Abstract 2422: Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples
Authors: Houghton A. McGarry , Meredith Gavin , Kargl Julia , McKay-Fleisch Jill , Ross P. Martin , Kharkia Anisha , Mashadi-Hossein Afshin , Kim Dae , Beechem Joseph 
Published: July 2017

Worldwide, lung cancer is the most commonly diagnosed form of cancer with a survival rate among the lowest. Combined, somatic mutations (in the form of SNVs and InDels) and gene fusions, account for the majority of interpretable and actionable genomic alterations. Importantly, this typically requires the analysis of DNA and RNA from limited amounts of FFPE-preserved specimens. Currently, these analyses typically require complex sample pre-processing for assay on separate platforms or separate complex library preparation methods for assessment by high throughput sequencing. To provide a unified and simpler alternative, NanoString’s molecular barcoding technology has been modularized to permit simultaneous digital measurement of cancer-relevant targets that span these two analyte classes. Novel ‘SNV’ probes enable sensitive and specific identification of DNA mutant allele sequences down to a level of detection of ≤ 5% from 5 ng of FFPE-extracted genomic DNA. Fusion transcripts are detected with 5’/3’ imbalance probes and toehold-mediated junction probes. This dual analyte workflow requires just a single 5-10 micron section of FFPE tissue and provides to sample-to-answer results with approximately 5 minutes of hands-on time per sample after nucleic acid extraction.

To demonstrate utility, 37 lung cancer samples were assayed simultaneously with an SNV panel that targets &gt;100 solid tumor somatic mutations and a lung cancer fusion gene panel that provides general evidence of ALK, RET, and ROS1 gene fusion events along with specific detection of 35 unique fusion transcripts that correspond to known break-points. In this particular cohort, 16 samples were positive for activating KRAS SNVs (one of which was also positive for an activating STK11 variant), 3 were positive for activating EGFR mutations including two SNVs and an 18-base InDel and one was positive for an activating KIF5B16:RET12 fusion transcript. Positive mutation calls obtained with the SNV panel could only be confirmed by whole-exome sequencing (average depth of 100X) for 13 of 20 variants detected; however, ultra-deep (average depth of 4400X) targeted sequencing revealed that the 7 additional panel-detected mutations were, in fact, present. Measured against the sequencing datasets, the SNV panel provided 100% sensitivity, specificity, accuracy and precision for all variants present at 5% or greater allele frequency. The KIF5B16:RET12 fusion event was also confirmed by sequencing. Combined, these results show that these two important classes of activating mutations can be readily and efficiently assayed together on a NanoString nCounter® system (for research use only).

Citation Format: A. McGarry Houghton, Gavin Meredith, Julia Kargl, Jill McKay-Fleisch, P. Martin Ross, Anisha Kharkia, Afshin Mashadi-Hossein, Dae Kim, Joseph Beechem. Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2017-2422