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

Ian A. Cree,Varun Bagai,Bastiaan B.j. Tops ,Anna Maria Rachiglio,Delphine Le Corre,Rosella Petraroli,Cecily P. Vaughn,Harriet Feilotter ,Kazuko Sakai,Susan Crocker,Henriette Kurth ,Andrea Mafficini,Hélène Blons,Roy R. L. Bastien,José Luís Costa ,Anne Reiman,Kelli Bramlett,Michel Bihl,Pierre Laurent‐Puig ,Orla Sheils,Federica Zito Marino,Aldo Scarpa,Nicola Normanno,Marjolijn J. L. Ligtenberg,Astrid Hirschmann,Alexander H. Boag ,Kazuto Nishio

doi:10.1186/s12885-018-4736-4

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.

Highlights

Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma
Cell lines Using the cocktail of RNA from the ALK, ROS1, and RET fusion-positive cell lines, each of the ten participating laboratories successfully detected all three rearrangements using the AmpliSeq Fusion Lung Panel assay
The advent of therapies targeting the fusion proteins arising from ALK, ROS1, and RET gene fusions makes the routine detection of these events important in patients with lung adenocarcinoma

Summary

Introduction

Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. Non-small cell lung carcinoma (NSCLC) has been categorized into several distinct entities by molecular characterization of genetic alterations occurring during epithelial cell transformation. Chromosomal rearrangements involving the tyrosine kinase receptor genes ALK, [4] ROS1, [5] RET, [6,7,8] and NTRK1, [9] have been more recently described, extending the repertoire of molecular alterations found in NSCLC These fusion events, involving a variety of partner genes, result in the formation of chimeric fusion kinases capable of oncogenic transformation and induction of oncogene dependency within the neoplastic cells. The prevalence of each of these chromosomal rearrangements individually is 1–7% in NSCLC [4, 6, 10, 11], and altogether can be identified in approximately 5–9% of NSCLC [7, 12, 13]

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Discussion

Conclusion