211P Cost-efficient Detection of NTRK1, NTRK2 and NTRK3 Gene Rearrangements Using the Test for 5’/3’-End Unbalanced Expression: the Analysis of 8075 Patients

The majority of NTRK1, NTRK2 and NTRK3 rearrangements result in the increased expression of the kinase portion of the gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5’-/3’-end expression imbalance is potentially capable of detecting the entire spectrum of NTRK gene fusions. Formalin-fixed tissue specimens were subjected to manual dissection of tumor cells, followed by DNA/RNA isolation and cDNA synthesis. 5’-/3’-end expression imbalance in NTRK genes was analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific multiplexed PCR for 42 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). The study initially included 8075 tissue specimens; 651 (8.1%) of these samples failed to pass the quality control. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.8%). Relatively high frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%) and salivary gland carcinomas (5/93, 5.4%). None of 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5’/3’-end expression imbalance. Variant-specific PCR was performed for 744 tumors with normal 5’/3’-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while NTRK fusions were detected in 2/447 (0.4%) non-lung adult malignancies. This study describes a robust pipeline for the detection of NTRK1, NTRK2 and NTRK3 gene fusions, which may be considered as a cost-efficient alternative to conventional methods of NTRK analysis.