Comprehensive Genetic Profiling Of Chromosomal Translocations In Lung Cancer Tumors: Development And Validation Of A Next-Generation Sequencing Panel In An International Multicenter Study

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. While tumors harboring ALK fusions are highly sensitive to crizotinib, emerging data have demonstrated that individuals with ROS1 or RET fusions may also benefit from inhibitors targeting these two kinases. A new oncogenic fusion involving NTRK1 that can be targeted by kinase inhibitors has been recently described. The two main methods to identify translocations, fluorescent in situ hybridization (FISH) and Immunohistochemistry (IHC) are labor intensive and sensitivity is rather low. Common sample types for lung cancer analysis are biopsies making the study of more than one fusion gene rather difficult. In this study we aimed to develop and validate a workflow based on AmpliSeq™ technology to comprehensively profile ALK, ROS1, RET and NTRK1 chromosomal translocations in lung tumors. Two hundred lung cancer retrospective samples, including tumor biopsy, were collected from 12 laboratories - the OncoNetwork Consortium. All the samples were previously characterized by orthologous techniques (FISH, IHC, RT-PCR and/or MassArray). More than 30 ALK positive samples were selected. The panel targets over 70 fusion transcripts associated with ALK, RET, ROS1, and NTRK1 genes. The panel also includes 5′ and 3′ gene expression assays for each gene as indicators of a translocation event and assays for 5 internal control genes. The workflow is compatible with formalin fixed paraffin embedded (FFPE) tissue, requiring only 10ng of total RNA and able to multiplex up to 16 libraries on a single Ion 318™ chip. A dedicated data analysis pipeline using the Ion Reporter software 4.2 was evaluated. Serial dilutions of cell lines RNA harboring known fusion events, in normal RNA showed that the methodology had 1% mutant RNA limit of detection. A second dilution experiment using RNA extracted from FFPE lung fusion positive samples diluted in a negative FFPE sample showed a detection limit of 15%. The use of a common control fusion positive RNA samples among all the consortia laboratories showed 100% reproducibility. Concordance study was performed using 200 FFPE samples previously characterized with standard method. A concordance of u003e98% was obtained between the methodologies. The discordant results are currently under study. A negative FFPE samples was run as single test on an Ion 318™ chip on to evaluate the possibility of false positive results due to high number of reads. No false positive was detected in this experiment. In this study we present a workflow that provides a robust, reproducible and accurate comprehensive genetic screening tool well suited for FFPE lung tumor biopsies, in a fast and cost-efficient manner. This may provide a valuable tool for reducing turn-around-time and expense in lung cancer analysis. Citation Format: Pierre Laurent - Puig, Jose Louis Costa, Orla Sheils, Bastiaan Tops, Andrea Mafficini, Delphine Le Corre, Henriette Kurth, Anna Maria Rachiglio, Helene Blons, Eliana Amato, Christoph Noppen, Renato Franco, Anne Reiman, Roy Bastien, Noah Welker, Jose Carlos Machado, Ian Cree, Harriet Feilotter, Marjolijn Ligtenberg, Aldo Scarpa, Nicola Normanno, Kazuto Nishio, Cecily Vaughn. Comprehensive genetic profiling of chromosomal translocations in lung cancer tumors: development and validation of a next-generation sequencing panel in an international multicenter study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4891. doi:10.1158/1538-7445.AM2015-4891