Current practices and guidelines for clinical next-generation sequencing oncology testing

Abstract

Next-generation sequencing (NGS) has been rapidly integrated into molecular pathology, dramatically increasing the breadth genomic of information available to oncologists and their patients. This review will explore the ways in which this new technology is currently applied to bolster care for patients with solid tumors and hematological malignancies, focusing on practices and guidelines for assessing the technical validity and clinical utility of DNA variants identified during clinical NGS oncology testing.

Keywords: Cancer genomics; molecular diagnostics; next-generation sequencing.

Figure1

Summary of technical validity and clinical utility assessment for cancer NGS. (A) NGS basecalling, wherein a DNA sequence and corresponding confidence score is generated from a nuclear genomic DNA template. (B) The next step, which compares all available data to the reference and each other. Variant calling is then performed (underlined bases in panel B), comparing base calls across many reads; many false positive variant calls (x'ed out bases) can be filtered, while true positives (circled bases) should generate a strong signal. (C) Multiple quality metrics are generated during variant calling, which can be compared to cutoffs established during assay validation (dashed lines). (D) Detailed review of available databases and literature (left side) and comparison to clinical history and tumor pathology (right side) to assess clinical utility. VAF, variant allele frequency; QUAL, variant call quality; COSMIC, Catalogue of Somatic Mutations in Cancer; TKIs, tyrosine kinase inhibitor therapies.

Figure2

Alignment views of four variants detected by NGS. Each panel depicts a representative set of variant reads for single nucleotide variant (A and B) or insertion/deletion variants (C and D) with either high quality (A and C) or low quality (B and D). The human genome reference sequence is the string of bases along the top of each panel. Aligned basecalls matching the reference are listed as dots (plus strand) or commas (minus strand). High quality variants typically have higher variant allele frequencies (VAF), and variants reads have fewer additional variants. Panel D depicts known false positive locus, likely due to the "homopolymer problem" where certain NGS technologies overcall insertion/deletion variants where the reference sequence has five or more of the same nucleotides in a row (in this case, 8 guanines).