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.

Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3′ Rapid Amplification of cDNA Ends (3′ RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine.

Introduction. Long-term outcomes of metastatic urothelial cancer remain unsatisfactory: the 5-year survival rate for this disease is less than 15 %. It is important to develop and introduce into clinical practice new drugs, including targeted agents, and the individualization of systemic therapy for urothelial cancer based on molecular genetic analysis of tumor tissue. Erdafitinib, a new drug developed for the treatment of locally advanced and metastatic urothelial cancer with certain genetic alterations in the FGFR2 and FGFR3 receptors, was registered in Russia in 2023. Materials and Methods. This work investigated the distribution of clinically relevant mutations/translocations affecting the FGFR2/3 genes in a large number urothelial cancer patients in Russia using a new test system developed on the basis of highly sensitive methods, such as digital droplet PCR and real-time PCR. Results and Conclusion. 34 out of 182 patients (18.7 %) presented genetic aberrations. These aberrations included mutations R248C (n = 4), S249C (n = 16), G370C (n = 4) and Y373C (n = 8) in the FGFR3 gene, and translocation FGFR3-TACC3 (F17;T11) (n = 2). FGFR3 gene alterations were more frequent in upper urinary tract tumors: mutations/translocations were found in 5/8 (62.5 %) renal pelvic cancer patients, in 2/6 (33.3 %) ureteral cancer patients and in 21/120 (17.5 %) bladder cancer patients (p = 0,008). There was also an association with patient age and a tendency for more frequent detection of mutations in the localized form of the disease (stages 1-2). Conclusion. The developed methods of mutation and translocation testing can be further used in clinical practice to determine the sensitivity of tumours to erdafitinib treatment. Введение. Долговременные результаты лечения метастатического уротелиального рака остаются неудовлетворительными: 5-летняя выживаемость при этом заболевании составляет менее 15 %. Разработка и внедрение в клиническую практику новых, в том числе таргетных препаратов, а также индивидуализация системной терапии уротелиального рака на основе молекулярно-генетического анализа опухолевой ткани являются актуальными задачами. Эрдафитиниб — новый препарат для лечения местнораспространённого и метастатического уротелиального рака с определёнными генетическими изменениями в рецепторах FGFR2 и FGFR3, был зарегистрирован в России в 2023 г. Материалы и методы. В настоящей работе нами было изучено распределение клинически значимых мутаций/транслокаций генов FGFR2/3 на значительной по объёму выборке российских пациентов с уротелиальным раком при помощи собственной тест-системы, разработанной на основе высокочувствительных методов — цифровой капельной ПЦР и ПЦР в реальном времени. Результаты. Генетические нарушения были обнаружены у 34 из 182 (18,7 %) пациентов. Были выявлены следующие аберрации: мутации в гене FGFR3 R248C (n = 4), S249C (n = 16), G370C (n = 4) и Y373C (n = 8), а также транслокации FGFR3-TACC3 (F17;T11) (n = 2). Данные изменения чаще выявлялись в опухолях верхних мочевых путей: мутации/транслокации были найдены у 5/8 (62,5 %) пациентов с раком почечной лоханки, у 2/6 (33,3 %) пациентов с раком мочеточника и у 21/120 (17,5 %) пациентов с раком мочевого пузыря (p = 0,008). Также наблюдалась ассоциация с возрастом пациентов и тенденция к более частому обнаружению мутаций при локализованной форме заболевания (стадии 1-2). Выводы. Разработанные методы тестирования мутаций и транслокаций в дальнейшем могут быть использованы в клинической практике для определения чувствительности опухолей к лечению эрдафитинибом.