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

The technique 3' rapid amplification of cDNA ends (3' RACE) allows for detection of translocations with unknown gene partners located at the 3' end of the chimeric transcript. We composed a 3' RACE-based RNA sequencing panel for the analysis of FGFR1-4 gene rearrangements, detection of activating mutations located within FGFR1-4, IDH1/2, ERBB2 (HER2), KRAS, NRAS, BRAF, and PIK3CA genes, and measurement of the expression of ERBB2, PD-L1, and FGFR1-4 transcripts. This NGS panel was utilized for the molecular profiling of 168 biliary tract carcinomas (BTCs), including 83 intrahepatic cholangiocarcinomas (iCCAs), 44 extrahepatic cholangiocarcinomas (eCCAs), and 41 gallbladder adenocarcinomas (GBAs). The NGS failure rate was 3/168 (1.8%). iCCAs, but not other categories of BTCs, were characterized by frequent FGFR2 alterations (17/82, 20.7%) and IDH1/2 mutations (23/82, 28%). Other potentially druggable events included ERBB2 amplifications or mutations (7/165, 4.2% of all successfully analyzed BTCs) and BRAF p.V600E mutations (3/165, 1.8%). In addition to NGS, we analyzed microsatellite instability (MSI) using the standard five markers and revealed this event in 3/158 (1.9%) BTCs. There were no instances of ALK, ROS1, RET, and NTRK1-3 gene rearrangements or MET exon 14 skipping mutations. Parallel analysis of 47 iCCA samples with the Illumina TruSight Tumor 170 kit confirmed good performance of our NGS panel. In conclusion, targeted RNA sequencing coupled with the 3' RACE technology is an efficient tool for the molecular diagnostics of BTCs.

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.

Content of circulating tumor DNA depends on the tumor type and the dynamics of tumor size, but is not influenced significantly by physical exercise, time of the day or recent meal.

PURPOSE: This study aimed to investigate factors, which influence the content of circulating tumor DNA (ctDNA). METHODS: 398 serial plasma samples were collected within 1-7 consecutive days from patients with EGFR-mutated lung cancer (n = 13), RAS/RAF-mutated colorectal cancer (n = 54) and BRAF-mutated melanoma (n = 17), who presented with measurable tumor disease. The amount of ctDNA was determined by ddPCR. RESULTS: Among 82 patients, who donated 2-6 serial plasma samples, 42 subjects were classified as ctDNA-positive; only 22% cases were mutation-positive across all consecutive tests, while 24/82 (29%) patients showed presence of mutated ctDNA in some but not all blood draws. Subjects with progressing tumors had higher probability of being detected ctDNA-positive as compared to patients, who responded to therapy or had stable disease (39/55 (71%) vs. 4/24 (17%); p = 0.0001). Our study failed to reveal the impact of the time of the day, recent meal or prior physical exercise on the results of ctDNA testing. CONCLUSIONS: Presence of ctDNA in plasma is particularly characteristic for patients, who experience clinical progression of tumor disease. Consecutive plasma tests may occasionally provide discordant data; thus, the repetition of analysis may be advised in certain cases in order to ensure the validity of negative ctDNA result.