Search

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers.Significance:Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring.See related commentary by Liggett and Sankaran, p. 889.This article is highlighted in the In This Issue feature, p. 873

Supplementary Data from Cancer-Causative Mutations Occurring in Early Embryogenesis

Supplementary Figure Legends

Detected RTK alterations and MAP2K1 mutations in sequenced colorectal carcinomas.

Comparative mRNA expression of MET exons 2-3, 14, and 20-21 from 5 patients with MET exon 14 splicing variants. All tumor samples tested had significantly and markedly lower expression of exon 14 in keeping with the transcriptional effects of the splice site mutations.

Representative photomicrographs from 3 patient samples demonstrating tumor localization on the left (H&E staining) and MET protein expression by IHC on the right. High membranous MET protein expression by IHC is present in all samples (H-score = 300).

nanoString probe sequences for MET exons 3, 4, 14, 20, and 21.

Supplemental Figure 1. The F53L, Q56P, K57N and D67N mutants were sensitive to MEK inhibition when treated with the 1 μM of the MEK inhibitor Selumetinib (AZD6244). ERK activation decreased significantly after 30 minutes of treatment.

Supplementary Methods and Figure Legends

Repertoire of non-synonymous somatic mutations in tumors and plasma DNA derived from ovarian cancer patients with BRCA1/2 germline mutations resistant/ refractory to platinum-based chemotherapy.

Supplemental Tables 1-11

List of 143 genes included in the targeted capture massively parallel sequencing assay.

Supplemental Methods 2

IMPACT_Gene_List_4_platforms

Repertoire of non-synonymous somatic mutations in plasma DNA derived from breast cancer patients with BRCA1/2 germline mutations previously treated with platinum-based chemotherapy and/or PARP inhibitors.

Sequencing statistics and non-synonymous somatic mutations identified in cfDNA and tumor tissue of ovarian and breast cancer patients included in this study.

Supplemental Figure 1. Consort diagram summarizing the molecular workflow of cases.

Variant allele frequencies of somatic mutations in the longitudinal plasma samples from breast cancer patient L031 as defined by amplicon re-sequencing.

Supplemental Figure 3. Comparison of overall and recurrence-free survival in TP53-wild type and TP53-mutant IMAs

CHS Mutations and CNA

Supplemental Methods 1

Purpose:Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.Experimental Design:A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).Results:Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R–NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001).Conclusions:This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Purpose: Genetic alterations affecting the MAPK/ERK pathway are common in lung adenocarcinoma (LAD). Early steps of the signaling pathway are most often affected with EGFR, KRAS, and BRAF mutations encompassing more than 70% of all alterations. Somatic mutations in MEK1, located downstream of BRAF, are rare and remain poorly defined as a distinct molecular subset.Experimental Design: Tumors harboring MEK1 mutations were identified through targeted screening of a large LAD cohort concurrently interrogated for recurrent mutations in MEK1, EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, and AKT. Additional cases were identified through a search of publically available cancer genomic datasets. Mutations were correlated with patient characteristics and treatment outcomes. Overall survival was compared with stage-matched patients with KRAS- and EGFR-mutant LADs.Results: We identified 36 MEK1-mutated cases among 6,024 LAD (0.6%; 95% confidence interval, 0.42–0.85). The majority of patients were smokers (97%, n = 35/36). There was no association with age, sex, race, or stage. The most common mutations were K57N (64%, 23/36) followed by Q56P (19%, 7/36), all mutually exclusive with other driver mutations in the targeted panel. Transversions G:C>T:A were predominant (89%, 31/35), in keeping with smoking-associated DNA damage. Additional less common somatic mutations were identified in the kinase domain, all of which are predicted to converge into a single interaction area based on in silico 3D modeling.Conclusions: MEK1 mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Mutations are predominantly transversions, in keeping with a strong association with smoking. Clin Cancer Res; 21(8); 1935–43. ©2014 AACR.

