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.

Figure 1

Scheme showing the data analysis pipeline. QA, quality assurance.

Figure 2

Copy number variants (CNVs) detection directly by UW-OncoPlex sequencing. Adjusted log₂ ratio of read depth of sequencing data are plotted for individual baits (y axis) across captured genomic regions (x axis). Overlaid horizontal black lines indicate genomic microarray results in the target regions for the same sample, illustrating high concordance of CNV detection between UW-OncoPlex and genomic microarray. Amplifications or deletions of specific genes are indicated (boxes), with corresponding array CGH calls highlighted (arrowheads). Depicted are examples from a melanoma (FFPE06) and colon cancer sample (FFPE02).

Figure 3

Novel gene rearrangements detected. Chromosomal ideograms, gene-level schematics of events, and confirmatory bidirectional Sanger sequencing are displayed for three chromosomal rearrangements. Predicted sequences of rearrangements inferred from genomic sequence data are displayed above electropherograms, with exact chromosomal breakpoints indicated (Hg19 coordinates). UTR, untranslated region.

Figure 4

Scheme showing the framework for variant interpretation and reporting.