Rmal DNA. Three variants were bone fide somatic mutations, present in the tumor DNA but absent from the matched normal DNA. The somatically mutated genes were ESCO1 (establishment of cohesion 1 homolog 1 (S. cerevisiae)), CHTF18 (chromosome transmission fidelity factor 18 homolog (S. cerevisiae)), and MRE11A (meiotic recombination 11 homolog A (S. cerevisiae)); each gene was mutated in 4 (1 of 24) of NEECs in the CI 1011 discovery screen. Although we found no evidence for somatic mutations in the remaining 18 candidate CIN genes, it is important to acknowledge that our discovery screen has insufficient power to detect all somatic mutations present in NEECs. We estimate that in a screen of 24 NEECs, the power to detect genes that are somatically mutated in 5 , 10 or 15 of all NEECs is 71 , 92 , and 98 respectively. We next sought to more precisely determine the frequency and spectrum of somatic mutations in ESCO1, CHTF18, and MRE11A in endometrial cancer. To do this, we performed a prevalence screen in which we resequenced the coding exons and splice sites of the three genes from an additional 28 serous tumors, 13 clear cell tumors, and 42 endometrioid tumors, unselected for MSI status. In the combined discovery and prevalence screens, we uncovered nonsynonymous somatic mutations within ESCO1, CHTF18, and MRE11A in, respectively, 3.7 (4 of 107), 1.9 (2 of 107), and 1.9 (2 of 107) of endometrial tumors (Table 2 and Figure S2). Overall, 7.7 (5 of 65) of NEECs and 2.4 (1 of 42) of EECs had somatic mutations in one or more of the three genes. Compared 18204824 to known MedChemExpress Madrasin consensus cancer genes with established roles in endometrial cancer, and to significantly mutated cancer genes, ESCO1, CHTF18, and MRE11A were infrequently mutated (Figure S3, Figure S4, Figure S5) [44], [52], [53], [54], suggesting that these three genes are either rare pathogenic driver genes for endometrial cancer or that they are non-pathogenic genes that have acquired passenger mutations. Immunoblotting confirmed the expression of MRE11A and CHTF18 in panel ofPrimer design and PCR amplificationPrimer pairs were designed, using published methods [47], to target 97.4 (458 of 470) of all exons of the 21 genes in the mutation discovery screen (Table S2), and all exons of the three genes in the mutation prevalence screen (Table S3). PCR conditions are available on request.Nucleotide sequencingPCR products were subjected to bidirectional Sanger sequencing using M13 primers and the BigDye Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were run on ABI 3730xl DNA Analyzers (Applied Biosystems). Sequence trace quality was assessed with the base-calling program, Phred [48], [49]. All traces were included in the subsequent analysis, since deletion-insertion polymorphisms can mimic poor quality data from a Phred-quality measure, but may contain valid sequence data. All sequences for a given primer pair were assembled using Consed [50]; overlapping amplimers were assembled separately to allow independent cross-validation of calls in overlapping regions. Sequence variants, including singlenucleotide differences and short (,100 base pair) insertions and deletions, were identified using PolyPhred v6.11 [51] and an inhouse algorithm (DIPDetector) optimized for improved sensitivity in finding insertions and deletions from aligned trace data. DIPDetector analyzes Sanger sequencing traces and predicts insertions and deletions by first examining read alignments.Rmal DNA. Three variants were bone fide somatic mutations, present in the tumor DNA but absent from the matched normal DNA. The somatically mutated genes were ESCO1 (establishment of cohesion 1 homolog 1 (S. cerevisiae)), CHTF18 (chromosome transmission fidelity factor 18 homolog (S. cerevisiae)), and MRE11A (meiotic recombination 11 homolog A (S. cerevisiae)); each gene was mutated in 4 (1 of 24) of NEECs in the discovery screen. Although we found no evidence for somatic mutations in the remaining 18 candidate CIN genes, it is important to acknowledge that our discovery screen has insufficient power to detect all somatic mutations present in NEECs. We estimate that in a screen of 24 NEECs, the power to detect genes that are somatically mutated in 5 , 10 or 15 of all NEECs is 71 , 92 , and 98 respectively. We next sought to more precisely determine the frequency and spectrum of somatic mutations in ESCO1, CHTF18, and MRE11A in endometrial cancer. To do this, we performed a prevalence screen in which we resequenced the coding exons and splice sites of the three genes from an additional 28 serous tumors, 13 clear cell tumors, and 42 endometrioid tumors, unselected for MSI status. In the combined discovery and prevalence screens, we uncovered nonsynonymous somatic mutations within ESCO1, CHTF18, and MRE11A in, respectively, 3.7 (4 of 107), 1.9 (2 of 107), and 1.9 (2 of 107) of endometrial tumors (Table 2 and Figure S2). Overall, 7.7 (5 of 65) of NEECs and 2.4 (1 of 42) of EECs had somatic mutations in one or more of the three genes. Compared 18204824 to known consensus cancer genes with established roles in endometrial cancer, and to significantly mutated cancer genes, ESCO1, CHTF18, and MRE11A were infrequently mutated (Figure S3, Figure S4, Figure S5) [44], [52], [53], [54], suggesting that these three genes are either rare pathogenic driver genes for endometrial cancer or that they are non-pathogenic genes that have acquired passenger mutations. Immunoblotting confirmed the expression of MRE11A and CHTF18 in panel ofPrimer design and PCR amplificationPrimer pairs were designed, using published methods [47], to target 97.4 (458 of 470) of all exons of the 21 genes in the mutation discovery screen (Table S2), and all exons of the three genes in the mutation prevalence screen (Table S3). PCR conditions are available on request.Nucleotide sequencingPCR products were subjected to bidirectional Sanger sequencing using M13 primers and the BigDye Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were run on ABI 3730xl DNA Analyzers (Applied Biosystems). Sequence trace quality was assessed with the base-calling program, Phred [48], [49]. All traces were included in the subsequent analysis, since deletion-insertion polymorphisms can mimic poor quality data from a Phred-quality measure, but may contain valid sequence data. All sequences for a given primer pair were assembled using Consed [50]; overlapping amplimers were assembled separately to allow independent cross-validation of calls in overlapping regions. Sequence variants, including singlenucleotide differences and short (,100 base pair) insertions and deletions, were identified using PolyPhred v6.11 [51] and an inhouse algorithm (DIPDetector) optimized for improved sensitivity in finding insertions and deletions from aligned trace data. DIPDetector analyzes Sanger sequencing traces and predicts insertions and deletions by first examining read alignments.
