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

Abstract

BackgroundGene 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.MethodsWe describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples.ResultsReproducibility 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.ConclusionThis 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 (10 ng) 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.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4736-4) contains supplementary material, which is available to authorized users.

GDNF and the RET Receptor in Cancer: New Insights and Therapeutic Potential

“…While RET receptor mutations are well-characterized mechanisms of carcinogenesis, the much broader implications of GFL-mediated RET signaling in cancer are only beginning to be recognized. State-of-the-art diagnostic approaches, such as liquid biopsies coupled to Next Generation Sequencing, or high through-put mutational screening panels to assess circulating tumor cells or cell free tumor DNA (Hench et al, 2018; Vaughn et al, 2018), are increasing our abilities to recognize RET-targetable cancers or recurrent disease, without need for more invasive tumor biopsies (Reckamp et al, 2018).…”

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material

“…Nevertheless, the PATH panel does not prevent additional predictive analyses. Immunohistochemistry of PD-L1, ALK and ROS1, or mRNA based analysis to detect relevant fusion genes involving ALK, ROS1, RET, or NTRK genes cannot be replaced by the PATH panel [56]. In addition, tumor mutational burden, associated with a favorable response to immune checkpoint inhibitors [57,58] cannot be performed by a small NGS panel.…”

Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples

“…Previous data have shown the possibility of identifying the lung cancer fusions using advanced molecular technologies that allows a rapid massively detection of chromosome rearrangements in a large set of genes by targeted sequencing of the fusion junctions or by paired-end mapping methods 20…”