Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors

• Published in: Neoplasia (New York, N.Y.) Vol. 19; no. 3; pp. 196 - 206
• Main Authors: Paasinen-Sohns, Aino, Koelzer, Viktor H, Frank, Angela, Schafroth, Julian, Gisler, Aline, Sachs, Melanie, Graber, Anne, Rothschild, Sacha I, Wicki, Andreas, Cathomas, Gieri, Mertz, Kirsten D
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-03-2017; Neoplasia Press; Elsevier
• Subjects: Adult; Aged; Aged, 80 and over; Biomarkers; DNA Copy Number Variations; Female; Genetic Variation; Genomics - methods; High-Throughput Nucleotide Sequencing; Humans; Immunohistochemistry; Male; Melanoma - genetics; Melanoma - metabolism; Middle Aged; Mutation; Neoplasms - genetics; Neoplasms - metabolism; Oncology; Original article; Reproducibility of Results; non–small cell lung cancer, squamous cell carcinoma; ribonucleic acid; RNA; IHC; non–small cell lung cancer, pleomorphic carcinoma; tumor stage as determined by pathological assessment; superficially spreading melanoma; OFA; CLPv2; Ion AmpliSeq Colon and Lung Cancer Research Panel v2; deoxyribonucleic acid; FFPE; Oncomine Focus Assay; formalin-fixed,paraffin-embedded; immunohistochemistry; hematoxylin and eosin; NSCLC (NOS); NSCLC (pleo); colorectal cancer; NSCLC; pT; somatic copy number variation; H&E; next generation sequencing; non–small cell lung cancer; NSCLC (adeno); non–small cell lung cancer, adenocarcinoma; NSCLC (squamous); SSM; CRC; DNA; NGS; SCNV; non–small cell lung cancer, not otherwise specified
• ISSN: 1476-5586; 1522-8002; 1476-5586
• DOI: 10.1016/j.neo.2017.01.003

Abstract Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15 years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15 years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology.