Dataset Information

MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

ABSTRACT: Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss of MMR. We performed extensive Sanger sequencing to validate both clonal and subclonal MonoSeq mutation calls. Eighty-one regions containing mononucleotide runs were sequenced in 540 primary endometrial cancers (223 with defective MMR). Our analyses revealed that the overall mutation rate in MMR-deficient tumors was 20-30-fold higher than in MMR-normal tumors. MonoSeq analysis identified several previously unreported mutations, including a novel hotspot in an A7 run in the terminal exon of ARID5B.The ARID5B indel mutations were seen in both MMR-deficient and MMR-normal tumors, suggesting biologic selection. The analysis of tumor mRNAs revealed the presence of mutant transcripts that could result in translation of neopeptides. Improved detection of mononucleotide run strand-slippage mutations has clear implications for comprehensive mutation detection in tumors with defective MMR. Indel frameshift mutations and the resultant antigenic peptides could help guide immunotherapy strategies.

SUBMITTER: Walker CJ

PROVIDER: S-EPMC5021604 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

Walker Christopher J CJ Miranda Mario A MA O'Hern Matthew J MJ Blachly James S JS Moyer Cassandra L CL Ivanovich Jennifer J Kroll Karl W KW Eisfeld Ann-Kathrin AK Sapp Caroline E CE Mutch David G DG Cohn David E DE Bundschuh Ralf R Goodfellow Paul J PJ

Human mutation 20160808 10

Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss o ...[more]

PMID: 27346418

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Project description:BACKGROUND:To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. METHODS:To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4 bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. RESULTS:Using the criteria of ≥ 1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI) = 91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI = 91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI = 80.6-97.5%) and a specificity of 97.9% (95% CI = 92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs). CONCLUSIONS:Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

Project description:BackgroundVarious approaches to calling single-nucleotide variants (SNVs) or insertion-or-deletion (indel) mutations have been developed based on next-generation sequencing (NGS). However, most of them are dedicated to a particular type of mutation, e.g. germline SNVs in normal cells, somatic SNVs in cancer/tumor cells, or indels only. In the literature, efficient and integrated callers for both germline and somatic SNVs/indels have not yet been extensively investigated.ResultsWe present SNVSniffer, an efficient and integrated caller identifying both germline and somatic SNVs/indels from NGS data. In this algorithm, we propose the use of Bayesian probabilistic models to identify SNVs and investigate a multiple ungapped alignment approach to call indels. For germline variant calling, we model allele counts per site to follow a multinomial conditional distribution. For somatic variant calling, we rely on paired tumor-normal pairs from identical individuals and introduce a hybrid subtraction and joint sample analysis approach by modeling tumor-normal allele counts per site to follow a joint multinomial conditional distribution. A comprehensive performance evaluation has been conducted using a diversity of variant calling benchmarks. For germline variant calling, SNVSniffer demonstrates highly competitive accuracy with superior speed in comparison with the state-of-the-art FaSD, GATK and SAMtools. For somatic variant calling, our algorithm achieves comparable or even better accuracy, at fast speed, than the leading VarScan2, SomaticSniper, JointSNVMix2 and MuTect.ConclusionsSNVSniffers demonstrates the feasibility to develop integrated solutions to fast and efficient identification of germline and somatic variants. Nonetheless, accurate discovery of genetic variations is critical yet challenging, and still requires substantially more research efforts being devoted. SNVSniffer and synthetic samples are publicly available at http://snvsniffer.sourceforge.net .

