Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

Abstract

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Novel MXD4–NUTM1 fusion transcript identified in primary ovarian undifferentiated small round cell sarcoma

“…The sites of the germline variants identified in the matched normal sample were examined for beta allele frequency (BAF) profiles. A threshold of ≥6 copies for gene amplification was used 23. Cancer‐associated genes that were annotated in OncoKB24 were focused in this study.…”

The Spectrum of Clinical Utilities in Molecular Pathology Testing Procedures for Inherited Conditions and Cancer

“…Now, mutation status has become a part of the disease name of an increasing number of cancers because they predict recurrence or have implications for targeted therapy, sometimes for multiple mutations (e.g., BRAF, MEK, RAS-mutant melanoma [28][29][30] ). MPS can interrogate multiple gene regions that have been characterized as mutational hot spots, providing an efficient method for identifying a number of somatic mutations known to be important cancer drivers 17,[31][32][33] . Just as important as diagnostic efficiency is the limitation presented by specimen type, which can impact the number of tumor cells and/or quantity of DNA available for analysis.…”

Assessing Copy Number Alterations in Targeted, Amplicon-Based Next-Generation Sequencing Data

“…Algorithms for assessing CNAs have been developed for NGS protocols that are based on hybridization-capture, whether in the setting of whole-exome sequencing 12 or targeted sequencing. 1,3,5,8 In contrast, little work has been done on CNA assessment in NGS data from amplicon-based libraries.…”