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

Abstract

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.

Response to trametinib, hydroxychloroquine, and bevacizumab in a young woman with NRAS-mutated metastatic intrahepatic cholangiocarcinoma: a case report

“…The analysis of FGFR1-3 , KRAS , NRAS , BRAF , HER2 , and IDH1 genes was done with custom next-generation RNA sequencing [ 14 ]. Receptor tyrosine kinase rearrangements ( RET and NTRK1–3 ) were analyzed by polymerase chain reaction (PCR) tests [ 21 , 22 ].…”

“…iCCAs often carry driver genetic mutations, particularly FGFR2 fusions or mutations, HER2 amplification, BRAF V600E substitutions, and hotspot alterations affecting IDH1 , KRAS , and NRAS genes [ 14 ]. While activating events involving FGFR2 , IDH1 , HER2 , and BRAF oncogenes are druggable, targeting RAS -mutated tumors remains a challenge [ 15 ].…”