Whole Exome Analysis of Myelodysplastic Syndromes Using Next-Generation Resequencing Technology

Abstract 295

Myelodysplastic syndromes (MDS) are a hetreogenous groups of myeloid neoplasms characterized by cytopenia of varying degrees and transition to acute myeloid leukemia (AML). MDS is one of the most frequent hematopoietic malignancies, particularly in the elderly. At present, allogeneic hematopoietic stem-cell transplantation is the only treatment that can induce long-term remission in MDS, but it is not applicable to most patients because of their advanced age and is associated with a high rate of treatment-related death and many complications such as chronic graft-versus-host disease. International Prognostic Scoring System (IPSS) is commonly used as a prognostic tool, but it is unsatisfactory from the point of view of genetic changes in MDS. Identification of the underlying genetic aberrations in MDS and the development of proper classification and targeted therapy are anticipated. To date, a number of gene mutations have been identified and implicated in the pathogenesis of MDS, including NRAS, TP53, RUNX1, cFMS, c-CBL, TET2, ASXL1, and more recently, IDH1, IDH2 and EZH2. However, only a part of MDS cases are able to be associated with these genetic changes. There are some remaining areas where copy number alterations and aUPDs are commonly observed and target genes have not been identified, and our knowledge about the genetic basis of MDS is thought to be still incomplete. Recently, next-generation resequencing technologies have been shown to be effective for the identification of disease-related gene and been successfully used to determine the genetic basis of some neoplastic disorders, such as AML and diffuse large B-cell lymphoma. More recently, the resequencing technology targeted for all protein-coding subsequences (i.e., whole exome analysis) has enabled cost-effective comprehensive mutation analysis of coding sequences, and has been successfully applied to identifying some Mendelian disorders. In this study, we performed a whole exome analysis of ten MDS patients in order to obtain a comprehensive registry of genetic lesions in MDS. Entire exon sequences were enriched by using SureSelect Human All Exon kit (Agilent Technologies) and were subjected to resequencing analysis using Illumina Genome Analizer IIx. On average, 12 gigabases (Gb) of sequence were generated per one tumor sample, in which more than 60% of mapped reads contained exon sequences. > 80% of exons were sequenced at the depth of >20 and average fold-coverage was >50 times. Because remission samples were difficult to obtain in MDS patients, paired CD3-positive T cells were used as a normal control. By comparing sequences in tumors and paired T cells, a number of candidate gene mutations and insertions-deletions, including those in IDH2, CKAP, TMEM146, CLEC1A, and other genes, which were validated by Sanger sequencing. Now, we are performing Sanger sequencing for some candidate genes, which were commonly mutated in more than one resequenced patients and were located within the regions of recurrent aUPDs in a cohort of 170 MDS subjects, assessing their prevalence in MDS. Our results suggested that target-capture resequencing technology is a powerful method to identify new gene mutations that are implicated in the pathogenesis of MDS.