Abstract
BackgroundGermline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques.MethodsTwenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA).ResultsNGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel.ConclusionThis study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.
Highlights
Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer
Development of the 25-gene panel Twenty-five genes associated with hereditary cancer syndromes [9] were selected for development of a panel to screen for syndromes associated with 8 primary types of cancer using next generation sequencing (NGS)
Initial sensitivity and specificity assessment of BRCA1 and BRCA2 sequencing For the initial sequencing assessment, NGS identified 15,877 variants, while prior Sanger sequencing identified 15,878 variants among 1864 anonymized samples from patients who had previously undergone BRCA1 and BRCA2 testing
Summary
Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. The detection of germline mutations in blood samples from patients can be extremely useful for identifying patients at high risk of developing a malignancy. This genetic information can be used to guide treatment discussion and genetic counseling for at-risk family members. The utility of the Sanger method is limited when analyzing multiple genes from several patients simultaneously because tests for targeted genes often need to be conducted serially instead of simultaneously [5] This upfront selectivity combined with incomplete follow-through in reflex testing may reduce the sensitivity of the testing overall
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