Tumor and germline next generation sequencing in high grade serous cancer: experience from a large population-based testing program.
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ABSTRACT:
SUBMITTER: Care M
PROVIDER: S-EPMC7782089 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPathogenic germline mutations in BRCA1/2 constitute the majority of hereditary breast and/or ovarian cancers worldwide. Incidence and mortality rate of breast and ovarian cancers in Pakistani women is high. Thus, to establish the diagnosis for targeted therapy in Pakistan, we conducted Next-generation sequencing-based germline testing for the detection of BRCA1/2 oncogenic variants associated with breast and ovarian cancer subtype.MethodsPeripheral blood of 24 women, diagnosed with breast and epithelial ovarian cancers, was taken from the recruited cases with the consent of performing germline genetic testing. DNA was isolated from the peripheral blood and subjected to indexed BRCA Panel libraries. Targeted NGS was performed for all coding regions and splicing sites of BRCA1 and BRCA2 genes using AmpliSeq for Illumina BRCA Panel and Illumina MiSeq sequencer (placed at AFIP). Analysis of the sequencing results has been done by using Illumina bioinformatics tools.ResultsWe detected 421 variants having a quality score of 100 in all cases under study. The list of identified variants in BRCA1 and BRCA2 genes was narrowed down after filtering out those which did not pass q30 and those with a minor allele frequency (MAF) > 0.05 based on gnomAD browser. To classify these variants, clinical significance was predicted using external curated databases. As a result, we interpreted (n = 4) 16.7% pathogenic variants in BRCA1 and (n = 6) 25% variants of uncertain significance (VUS) in both genes. Descriptive statistics depicted that the age and BMI of BRCA positive cases are less than BRCA negative cases.ConclusionOur findings exhibit an initial report for the NGS based cancer genetic testing in Pakistan. This will enable us to pursue screening and diagnosis of hereditary BRCA mutation utilizing the latest state-of-the-art technique locally available in Pakistan ultimately resulting in targeted cancer therapy.
| S-EPMC8286662 | biostudies-literature
Project description:BACKGROUND:Mutation analysis for personalized treatment has become increasingly important in the management of different types of cancer. The advent of new DNA extraction protocols and sequencing platforms with reduced DNA input requirements might allow the use of cytology specimens for high-throughput mutation analysis. In this study, the authors evaluated the use of effusion fluid for next-generation sequencing-based, multigene mutation profiling. METHODS:Four specimens from each of 5 patients who had at least stage III, high-grade serous ovarian carcinoma were selected: effusion fluid; frozen tumor; formalin-fixed, paraffin embedded tumor; and matched normal blood. Frozen tumors from each patient were previously characterized by The Cancer Genomic Atlas (TCGA). DNA was extracted from all specimens and was sequenced using a custom hybridization capture-based assay. Genomic alterations were compared among all specimens from each patient as well as with mutations reported in TCGA for the same tumors. RESULTS:In total, 17 distinct somatic mutations were identified in the cohort. Ten of 17 mutations were reported in TCGA and were called in all 3 malignant specimens procured from the patients. Of the remaining 7 mutations, 2 were called in all 3 specimens, and the other 5 were sample-specific. Two mutations were detected only in the cytology specimens. Copy number profiles were concordant among the tumors analyzed. CONCLUSIONS:Cytology specimens represent suitable material for high-throughput sequencing, because all mutations described by TCGA were independently identified in the effusion fluid. Differences in mutations detected in samples procured from the same patient may reflect tumor heterogeneity.
| S-EPMC4992943 | biostudies-literature
Project description:The introduction of next generation sequencing (NGS) technologies has revolutionized the practice of Medical Genetics, and despite initial reticence in its application to prenatal genetics (PG), it is becoming gradually routine, subject to availability. Guidance for the clinical implementation of NGS in PG, in particular whole exome sequencing (ES), has been provided by several professional societies with multiple clinical studies quoting a wide range of testing yields. ES was introduced in our tertiary care center in 2017; however, its use in relation to prenatally assessed cases has been limited to the postnatal period. In this study, we review our approach to prenatal testing including the use of microarray (CMA), and NGS technology (gene panels, ES) over a period of three years. The overall diagnostic yield was 30.4%, with 43.2% of those diagnoses being obtained through CMA, and the majority by using NGS technology (42% through gene panels and 16.6% by ES testing, respectively). Of these, 43.4% of the diagnoses were obtained during ongoing pregnancies. Seventy percent of the abnormal pregnancies tested went undiagnosed. We are providing a contemporary, one tertiary care center retrospective view of a real-life PG practice in the context of an evolving use of NGS within a Canadian public health care system that may apply to many similar jurisdictions around the world.
