Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting

• Published in: Pathology oncology research Vol. 30; p. 1611590
• Main Authors: Kumbrink, Jörg, Demes, Melanie-Christin, Jeroch, Jan, Bräuninger, Andreas, Hartung, Kristin, Gerstenmaier, Uwe, Marienfeld, Ralf, Hillmer, Axel, Bohn, Nadine, Lehning, Christina, Ferch, Ferdinand, Wild, Peter, Gattenlöhner, Stefan, Möller, Peter, Klauschen, Frederick, Jung, Andreas
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 28.03.2024
• Subjects: actionable mutations; anchored multiplex PCR; lung cancer; next-generation sequencing panel; Pathology and Oncology Archive; targeted therapy; anchored multiplex PCR; lung cancer; targeted therapy; actionable mutations; next-generation sequencing panel
• ISSN: 1532-2807; 1219-4956; 1532-2807
• DOI: 10.3389/pore.2024.1611590

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples ( = 90). With default filter settings, all but two potential exon 14 skipping variants were identified at similar allele frequencies. Both variants were found with an adapted calling filter. Three additional variants ( , , ) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.