Project description:BackgroundMonoclonal antibodies targeting programmed death ligand 1 (PD-L1) signaling currently approved for defective mismatch repair (dMMR)/microsatellite instability high (MSI-H) tumors must be delivered by intravenous infusion. Envafolimab, a humanized single-domain anti-PD-L1 antibody fused to an Fc fragment, represents a potential advance because it can be conveniently administered subcutaneously.MethodsThis open-label, single-arm, phase 2 study evaluated the efficacy and safety of envafolimab in patients with previously treated advanced dMMR/MSI-H tumors from 25 clinical sites across China. Adults with histologically confirmed locally advanced or metastatic malignant dMMR/MSI-H solid tumors received weekly 150 mg subcutaneous envafolimab injections in a 28-day treatment cycle. The primary efficacy endpoint was the objective response rate (assessed by a blinded independent review committee). Secondary efficacy outcomes were disease control rate, duration of response, progression-free survival, and overall survival.ResultsOne hundred and three patients (65 with colorectal cancer, 18 with gastric cancer, and 20 with other solid tumors) were enrolled. Median follow-up was 11.5 months. The objective response rate was 42.7% (95% confidence interval [CI] 33.0-52.8), and the disease control rate was 66.0% (95% CI 56.0-75.1). Median duration of response was not reached; the duration of response rate at 12 months was 92.2% (95% CI 77.5-97.4). Median progression-free survival was 11.1 months (95% CI 5.5 to not evaluable). Overall survival at 12 months was 74.6% (95% CI 64.7-82.1). Sixteen patients (16%) had at least one grade 3 or 4 related treatment-emergent adverse event. No grade 5 treatment-emergent adverse events related to envafolimab were reported. Injection site reactions, all grade 1-2, were reported in nine patients (9%), but there were no infusion reactions. Eight patients (8%) had grade 3 or 4 immune-related adverse events.ConclusionsThis is the first pivotal phase 2 study to examine the efficacy and safety of a single-domain immune checkpoint antibody in the treatment of cancer. Envafolimab was effective and had acceptable safety in the treatment of previously treated advanced dMMR/MSI-H solid tumors. As the first single-domain PD-L1-targeting antibody administered by rapid subcutaneous injection, envafolimab has the potential to be a significant advance in the treatment of cancer. Trial registration ClinicalTrials.gov, NCT03667170. Registered 10 September 2018-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03667170 .

Project description:Background & aimsSome colorectal and endometrial tumors with microsatellite instability not attributable to MLH1 hypermethylation or germline mutations contain 2 or more somatic mutations in genes encoding mismatch repair (MMR) proteins. We sought to define the molecular phenotype of this newly recognized tumor subtype.MethodsFrom 2 prospective studies of the efficacy of screening for Lynch syndrome, we identified patients with colorectal and endometrial tumors who had 2 or more somatic (but not germline) mutations in genes encoding MMR proteins (double somatic). We determined the frequencies of tumor mutations in PIK3CA, BRAF, KRAS, NRAS, and PTEN by targeted next-generation sequencing and used logistic-regression models to compare them with those from patients with Lynch syndrome, MLH1-hypermethylated, or microsatellite-stable tumors. We validated our findings using independent data sets from The Cancer Genome Atlas.ResultsAmong colorectal cancer cases, we found that 14 of 21 (67%) patients with double somatic tumors also had PIK3CA mutations, compared with 4 of 18 (22%) tumors from patients with Lynch syndrome, 2 of 10 (20%) tumors with MLH1 hypermethylation, and 12 of 78 (15%) tumors with microsatellite stability (P < .0001 for patients with double somatic tumors vs other subgroups). Mutations in PIK3CA were detected in all 13 patients with double somatic endometrial cancers (P = .04 compared with other subgroups). We did not detect BRAF mutations in patients with double somatic colorectal tumors or Lynch syndrome. We found highly similar results in a validation cohort from The Cancer Genome Atlas (113 patients with colorectal tumors, 178 endometrial tumors); 100% of double somatic cases had a somatic mutation in PIK3CA (P < .0001 compared with other subgroups).ConclusionsMost patients with colorectal or endometrial tumors with 2 or more somatic (but not germline) mutations in MMR proteins also have mutations in PIK3CA; mutations in PIK3CA are detected at substantially higher frequencies in these double somatic tumors than in other microsatellite-instability subgroups. PIK3CA mutation status might be used to identify a specific group of colorectal tumors, and to select treatment or determine prognosis.

Dataset Information

MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

Publications

MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

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