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

Overview

Journal Diagnostics (Basel)

Specialty Radiology

Date 2023 Oct 28

PMID 37891989

Authors

Natalia V Mitiushkina

Vladislav I Tiurin

Aleksandra A Anuskina

Natalia A Bordovskaya

Anna D Shestakova

Aleksandr S Martianov

Mikhail G Bubnov

Anna S Shishkina

Maria V Semina

Aleksandr A Romanko

Ekaterina S Kuligina

Evgeny N Imyanitov

Affiliations

Soon will be listed here.

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 gene rearrangements, detection of activating mutations located within , and genes, and measurement of the expression of and 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 alterations (17/82, 20.7%) and mutations (23/82, 28%). Other potentially druggable events included amplifications or mutations (7/165, 4.2% of all successfully analyzed BTCs) and 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 and gene rearrangements or 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.

Citing Articles

Use of 3' Rapid Amplification of cDNA Ends (3' RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic Alterations in Urothelial Carcinomas.

Mitiushkina N, Tiurin V, Anuskina A, Bordovskaya N, Nalivalkina E, Terina D Int J Mol Sci. 2024; 25(22).

PMID: 39596194 PMC: 11594887. DOI: 10.3390/ijms252212126.

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

Musaelyan A, Anokhina E, Turdubaeva A, Mitiushkina N, Ershova A, Shestakova A Explor Target Antitumor Ther. 2024; 5(3):780-788.

PMID: 38966164 PMC: 11220291. DOI: 10.37349/etat.2024.00246.

References

Zingg D, Bhin J, Yemelyanenko J, Kas S, Rolfs F, Lutz C . Truncated FGFR2 is a clinically actionable oncogene in multiple cancers. Nature. 2022; 608(7923):609-617. PMC: 9436779. DOI: 10.1038/s41586-022-05066-5. View

Yamashita K, Iwatsuki M, Yasuda-Yoshihara N, Morinaga T, Nakao Y, Harada K . Trastuzumab upregulates programmed death ligand-1 expression through interaction with NK cells in gastric cancer. Br J Cancer. 2020; 124(3):595-603. PMC: 7851117. DOI: 10.1038/s41416-020-01138-3. View

Subbiah V, Cassier P, Siena S, Garralda E, Paz-Ares L, Garrido P . Pan-cancer efficacy of pralsetinib in patients with RET fusion-positive solid tumors from the phase 1/2 ARROW trial. Nat Med. 2022; 28(8):1640-1645. PMC: 9388374. DOI: 10.1038/s41591-022-01931-y. View

Singh R . Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology. Diagnostics (Basel). 2022; 12(7). PMC: 9318977. DOI: 10.3390/diagnostics12071539. View

Matter M, Chijioke O, Savic S, Bubendorf L . Narrative review of molecular pathways of kinase fusions and diagnostic approaches for their detection in non-small cell lung carcinomas. Transl Lung Cancer Res. 2021; 9(6):2645-2655. PMC: 7815372. DOI: 10.21037/tlcr-20-676. View

Lopes Pinto F, Lindblad P . A guide for in-house design of template-switch-based 5' rapid amplification of cDNA ends systems. Anal Biochem. 2009; 397(2):227-32. DOI: 10.1016/j.ab.2009.10.022. View

Smith T, Heger A, Sudbery I . UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Res. 2017; 27(3):491-499. PMC: 5340976. DOI: 10.1101/gr.209601.116. View

Mitiushkina N, Romanko A, Preobrazhenskaya E, Tiurin V, Ermachenkova T, Martianov A . Comprehensive evaluation of the test for 5'-/3'-end mRNA unbalanced expression as a screening tool for ALK and ROS1 fusions in lung cancer. Cancer Med. 2022; 11(17):3226-3237. PMC: 9468436. DOI: 10.1002/cam4.4686. View

Benayed R, Offin M, Mullaney K, Sukhadia P, Rios K, Desmeules P . High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden. Clin Cancer Res. 2019; 25(15):4712-4722. PMC: 6679790. DOI: 10.1158/1078-0432.CCR-19-0225. View

10.

Tiurin V, Preobrazhenskaya E, Mitiushkina N, Romanko A, Anuskina A, Mulkidjan R . Rapid and Cost-Efficient Detection of Rearrangements in a Large Consecutive Series of Lung Carcinomas. Int J Mol Sci. 2023; 24(13). PMC: 10342123. DOI: 10.3390/ijms241310530. View

11.

Heydt C, Wolwer C, Velazquez Camacho O, Wagener-Ryczek S, Pappesch R, Siemanowski J . Detection of gene fusions using targeted next-generation sequencing: a comparative evaluation. BMC Med Genomics. 2021; 14(1):62. PMC: 7912891. DOI: 10.1186/s12920-021-00909-y. View

12.

Abou-Alfa G, Macarulla T, Javle M, Kelley R, Lubner S, Adeva J . Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol. 2020; 21(6):796-807. PMC: 7523268. DOI: 10.1016/S1470-2045(20)30157-1. View

13.

Ozawa T, Kondo M, Isobe M . 3' rapid amplification of cDNA ends (RACE) walking for rapid structural analysis of large transcripts. J Hum Genet. 2004; 49(2):102-105. DOI: 10.1007/s10038-003-0109-0. View

14.

Kongpetch S, Jusakul A, Lim J, Ng C, Chan J, Rajasegaran V . Lack of Targetable FGFR2 Fusions in Endemic Fluke-Associated Cholangiocarcinoma. JCO Glob Oncol. 2020; 6:628-638. PMC: 7193781. DOI: 10.1200/GO.20.00030. View

15.

Haas B, Dobin A, Li B, Stransky N, Pochet N, Regev A . Accuracy assessment of fusion transcript detection via read-mapping and de novo fusion transcript assembly-based methods. Genome Biol. 2019; 20(1):213. PMC: 6802306. DOI: 10.1186/s13059-019-1842-9. View

16.

Crotty R, Hu K, Stevenson K, Pontius M, Sohani A, Ryan R . Simultaneous Identification of Cell of Origin, Translocations, and Hotspot Mutations in Diffuse Large B-Cell Lymphoma Using a Single RNA-Sequencing Assay. Am J Clin Pathol. 2020; 155(5):748-754. PMC: 8244124. DOI: 10.1093/ajcp/aqaa185. View

17.

Arai Y, Totoki Y, Hosoda F, Shirota T, Hama N, Nakamura H . Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma. Hepatology. 2013; 59(4):1427-34. DOI: 10.1002/hep.26890. View

18.

Park H, Baek I, Cheang G, Solomon J, Song W . Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions. J Mol Diagn. 2021; 23(11):1443-1451. DOI: 10.1016/j.jmoldx.2021.07.027. View

19.

Goeppert B, Frauenschuh L, Renner M, Roessler S, Stenzinger A, Klauschen F . BRAF V600E-specific immunohistochemistry reveals low mutation rates in biliary tract cancer and restriction to intrahepatic cholangiocarcinoma. Mod Pathol. 2013; 27(7):1028-34. DOI: 10.1038/modpathol.2013.206. View

20.

Gilani A, Donson A, Davies K, Whiteway S, Lake J, DeSisto J . Targetable molecular alterations in congenital glioblastoma. J Neurooncol. 2019; 146(2):247-252. DOI: 10.1007/s11060-019-03377-8. View