CHS clinical data

Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT).Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing.Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively.Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

Purpose:Chondrosarcomas are the second most common primary malignant bone tumors. Although histologic grade is the most important factor predicting the clinical outcome of chondrosarcoma, it is subject to interobserver variability. Isocitrate dehydrogenase 1 (IDH1) and IDH2 hotspot mutations were recently found to be frequently mutated in central chondrosarcomas. However, a few published articles have been controversial regarding the association between IDH1/IDH2 mutation status and clinical outcomes in chondrosarcomas.Experimental Design:We performed hotspot sequencing of IDH1 and IDH2 genes in 89 central chondrosarcomas and targeted next-generation sequencing in 54 of them, and then correlated the IDH1/IDH2 mutation status with the patient's clinical outcome.Results:Although no association was discovered between IDH mutation status and the patient's overall survival, IDH1/IDH2 mutation was found to be associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas. Genomic profiling reveals TERT gene amplification and ATRX mutation, for the first time, in addition to TERT promoter mutation in a subset (6/30, 20%) of high-grade and dedifferentiated chondrosarcomas. These abnormalities in telomere genes are concurrent with IDH1/IDH2 mutation and with CDKN2A/2B deletion or TP53 mutation, suggesting a possible association and synergy among these genes in chondrosarcoma progression. We found 21% of patients with chondrosarcoma also had histories of second malignancies unrelated to cartilaginous tumors, suggesting possible unknown genetic susceptibility to chondrosarcoma.Conclusions:IDH1/IDH2 mutations are associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas, and they tend to co-occur with TERT mutations and with CDKN2A/2B and TP53 alterations in a subset of high-grade chondrosarcomas.

Putative BRCA1/2 reversion mutations identified in cfDNA of ovarian and breast cancer patients using targeted massively parallel sequencing and results of amplicon re-sequencing of putative BRCA2 reversion mutations and other somatic mutations in cfDNA and tumor tissue of metastatic breast cancers.

PDF file - 97K

Purpose: Resistance to platinum-based chemotherapy or PARP inhibition in germline BRCA1 or BRCA2 mutation carriers may occur through somatic reversion mutations or intragenic deletions that restore BRCA1 or BRCA2 function. We assessed whether BRCA1/2 reversion mutations could be identified in circulating cell-free DNA (cfDNA) of patients with ovarian or breast cancer previously treated with platinum and/or PARP inhibitors.Experimental Design: cfDNA from 24 prospectively accrued patients with germline BRCA1 or BRCA2 mutations, including 19 patients with platinum-resistant/refractory ovarian cancer and five patients with platinum and/or PARP inhibitor pretreated metastatic breast cancer, was subjected to massively parallel sequencing targeting all exons of 141 genes and all exons and introns of BRCA1 and BRCA2. Functional studies were performed to assess the impact of the putative BRCA1/2 reversion mutations on BRCA1/2 function.Results: Diverse and often polyclonal putative BRCA1 or BRCA2 reversion mutations were identified in cfDNA from four patients with ovarian cancer (21%) and from two patients with breast cancer (40%). BRCA2 reversion mutations were detected in cfDNA prior to PARP inhibitor treatment in a patient with breast cancer who did not respond to treatment and were enriched in plasma samples after PARP inhibitor therapy. Foci formation and immunoprecipitation assays suggest that a subset of the putative reversion mutations restored BRCA1/2 function.Conclusions: Putative BRCA1/2 reversion mutations can be detected by cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and to aid the selection of patients for PARP inhibition therapy. Clin Cancer Res; 23(21); 6708–20. ©2017 AACR.

Supplemental Figure 4. Comparison of overall and recurrence-free survival in CDKN2A-wild type and CDKN2A-mutant IMAs.

Supplemental Figure 2. Detailed graphical visualizations of the F11R-NRG2 fusion.

Transcriptional activity of the androgen receptor (AR) is crucial for growth and survival of prostate cancer even upon development of resistance to androgen ablation and antiandrogen therapies. Therefore, novel therapies that can suppress AR transcriptional activity when conventional hormone therapies fail are needed. Here, we show that histone deacetylase (HDAC) inhibitors, including SAHA (vorinostat) and LBH589, which are currently being tested in clinic, could be such a therapy. HDAC inhibitors block the AR-mediated transcriptional activation of many genes, including the TMPRSS2 gene involved in fusion with ETS family members in a majority of prostate cancers. Genetic knockdown of either HDAC1 or HDAC3 can also suppress expression of AR-regulated genes, recapitulating the effect of HDAC inhibitor treatment. Whereas HDAC inhibitor treatment can lower androgen receptor protein levels in prostate cancer cells, we show that independent of AR protein levels, HDAC inhibitors block AR activity through inhibiting the assembly of coactivator/RNA polymerase II complex after AR binds to the enhancers of target genes. Failed complex assembly is associated with a phase shift in the cyclical wave of AR recruitment that typically occurs in response to ligand treatment. HDAC inhibitors retain the ability to block AR activity in castration-resistant prostate cancer models and, therefore, merit clinical investigation in this setting. The HDAC-regulated AR target genes defined here can serve as biomarkers to ensure sufficient levels of HDAC inhibition. [Cancer Res 2009;69(3):958–66]

