Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: A multi-institutional research study

Background: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. Methods: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. Results: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. Conclusion: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.This work resulted from projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), “GenomePT” (POCI-01-0145-FEDER-022184), “Advancing cancer research: from knowledge to application” (NORTE-01-0145-FEDER-000029) supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Norte Portugal Regional Programme (NORTE 2020), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT - Fundação para a Ciência e a Tecnologia (PTDC/DTP-PIC/2500/2014). This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to N. Normanno (Grant number: IG17135)