Abstract
Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.
Highlights
The value of specific gene alterations to match individuals with cancer to molecularly targeted agents is clear in many tumour types [1–18]
The OCAv3 is the assay of choice in the on-going Adult and Pediatric NCI- Molecular Analysis for Therapy Choice (MATCH) programs (NCT02465060/ NCT03155620) [24, 25], to allocate metastatic patients to novel targeted therapeutics
Twelve samples failed the original RNA read length metric (Table 5), including 6 from Laboratory B, this was reduced to 5 failed samples using the newer minimum read length requirement of 60 bases
Summary
The value of specific gene alterations to match individuals with cancer to molecularly targeted agents is clear in many tumour types [1–18]. Broad genomic profiling, using whole genome or exome sequencing and whole transcriptome sequencing, is used in clinical trials of metastatic cancer to attempt to match patients to targeted therapeutics based on genomic features of their tumours [19–23] Many of these trials highlight the complexity of delivering broad genomic analyses from small diagnostic samples in a timely fashion. The OFA is a smaller and more economical version of the OCAv3, and, like OCAv3, has the advantage of being appropriate for small amounts of DNA/RNA extracted from formalin fixed paraffin embedded (FFPE) diagnostic samples Both OCAv3 and OFA can be readily adopted in routine clinical settings combining deep coverage of key genes using generation sequencing methodology and a curated informatics reporting pipeline. In order to assess the accuracy and reproducibility of the OFA for detection of mutations, copy number alterations and fusions, we performed a ring study that included two research and five clinical molecular diagnostic laboratories in Ontario and compared results using a small series of solid tumour and control specimens
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