Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

Endometrial cancer is one of the tumor types in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur. It is known to possess mutations frequently in the Ras-PI3K (phosphatidylinositol 3 0 -kinase) pathway. We performed a comprehensive genomic survey in 31 endometrial carcinomas with paired DNA for chromosomal imbalances (25 by the 50K and 6 by the 250K single-nucleotide polymorphism (SNP) array), and screened 25 of the 31 samples for MSI status and mutational status in the Ras-PI3K pathway genes. We detected five or more copy number changes (classified as CIN-extensive) in 9 (29%), 1 to 4 changes (CINintermediate) in 17 (55%) and no changes (CIN-negative) in 5 (16%) tumors. Positive MSI was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%), and multivariate analysis showed that CIN-extensive is an independent poor prognostic factor. SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN. In addition to eight (26%) tumors with PTEN deletions, we detected chromosomal imbalances of NF1, K-Ras and PIK3CA in four (13%), four (13%) and six (19%) tumors, respectively. In all, 7 of the 9 CIN-extensive tumors harbor deletions in the loci of PTEN and/or NF1, whereas all the 10 MSI-positive tumors possess PTEN, PIK3CA and/or K-Ras mutations. Our results showed that genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability, and suggest that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas.

Genome-wide genotyping diagnosis may represent a useful approach for distinguishing between SPM and DP in synchronous endometrial and ovarian carcinomas.

[Objectives]: Activation of Ras/PI3-kinase (PI3K) pathway plays a direct causal role in human cancer. Oncogenic mutant Ras remains in active state, and activates various Ras effectors, including PI3K pathway. Ras/PI3K pathway activating mutations are very frequent in endometrial cancer, as reported at 35-55% for PTEN, 25-36% for PIK3CA and 10-20% for K-Ras. NF1 is a negative regulator of Ras and its alterations have been reported in several types of tumors. Although LOH at PTEN locus (10q23.3) is reported at 15-30%, other chromosomal imbalances involved in the Ras/PI3K pathway are not well characterized in endometrial cancer. In this study, we focused on chromosomal copy number alterations (CNAs), including copy number neutral (CNN) LOH, involved in the Ras/PI3K pathway in endometrial carcinomas. [Materials and Methods]: Under the approval of institutional ethical committees, we performed 250K or 50K Single Nucleotide Polymorphism (SNP) arrays in 31 endometrial carcinomas with paired DNA and examined allele-specific CNAs at K-Ras, NF1, PIK3CA and PTEN. [Results]: We detected deletions of PTEN locus in eight out of 31 (26%) tumors; one with homozygous deletions (HD), four with CNN LOH and three with LOH without gain of the opposite allele. In addition to one HD (3%) and three LOH (10%) at NF1 locus, we found gains at the loci of PIK3CA (3q26.3) in four (13%) tumors and K-Ras (12p12.1) in four (13%) tumors. Taken together, we detected 12 (39%) tumors with one or more CNAs involved in the Ras/PI3K pathway. All the four tumors with NF1 deletions coexist with other CNAs involved in the Ras/PI3K pathway (Table). [Conclusion]: Our data unveiled CNN LOH of PTEN and homozygous deletions of PTEN and NF1, which had not been reported by conventional CGH analysis. Various types of CNAs might play roles in activation of the Ras/PI3K pathway and targeting the Ras-PI3K pathway might be effective in endometrial carcinomas with the Ras-PI3K related CNAs.Table.Coexistent CN alterations in the Ras/PI3K pathwayPTEN (Loss)PIK3CA (Gain)K-Ras (Gain)NF1 (Loss)LOH Gain LOH  LOH Gain LOHLOHGain HDLOHGainGainLOH Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3139.

[Objectives]: Endometrial cancer is one of the tumor types, in which either chromosomal instability (CIN) or microsatellite instability (MSI) may occur, and is known to possess mutations frequently in Ras-PI3K (phosphatidylinositol 3’-kinase) pathway. In this study, we attempted to clarify prognostic impact of genomic instability, determined by CIN and MSI status, and figure out the relationship between alterations in the Ras-PI3K pathway genes and the status of genomic instability. [Materials and Methods]: Under the approval of institutional ethical committees, we analyzed allele-specific chromosomal copy number alterations by SNP typing arrays in 31 endometrioid endometrial adenocarcinomas with paired DNA (25 by the 50K and six by the 250K probes). We also screened 25 of the 31 samples for MSI status with five microsatellite markers, and 22 samples for mutations in PIK3CA (exon 9 and 20), PTEN (exons 1-8) and K-Ras (exon1 and 2). Survival curves were constructed using the Kaplan-Meier method and compared with a log-rank test. [Results]: We detected five or more copy number changes (classified as CIN-extensive) in nine (29%), one to four changes (CIN-intermediate) in 17 (55%) and no changes (CIN-negative) in five (16%) tumors. Positive MSI was detected in ten (40%) of the 25 tumors and was less common in CIN-extensive tumors (14%), compared with CIN-intermediate/negative tumors (50%). CIN-extensive was significantly poor prognostic (p=0.0034), and the prognosis was shown to be independent from any other clinicpopathological characteristics by multivariate analysis. In contrast, positive MSI showed a trend to be a favorable prognostic factor (p=0.069), in spite of the association with vascular invasion (p=0.049). SNP array analysis unveiled copy number neutral LOH at 54 loci in 13 tumors (42%), including four at the locus of PTEN, and homozygous deletions at five regions in three tumors, including the locus of PTEN and NF1. Totally we detected eight (26%) tumors with PTEN deletions, and four (13%) with NF1 deletions. We identified mutations of PTEN, PIK3CA and K-Ras in 16 (73%), 6 (27%) and 2 (9%) out of 22 tumors, respectively. Seven (78%) of the nine CIN-extensive tumors harbor deletions at the loci of PTEN and/or NF1, with less frequent PTEN mutations (22%). On the other hand, PTEN mutations were detected in all the ten MSI-positive tumors, five of which coexist with PIK3CA and/or K-Ras mutations. We detected no deletions of NF1 and only one (10%) LOH of PTEN in the ten MSI-positive tumors. [Conclusion]: Our results demonstrated that the degree of CIN is a useful biomarker for prognosis in endometrial carcinomas. In addition, genomic alterations in the Ras-PI3K pathway are remarkably widespread in endometrial carcinomas, regardless of the type of genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2152.