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About the bone marrow transplantation that high dose chemotherapy and total-body irradiation (TBI) are used for as conditioning regimen, a late toxicity may become the problem in the long-term survival patient. One of the toxicities which has been implied to be associated with TBI is secondary cacinogenesis. Between June 1995 and December 2010, 370 patients who were undergoing allogeneic hematopoietic stem cell transplantation using a TBI-based regimen at our department, were the subjects of this study. Eleven secondary cancers occurred in 10 patients. The median time from transplantation to diagnosis of a secondary cancer was 6.8 years. In this analysis, the cumulative incidence rate of secondary cancer at 5 and 10 years was 2.15% and 6.46%, respectively after TBI in our institution.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-424) contains supplementary material, which is available to authorized users.

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. Disclosures: No relevant conflicts of interest to declare.

4011 Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect-system® (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with >20 depths of reading. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations. Disclosures: No relevant conflicts of interest to declare.