Mismatch repair protein deficiency is a hallmark of cancers associated with Lynch syndrome and is a biomarker for response to immunotherapy. With the increasing adoption of cancer next-generation ...sequencing, there has been a movement to develop screening approaches that take advantage of the unique mutational signatures of mismatch repair-deficient tumors. Here, we develop a sequencing-based metric that distinguishes mismatch repair-deficient from mismatch repair-proficient colorectal adenocarcinomas with comparison to immunohistochemical staining. We find that a single criterion of three or more single base pair insertion or deletion mutations per megabase sequenced, occurring in mononucleotide repeat regions of four or more nucleotides, is sufficient to detect mismatch repair deficiency with 96% sensitivity and 100% specificity in a training set of 241 cancers and 96% sensitivity and 99% specificity in a validation set of 436 additional cancers. Using data from the same cohort, we also find that sequencing information from only three genes-ARID1A, KMT2D, and SOX9-is sufficient to detect mismatch repair-deficient colorectal adenocarcinomas with 76% sensitivity and 98% specificity in the validation set. These findings support the notion that targeted next-generation sequencing already being performed for clinical or research purposes can also be used to accurately detect mismatch repair deficiency in colorectal adenocarcinomas.
Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression ...alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.
Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation ...in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.
Abstract
Viruses are a major contributor to oncogenesis, causing 10-15% of human cancers. Molecular pathways involved in malignant transformation are frequently activated by genetic alterations, ...including but not limited to, somatic mutations, copy number aberrations, structural variants, and oncoviruses. Precision cancer medicine aims to classify tumors by site, histology, and molecular tests to determine an “individualized” profile of cancer alterations. However, clinical tests for these various alterations are sequential, time consuming, and use a lot of material, which is often quite limited (e.g., biopsies). Moreover, tests for the presence of viral sequence are generally performed separately to tests (such as massively parallel sequencing) to detect human genomic alterations. Here we present a hybrid capture and massively parallel sequencing approach to detect viral infection concurrently with targeted genomic analysis, which may decrease assay costs, increase sensitivity and scalability, and detect many types of alterations, thereby providing a more complete tumor genetic profile all from a single sample.
We have created a custom hybrid capture probeset for targeted Illumina sequencing to determine whether oncoviruses are present in tissue samples and also determine if the virus has integrated into the host’s genome. We have created both ‘detection’ and ‘integration’ baits for several oncoviruses, including polyomaviruses, human papilloma viruses, Epstein-Barr virus, human cytomegalovirus, Kaposi sarcoma herpesvirus, human T-lymphotropic virus, and hepatitis B virus. To distinguish between different strains of a single virus, strain-specific detection baits were created to bind to variable regions of viral genomes. The integration bait was designed to bind to regions of the viral genomes that are commonly integrated into the human genome. This baitset can also be combined with other capture panels targeting oncogenes to simultaneously determine infection and integration statuses, as well as somatic mutations, copy number and structural variants.
To detect virus presence, reads were aligned to a hybrid reference of both the human, and targeted virus genomes. Viral integration status and integration loci were determined by leveraging discordant read pairs that aligned to both the human genome and a viral genome. We have tested our techniques on tissue samples that were infected with either Merkel Cell Polyomavirus or Epstein-Barr virus, as determined using quantitative polymerase chain reaction (qPCR) or immunohistochemistry (IHC) techniques, and have successfully detected these viruses and identified viral integration loci. Overall, this viral hybrid capture probeset provides the ability to simultaneously determine a tissue sample’s infection and viral integration status alongside other somatic genomic analyses, saving both time and sample material.
Citation Format: Robert T. Burns, Samuel S. Hunter, Matthew D. Ducar, Aaron R. Thorner, James A. Decaprio, Paul Van Hummelen, Alexander Frieden, Anwesha Nag, Haley A. Coleman, Michael K. Slevin, Andrea Clapp, Samantha D. Drinan, Suzanne R. McShane, Edwin Thai, Priyanka Shivdasani, Joshua Bohannon, Johann Hoeftberger, Reuben Jacobs, Bruce M. Wollison, Neil A. Patel, Monica D. Manam, Phani Davineni, Matthew Meyerson, Laura E. MacConaill. Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2017-562
Loss of function variants in the PCDH15 gene can cause Usher syndrome type 1F, an autosomal recessive disease associated with profound congenital hearing loss, vestibular dysfunction, and retinitis ...pigmentosa. The Ashkenazi Jewish population has an increased incidence of Usher syndrome type 1F (founder variant p.Arg245X accounts for 75% of alleles), yet the variant spectrum in a panethnic population remains undetermined. We sequenced the coding region and intron-exon borders of PCDH15 using next-generation DNA sequencing technology in approximately 14,000 patients from fertility clinics. More than 600 unique PCDH15 variants (single nucleotide changes and small indels) were identified, including previously described pathogenic variants p.Arg3X, p.Arg245X (five patients), p.Arg643X, p.Arg929X, and p.Arg1106X. Novel truncating variants were also found, including one in the N-terminal extracellular domain (p.Leu877X), but all other novel truncating variants clustered in the exon 33 encoded C-terminal cytoplasmic domain (52 patients, 14 variants). One variant was observed predominantly in African Americans (carrier frequency of 2.3%). The high incidence of truncating exon 33 variants indicates that they are unlikely to cause Usher syndrome type 1F even though many remove a large portion of the gene. They may be tolerated because PCDH15 has several alternate cytoplasmic domain exons and differentially spliced isoforms may function redundantly. Effects of some PCDH15 truncating variants were addressed by deep sequencing of a panethnic population.
Stability of Disodium Adenosine Triphosphate Morell, S. A.; Lipton, S. H.; Frieden, Alexander
Science (American Association for the Advancement of Science),
09/1951, Letnik:
114, Številka:
2961
Journal Article
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