Modular Targeted Capture Assay Detects Clinically Significant Oncology Alterations

The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented.

The need to comprehensively assess clinical cancer specimens for an expanding list of alterations critical to therapeutic decision making led to the adoption of large “fixed-content” genetic panels that utilized massively parallel sequencing, more commonly referred to as next-generation sequencing (NGS).


Medical Laboratory Scientists at the University of Washington Medical Center (Seattle, WA, USA) used DNA samples for the validation of their OncoPlex Cancer Gene Panel version 6 (UW-OPXv6) were derived from 108 unique specimens from 29 different adult and pediatric neoplasms including central nervous system (CNS) malignancies, leukemia/lymphoma, melanoma, sarcoma, and carcinomas of the lung, breast, endometrium, bowel, and prostate, in addition to five germline samples. UW-OncoPlex is a multiplexed mutation assay for tumor tissue that assesses mutations >350 genes related to cancer treatment, prognosis, or diagnosis.

The team described the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes.

All samples had prior molecular characterization via orthogonal clinical tests including both laboratory-developed amplicon-based and hybrid-capture-based NGS assays and/or a custom commercial RNA sequencing assay (FusionPlex, ArcherDx, Boulder, CO, USA). DNA was extracted using one or more kits from Qiagen (Qiagen, Valencia, CA, USA) depending on specimen type and nucleic acid extraction desired. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms (Illumina, San Diego, CA, USA). Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results.

The scientists reported that the performance characteristics of OPXv6 are excellent for all tested variant classes (SNVs, Indels, SVs, and CNVs), both using standard protocols and in the setting of decreased DNA input and multiple methods of nucleic acid extraction. Accuracy was 99% for SNVs ≥5% allele fraction, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB. Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target.

The authors concluded that OPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to result. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology. The study was published on February 3, 2020 in the journal Practical Laboratory Medicine.

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University of Washington Medical Center
ArcherDx
Qiagen
Illumina