This study aimed to analyze clinical and regional factors influencing the distribution of actionable genetic alterations in a large consecutive series of colorectal carcinomas (CRCs). KRAS, NRAS and BRAF mutations, HER2 amplification and overexpression, and microsatellite instability (MSI) were tested in 8355 CRC samples. KRAS mutations were detected in 4137/8355 (49.5%) CRCs, with 3913 belonging to 10 common substitutions affecting codons 12/13/61/146, 174 being represented by 21 rare hot-spot variants, and 35 located outside the “hot” codons. KRAS Q61K substitution, which leads to the aberrant splicing of the gene, was accompanied by the second function-rescuing mutation in all 19 tumors analyzed. NRAS mutations were detected in 389/8355 (4.7%) CRCs (379 hot-spot and 10 non-hot-spot substitutions). BRAF mutations were identified in 556/8355 (6.7%) CRCs (codon 600: 510; codons 594–596: 38; codons 597–602: 8). The frequency of HER2 activation and MSI was 99/8008 (1.2%) and 432/8355 (5.2%), respectively. Some of the above events demonstrated differences in distribution according to patients’ age and gender. In contrast to other genetic alterations, BRAF mutation frequencies were subject to geographic variation, with a relatively low incidence in areas with an apparently warmer climate (83/1726 (4.8%) in Southern Russia and North Caucasus vs. 473/6629 (7.1%) in other regions of Russia, p = 0.0007). The simultaneous presence of two drug targets, BRAF mutation and MSI, was observed in 117/8355 cases (1.4%). Combined alterations of two driver genes were detected in 28/8355 (0.3%) tumors (KRAS/NRAS: 8; KRAS/BRAF: 4; KRAS/HER2: 12; NRAS/HER2: 4). This study demonstrates that a substantial portion of RAS alterations is represented by atypical mutations, KRAS Q61K substitution is always accompanied by the second gene-rescuing mutation, BRAF mutation frequency is a subject to geographical variations, and a small fraction of CRCs has simultaneous alterations in more than one driver gene.

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.

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.