ALK, ROS1, RET and NTRK1-3 Gene Fusions in Colorectal and Non-Colorectal Microsatellite-Unstable Cancers.

This study aimed to conduct a comprehensive analysis of actionable gene rearrangements in tumors with microsatellite instability (MSI). The detection of translocations involved tests for 5'/3'-end expression imbalance, variant-specific PCR and RNA-based next generation sequencing (NGS). Gene fusions were detected in 58/471 (12.3%) colorectal carcinomas (CRCs), 4/69 (5.8%) gastric cancers (GCs) and 3/65 (4.6%) endometrial cancers (ECs) (ALK: 8; RET: 12; NTRK1: 24; NTRK2: 2; NTRK3: 19), while none of these alterations were observed in five cervical carcinomas (CCs), four pancreatic cancers (PanCs), three cholangiocarcinomas (ChCs) and two ovarian cancers (OCs). The highest frequency of gene rearrangements was seen in KRAS/NRAS/BRAF wild-type colorectal carcinomas (53/204 (26%)). Surprisingly, as many as 5/267 (1.9%) KRAS/NRAS/BRAF-mutated CRCs also carried tyrosine kinase fusions. Droplet digital PCR (ddPCR) analysis of the fraction of KRAS/NRAS/BRAF mutated gene copies in kinase-rearranged tumors indicated that there was simultaneous co-occurrence of two activating events in cancer cells, but not genetic mosaicism. CRC patients aged above 50 years had a strikingly higher frequency of translocations as compared to younger subjects (56/365 (15.3%) vs. 2/106 (1.9%), p = 0.002), and this difference was particularly pronounced for tumors with normal KRAS/NRAS/BRAF status (52/150 (34.7%) vs. 1/54 (1.9%), p = 0.001). There were no instances of MSI in 56 non-colorectal tumors carrying ALK, ROS1, RET or NTRK1 rearrangements. An analysis of tyrosine kinase gene translocations is particularly feasible in KRAS/NRAS/BRAF wild-type microsatellite-unstable CRCs, although other categories of tumors with MSI also demonstrate moderate occurrence of these events.

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

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.

Rapid and Cost-Efficient Detection of RET Rearrangements in a Large Consecutive Series of Lung Carcinomas.

RET-kinase-activating gene rearrangements occur in approximately 1-2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5'- and 3'-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5'/3'-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5'/3'-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5'/3'-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5'/3'-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations.

Spectrum of kinase gene rearrangements in a large series of paediatric inflammatory myofibroblastic tumours.

INTRODUCTION: Inflammatory myofibroblastic tumours (IMTs), being an exceptionally rare category of paediatric neoplasms, often contain druggable gene rearrangements involving tyrosine kinases. METHODS AND RESULTS: This study presents a large consecutive series of IMTs which were analysed for the presence of translocations by the PCR test for 5'/3'-end ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 unbalanced expression, variant-specific PCR for 47 common gene fusions and NGS TruSight RNA fusion panel. Kinase gene rearrangements were detected in 71 of 82 (87%) IMTs (ALK: n = 47; ROS1: n = 20; NTRK3: n = 3; PDGFRb: n = 1). The test for unbalanced expression had 100% reliability in identifying tumours with ALK fusions, but failed to reveal ROS1 rearrangements in eight of 20 (40%) ROS1-driven IMTs; however, ROS1 alterations were detectable by variant-specific PCR in 19 of 20 (95%) cases. ALK rearrangements were particularly common in patients below 1 year of age (10 of 11 (91%) versus 37 of 71 (52%), P = 0.039). ROS1 fusions occurred more often in lung IMTs than in tumours of other organs (14 of 35 (40%) versus six of 47 (13%), P = 0.007). Among 11 IMTs with no kinase gene rearrangement identified, one tumour demonstrated ALK activation via gene amplification and overexpression, and another neoplasm carried COL1A1::USP6 translocation. CONCLUSIONS: PCR-based pipeline provides a highly efficient and non-expensive alternative for molecular testing of IMTs. IMTs with no detectable rearrangements need further studies.