Supplementary Figure 3 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure Legends 1-4 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure 1 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Table 1 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure 2 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Supplementary Figure 4 from Histone Deacetylases Are Required for Androgen Receptor Function in Hormone-Sensitive and Castrate-Resistant Prostate Cancer

Purpose: Chondrosarcomas are the second most common primary malignant bone tumors. Although histologic grade is the most important factor predicting the clinical outcome of chondrosarcoma, it is subject to interobserver variability. Isocitrate dehydrogenase 1 (IDH1) and IDH2 hotspot mutations were recently found to be frequently mutated in central chondrosarcomas. However, a few published articles have been controversial regarding the association between IDH1/IDH2 mutation status and clinical outcomes in chondrosarcomas. Experimental Design: We performed hotspot sequencing of IDH1 and IDH2 genes in 89 central chondrosarcomas and targeted next-generation sequencing in 54 of them, and then correlated the IDH1/IDH2 mutation status with the patient's clinical outcome. Results: Although no association was discovered between IDH mutation status and the patient's overall survival, IDH1/IDH2 mutation was found to be associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas. Genomic profiling reveals TERT gene amplification and ATRX mutation, for the first time, in addition to TERT promoter mutation in a subset (6/30, 20%) of high-grade and dedifferentiated chondrosarcomas. These abnormalities in telomere genes are concurrent with IDH1/IDH2 mutation and with CDKN2A/2B deletion or TP53 mutation, suggesting a possible association and synergy among these genes in chondrosarcoma progression. We found 21% of patients with chondrosarcoma also had histories of second malignancies unrelated to cartilaginous tumors, suggesting possible unknown genetic susceptibility to chondrosarcoma. Conclusions: IDH1/IDH2 mutations are associated with longer relapse-free and metastasis-free survival in high-grade chondrosarcomas, and they tend to co-occur with TERT mutations and with CDKN2A/2B and TP53 alterations in a subset of high-grade chondrosarcomas.

Targeted therapy for metastatic colorectal carcinoma consists of anti-EGFR therapy for patients with RAS/RAF wild-type tumors. However, the response rate remains low, suggesting the presence of alternative drivers possibly also representing potential therapeutic targets. We investigated receptor tyrosine kinase (RTK) alterations and MAP2K1 (MEK1) mutations in a large cohort of colorectal carcinoma patients studied by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and The Cancer Genome Atlas, focusing on amplifications, fusions, and hotspot mutations in RTK genes and MAP2K1. RTK gene amplifications were confirmed with FISH and immunohistochemical (IHC) staining. Among 751 colorectal carcinoma cases with next-generation sequencing data, 7% and 1% of colorectal carcinoma harbored RTK alterations and MAP2K1 hotspot mutations (n = 7), respectively. RTK-altered cases had fewer concurrent RAS/RAF mutations (P = 0.003) than RTK/MAP2K1 wild-type colorectal carcinoma. MAP2K1-mutated colorectal carcinoma showed no RAS/RAF mutations. ERBB2 (n = 32) and EGFR (n = 13) were the most frequently altered RTKs, both activated by amplification and/or hotspot mutations. Three RTK fusions were identified: NCOA4-RET, ERBB2-GRB7, and ETV6-NTRK3. Only 1 of 6 patients with an RTK or MAP2K1 alteration who received anti-EGFR and/or anti-ERBB2 therapy demonstrated stable disease; the rest progressed immediately. Overall, RTK alterations and MAP2K1 mutations occur in approximately 8% of colorectal carcinoma. In spite of the usual absence of RAS/RAF mutations, response to anti-EGFR and/or anti-ERBB2 therapy was poor in this limited group. Larger studies are warranted to further define these kinase alterations as novel therapeutic targets in colorectal carcinoma and as negative predictors of response to anti-EGFR therapy. Implications: Targetable kinase alterations were identified in a subset of advanced colorectal carcinoma patients, preferentially associated with wild-type RAS/RAF, and may predict poor response to standard anti-EGFR therapy. Mol Cancer Res; 14(3); 296–301. ©2015 AACR.

