Validation and Implementation of Targeted Capture and Sequencing for the Detection of Actionable Mutation, Copy Number Variation, and Gene Rearrangement in Clinical Cancer Specimens

Abstract

Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

“…We analyzed the sequencing depths at KRAS c. 35 (p.G12, routinely tested in NSCLC and GI malignancies), BRAF c.1799 (p.V600, routinely tested in melanoma and GI malignancies), and all bases in EGFR exon 19 (for exon 19 deletions) and EGFR c.2573 (p.L858), which were routinely tested in NSCLC. The minimum coverage of these four select routinely tested bases or regions in the pilot experiment was 41,909Â, 16,426Â, 8565Â, and 8687Â for BRAF c.1799, KRAS c.35, any base in EGFR exon 19, and EGFR c.2573, respectively.…”

“…Other NGS validation studies have shown high concordance between novel variants and other confirmatory methods. 35,36 Conversely, the true sensitivity of the TST NGS assay is difficult to define because one or more samples may have harbored masked variants. False-negative results can occur secondary to the capture methods or, more often, the alignment and variant calling algorithms.…”

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.…”

Review of Clinical Next-Generation Sequencing

“…This is especially important for any oncology assay in which tumor percentage and heterogeneity affect the allele frequency, but it is also relevant for the ability to reliably detect mosaicism in an assay to detect inherited disorders. 31,80,81 During the validation, metrics should be defined to assess the quality of a test run and criteria for repeated testing established. 61,[82][83][84] These metrics may include cutoffs for the insert sizes after library preparation; criteria for assessing adequate target enrichment; library concentration parameters for various steps; expected performance of controls; and metrics for sequencing performance such as clustering, base and mapping quality scores, error rates, GC bias, transition/ transversion ratios, total number of sequencing reads, and coverage.…”