Systematic Genomic Screen for Tyrosine Kinase Mutations in CLL.

Abstract

Genomic screens have identified activating somatic mutations in tyrosine kinases in a spectrum of acute and chronic hematologic malignancies. These mutations lead to increased cellular proliferation and/or impairment of apoptosis. Chronic lymphocytic leukemia is a common chronic leukemia of adults that is thought to result at least in part from failure of apoptosis. Recent data suggest that CLL cell turnover is higher than previously thought and may correlate with disease progression, suggesting that somatic mutations that activate signal transduction may contribute to CLL pathogenesis and/or progression. The purpose of this study was to look systematically for activating mutations in protein tyrosine kinases in CLL using high-throughput DNA resequencing. 95 CLL patients were selected to encompass the range of prognostic groups defined by somatic hypermutation of the immunoglobulin heavy chain gene, ZAP-70 status and FISH cytogenetics. DNA was isolated from Ficoll separated peripheral blood mononuclear cells, and germline DNA was obtained from buccal swabs and/or saliva samples. 70 tyrosine kinases were selected for resequencing based on their involvement in lymphocyte biology, known altered gene expression in CLL, or location at a site of chromosomal gain, loss or mutation in CLL. All coding exons of ZAP-70 were included. Nested M13 tailed primers were designed to amplify and sequence the exons encoding the activation loops and JM domains. Bidirectional sequence analysis was performed using Mutation Surveyor version 2.28 (SoftGenetics). Candidate mutations were reamplified and sequenced from the original CLL DNA sample for verification; in parallel, the same exon was amplified and resequenced from the paired germline sample to assess whether non-synonymous mutations not known to be SNPs were present in the germline. A total of 178 exons were sequenced per sample. Analysis of 6.21 megabases of sequence data identified nine novel non-synonymous alterations in seven different genes; at least seven of these nine alterations are present in the germline, with the other two presently undergoing analysis. Although these non-synonymous alleles may have functional relevance to the pathogenesis of CLL, these data indicate that a significant proportion of non-synonymous alleles identified in high-throughput genomic screens are non-synonymous polymorphisms. These findings further suggest that the rate of acquired somatic mutation in the tyrosine kinome in CLL is low, and demonstrate the importance of matched germline DNA to allow assessment of candidate non-synonymous mutations in human cancer samples.