The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K–mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. Implications: This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors. Mol Cancer Res; 13(6); 1003–8. ©2015 AACR.

Purpose: Genetic alterations affecting the MAPK/ERK pathway are common in lung adenocarcinoma (LAD). Early steps of the signaling pathway are most often affected with EGFR, KRAS, and BRAF mutations encompassing more than 70% of all alterations. Somatic mutations in MEK1, located downstream of BRAF, are rare and remain poorly defined as a distinct molecular subset. Experimental Design: Tumors harboring MEK1 mutations were identified through targeted screening of a large LAD cohort concurrently interrogated for recurrent mutations in MEK1, EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, and AKT. Additional cases were identified through a search of publically available cancer genomic datasets. Mutations were correlated with patient characteristics and treatment outcomes. Overall survival was compared with stage-matched patients with KRAS- and EGFR-mutant LADs. Results: We identified 36 MEK1-mutated cases among 6,024 LAD (0.6%; 95% confidence interval, 0.42–0.85). The majority of patients were smokers (97%, n = 35/36). There was no association with age, sex, race, or stage. The most common mutations were K57N (64%, 23/36) followed by Q56P (19%, 7/36), all mutually exclusive with other driver mutations in the targeted panel. Transversions G:C>T:A were predominant (89%, 31/35), in keeping with smoking-associated DNA damage. Additional less common somatic mutations were identified in the kinase domain, all of which are predicted to converge into a single interaction area based on in silico 3D modeling. Conclusions: MEK1 mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Mutations are predominantly transversions, in keeping with a strong association with smoking. Clin Cancer Res; 21(8); 1935–43. ©2014 AACR.

Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT). Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing. Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively. Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

As genomic data accumulate at an ever-increasing rate, it is becoming evident that a comprehensive description of molecular aberrations that define individual patients’ tumors will prove useful to determine optimal therapeutic strategies. Several platforms are currently in use for clinical molecular diagnostics testing, including PCR, Sanger sequencing, restriction fragment length polymorphism analysis, and mass spectrometric genotyping (Sequenom). Massively parallel sequencing (MPS) technology has the potential to expand upon Sequenom genotyping because it allows for the identification of mutations across entire exons (rather than single base pairs) as well as copy gains, losses and gene fusions. However, for MPS to be considered a viable clinical strategy, it must first be shown to be compatible with formalin-fixed, paraffin embedded (FFPE) tissue across a range of tumor types, sizes, and cellularities. Also, it must exhibit high concordance with mutation profiles derived using the current best diagnostic methods available. To explore the clinical utility of MPS, we selected 71 surgically resected FFPE tumors that had previously been tested for approximately 100 oncogenic mutations in 8 oncogenes by Sequenom genotyping and subjected them to targeted DNA sequencing of 189 cancer-related genes. DNA was extracted from four 10-micron unstained sections from the diagnostic FFPE block (yielding a minimum of 250 nanograms per case), followed by sequencing library construction and hybridization-based capture of 3230 exons and 37 intronic intervals. Deep sequencing was performed, yielding an average coverage of >750X for uniquely-mapping reads. Sequence data were analyzed for single nucleotide variants and small insertions and deletions. High concordance was noted between Sequenom and MPS: 62 and 65 mutations were called by the two technologies, respectively, at mutually tested sites, with 60 mutation calls in common. Notably, mutant allele frequencies in these concordant calls ranged as low as 4% by MPS, highlighting the sensitivity of detection enabled by both approaches. The few discordant mutation calls exhibited no or weak evidence in the other dataset, possibly due to local tumor heterogeneity. Further, MPS revealed 73 sequence variants at additional sites of known recurrent somatic mutations and 30 loss-of-function variants in key tumor suppressor genes not tested by Sequenom. Many of these variants represent plausibly actionable mutations that could influence treatment decisions. Thus, we conclude that: (1) MPS exhibits high concordance with current best methods and is a viable strategy for clinical diagnostics, and (2) MPS captures additional variants not typically interrogated in the current clinical setting but with potential implications for the selection of approved and/or experimental targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 965. doi:1538-7445.AM2012-965