer3 program through the As a positive control, sequencing of the FGFR3 kinase domain Canadian Bioinformatics Resource web portal (cbr-rbc.nrc- of MM cell lines confirmed the known K650E FGFR3 mutation cnrc.gc.ca/index_e.php). Aliquots of PCR products were se- in OPM1 and OPM2 (Figure 2c). Interestingly, however, we also quenced on 96-well plates without purification using nested identified a novel heterozygous non-synonymous mutation sequencing primers. Sequence files generated from ABI 3730XL S433C (AAG to GAG) in KMS12PE, a cell line derived from DNA analyzer were analyzed using SeqScape software (Applied pleural effusion of an MM patient (Figure 2c). This mutation is Biosystems, Foster City, CA, USA) (ABI) and sequence changes not present in the bone marrow-derived cell line from the same were visually and manually inspected.