Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples

Abstract

The majority of NTRK1, NTRK2, and NTRK3 rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5′/3′-end expression imbalances is potentially capable of detecting the entire spectrum of NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5′/3′-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. 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, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5′/3′-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a 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 NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1–3 analysis.

Molecular Analysis of Biliary Tract Cancers with the Custom 3′ RACE-Based NGS Panel

“…These types of translocations include targetable ALK, ROS1, RET, and NTRK1-3 genes fusions, which were detected in a small fraction of BTCs, primarily iCCAs, in some studies [48][49][50][51][52]. We assessed these translocations in available iCCA samples using either the Illumina TruSight Tumor 170 kit or the previously described PCR-based tests, including tests for unbalanced expression of the 5 /3 mRNA ends [29][30][31], but found no instances of such alterations.…”

“…Translocations involving known targetable genes, such as ALK, ROS1, RET, and NTRK1-3, were previously reported as rare targetable alterations in BTCs (mainly, in iCCA cases) [48][49][50][51][52]. We applied a panel of RT-PCR-based tests [28][29][30][31] to the iCCA cases that were not subjected to the analysis with Illumina TruSight Tumor 170 panel; however, no such rearrangements or the MET exon 14 skipping mutations, were found.…”

“…ERBB2 amplifications and MSI status (standard pentaplex panel: BAT25, BAT26, NR21, NR22, and NR24 mononucleotide markers) were determined as described previously [27]. For the iCCA samples that were not subjected to the NGS analysis using Illumina TruSight Tumor 170 kit, receptor tyrosine kinase rearrangements (ALK, RET, ROS1, RET, and NTRK1-3) and MET exon 14 skipping mutations were analyzed via PCR tests as described previously [28][29][30][31].…”

Agnostic Administration of Targeted Anticancer Drugs: Looking for a Balance between Hype and Caution

“…However, NTRK1-3 rearrangements are included in the majority of multigene diagnostic panels, so their detection is supported by the rapidly increasing utilization of NGS. While the incidence of NTRK activation in commonly occurring carcinomas affecting adults falls below 1:500-1:1000, NTRK1-3 translocations are detected at frequency of about 5% in pediatric tumors, sarcomas, and salivary gland carcinomas [53,54]. NTRK1-3 fusions are particularly common in microsatellite-unstable RAS/RAF mutation-negative colorectal carcinomas, while non-colorectal tumors with MSI rarely carry gene translocations [37].…”

“…Current clinical experience of the use of entrectinib and larotrectinib includes no more than a few hundred patients with very diverse tumor types. The data are particularly scarce for NTRK2 rearrangements, which are significantly less frequent than NTRK1 or NTRK3 fusions [48,49,53,54].…”