| S-EPMC9690880 | biostudies-literature
Project description:Microsatellite instability (MSI) is a useful marker for risk assessment, prediction of chemotherapy responsiveness and prognosis in patients with colorectal cancer. Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform. Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest. We sequenced a series of 44 colorectal tumours with normal controls for five MSI loci (BAT25, BAT26, BAT34c4, D18S55, D5S346) and a second series of six colorectal tumours (no control) with two mononucleotide loci (BAT25, BAT26). In the first series, we were able to determine 17 MSI-High, 1 MSI-Low and 26 microsatellite stable (MSS) tumours. In the second series, there were three MSI-High and three MSS tumours. Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method.
| S-EPMC4377833 | biostudies-other
Project description:Somatic mutations in TP53 are a hallmark of high-grade serous ovarian cancer (HGSOC), although their prognostic and predictive value as markers is not well defined. Next-generation sequencing (NGS) can identify novel mutations with high sensitivity, that may be repurposed as potential druggable anti-cancer targets and aid in therapeutic decisions. Here, a commercial NGS cancer panel comprising 26 genes, including TP53, was used to identify new genetic markers of platinum resistance and patient prognosis in a retrospective set of patients diagnosed with epithelial ovarian cancer. Six novel TP53 somatic mutations in untreated tumors from six distinct patients diagnosed with HGSOC were identified: TP53 c.728_739delTGGGCGGCATGA (p.Met243_Met247del, in-frame insertion or deletion (INDEL); TP53 c.795_809delGGGACGGAACAGCTT (p.Gly266_Phe270del, in-frame INDEL); TP53 c.826_827GC>AT (p.Ala276Ile, missense); TP53 c.1022insT (p.Arg342Profs*5, frameshift INDEL); TP53 c.1180delT (p.Ter394Aspfs*28, frameshift INDEL); and TP53 c.573insT (p.Gln192Serfs*17, frameshift INDEL). Novel TP53 variants were validated by classical sequencing methods and their impact on protein expression in tumors explored by immunohistochemistry. Further insights into the potential functional effect of the mutations were obtained by different in silico approaches, bioinformatics tools, and structural modeling. This discovery of previously unreported TP53 somatic mutations provides an opportunity to translate NGS technology into personalized medicine and identify new potential targets for therapeutic applications.
| S-EPMC5983728 | biostudies-literature
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.
2017-04-03 | PXD003804 | Pride
Project description:Next-generation sequencing (NGS) multigene panel testing has become widespread, including the Veterans Affairs (VA), through the VA National Precision Oncology Program (NPOP). The interpretation of genomic alterations remains a bottleneck for realizing precision medicine. We sought to examine the concordance for pathogenicity determination and clinical actionability of annotation services in NPOP. Unique gene variants were generated from NGS gene panel results using two sequencing services. For each unique gene variant, annotations were provided through N-of-One (NoO), IBM Watson for Genomics (WfG), and OncoKB. Annotations for pathogenicity (all three sources) and actionability (WfG and OncoKB) were examined for concordance. Cohen's kappa statistic was calculated to measure agreement between annotation services. Among 1,227 NGS results obtained between 2015 and 2017, 1,388 unique variants were identified in 117 genes. The genes with the largest number of variants included TP53 (270), STK11 (92), and CDKN2A (81). The most common cancer type was lung adenocarcinoma (440), followed by colon adenocarcinoma (113). For pathogenic and likely pathogenic variants, there was 30% agreement between WfG and NoO (kappa, -0.26), 76% agreement between WfG and OncoKB (kappa, 0.22), and 42% agreement between NoO and OncoKB (kappa, -0.07). For level 1 drug actionability of gene variant-diagnosis combinations, there was moderate agreement between WfG and OncoKB (96.9%; kappa, 0.44), with 27 combinations identified as level 1 by both services, 58 by WfG alone, and 6 variants by OncoKB alone. There is substantial variability in pathogenicity assessment of NGS variants in solid tumors by annotation services. In addition, there was only moderate agreement in level 1 therapeutic actionability recommendations between WfG and OncoKB. Improvement in the precision of NGS multigene panel annotation is needed.
| S-EPMC7446349 | biostudies-literature
Project description:Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.
| S-EPMC4820127 | biostudies-literature
Project description:BackgroundPreimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.MethodsPGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45-50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.ResultsAll nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.ConclusionsNGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.
| S-EPMC10239584 | biostudies-literature
Project description:We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10-3). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2, where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.
| S-EPMC5584218 | biostudies-literature