The mutational profile of muscle-invasive urothelial carcinoma and its association with the course of the disease

Study aim: The primary aim of the study was to evaluate the mutational profile of muscle-invasive urothelial car­cinoma (MIUC) using next generation sequencing (NGS). A secondary aim was to identify mutations that provide potential targets for anticancer therapy, while an exploratory aim was to identify the associations between the mutational profile and the course of the disease.

Materials: The study used tumor tissue and medical data from 50 patients with MIUC of the bladder (48 (96.0 %)) or renal pelvis (2 (4.0 %)). DNA and RNA alterations were studied in cells isolated from tumors with histologically confirmed invasive UC using NGS with a panel of 523 genes.

Results: The median age was 72 (51-87) years; the study sample included 43 men (86.0 %). MIUC was confirmed in all patients, either de novo (T2-T4a in 32 (64.0 %) patients, including 2 (4.0 %) patients with renal pelvis cancer) or as a result of progression of non-muscle-invasive bladder cancer (Tis-Tl in 18 (36.0 %) patients). Regional metastases were diagnosed in 8 (16.0 %) subjects, and distant metastases in 5 (10.0 %) patients. High grade UC was confirmed for 44 (88.0 %) samples (including concomitant carcinoma in situ in 4 (8.0 %) cases). The median tumor mutational burden (TMB) was 10.9 (0.0-49.6) muts / Mb (high TMB (> 10 muts / Mb) in 30 (60.0 %) out of 50 cases). The level of microsatellite instability was low in all samples; 244 therapeutically significant and oncogenic mutations in 84 genes were detected in 50 samples (median: 5 (1-11) mutations per sample). Level 1-2 pathogenic mutations were detected in 13 genes of 29 (58.0 %) samples (in > 1 gene in 13 cases (26.0 %)), with a frequency of>10% in the FGFR3 (9 (18.0 %)), TSC1 (9 (18.0 %)), PIK3CA (7 (14.0 %)), ERBB2 (6 (12.0 %)) genes. Level 3-4 mutations were identified in 12 genes of 33 (66.0 %) samples (in > 1 gene in 15 (10.0 %) cases), with a frequency of>10% in the KDM6A (19 (38.0 %)), ARID1A (12 (24.0%)) and MDM2 (7 (14.0%)) genes. Oncogenic mutations were detected in 63 genes of 46 (92.0%) samples (in > 1 gene in 37 (74.0 %) cases), with a frequency of >10% in the TP53 (25 (50.0 %)), FGF4 (5 (10.0 %)), RBI (6 (12.0 %)), CDKN1A, STAG2, FGF3, CCND1 genes (5 (10.0 %) samples with mutations for each). Invasive de novo UC is associated with a higher incidence of high TMB compared with recurrent UC (71.9 % vs. 38.9 %,p = 0.024) and a higher frequency of mutations in the PI3K signaling pathway genes (46.8 % vs. 16.7 %,p = 0.031).

Conclusion: MIUC is characterized by high TMB and low frequency of microsatellite instability. The most common mutations providing potential therapeutic targets are alterations of the FGFR3, TSC1, PIK3CA, and ERBB2 genes. De novo MIUC is associated with a higher frequency of high TMB and an increased frequency of mutations in the PI3K signaling pathway genes compared with invasive recurrence of non-muscle-invasive UC.

1. https://support.illumina.com/content/dam/illuminasupport/documents/documentation/software_documentation/rusight/trusight-oncology500/trusight-oncology-500-local-app-v2.2-user-guide-1000000137777-01.pdf

2. Gudmundsson S., Singer-Berk Мю, Watts N.A., et al. Variant interpretation using population databases: Lessons from gnomAD. Hum Mutat 2022;43(8):1012—1030. https://doi.org/10.1002/humu.24309

3. ClinVar aggregates information about genomic variation and its relationship to human health. Available at: https://www.ncbi.nlm.nih.gov/clinvar/

4. Li M.M., Datto M., Duncavage E.J., et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn 2017;19(l):4-23. https://doi.org/10.1016/).jmoldx.2016.10.002

5. Chakravarty D., Gao J., Phillips S.M., et al. OncoKB: A precision oncology knowledge base. JCO Precis Oncol 2017;2017:PO. 17.00011. https://doi.org/10.1200/PO.17.00011

6. Damrauer J.S., Beckabir W., Klomp J., et al. Collaborative study from the Bladder Cancer Advocacy Network for the genomic analysis of metastatic urothelial cancer. Nat Commun 2022;13(1):6658. https://doi.org/10.1038/s41467-022-33980-9

7. Robertson A.G., Kim J., Al-Ahmadie H., et al. Comprehensive molecular characterization of muscle-invasive bladder cancer. Cell 2018;174(4):1033. https://doi.Org/10.1016/j.cell.2018.07.036

8. Kamoun A., de Reynies A., Allory Y., et al. A Consensus Molecular Classification of Muscle-invasive Bladder Cancer. Eur Urol 2020;77(4):420—433). https://doi.Org/10.1016/j.eururo.2019.09.006

9. Tate J.G., Bamford S., Jubb H.C., et al. COSMIC: The catalogue of somatic mutations in cancer. Nucleic Acids Res 2019;47(D1):D941-D947. https://doi.org/10.1093/nar/gkyl015

10. Abu-Hashish H., Browne R., Zhu X. The impact of next generation sequencing on the management of urothelial cell carcinoma. J Clin Oncol 2023;41(16_suppl):el6576-el6576. https://doi.org/10.1200/JCO.2023.41.16_suppl.el6576

11. Nassar A.H., Umeton R., Kim J., et al. Mutational analysis of 472 urothelial carcinoma across grades and anatomic sites. Clin Cancer Res 2019;25(8):2458-2470. https://doi.org/10.1158/1078-0432.CCR-18-3147

12. Klempner S.J., Fabrizio D., Bane S., et al. Tumor mutational burden as a predictive biomarker for response to immune checkpoint inhibitors: a review of current evidence. Oncologist 2020;25(l):el47-el59. https://doi.org/10.1634/theoncologist.2019-0244

13. Powles T., van der Heijden M.S., Castellano D., et al. Durvalumab alone and durvalumab plus tremelimumab versus chemotherapy in previously untreated patients with unresectable, locally advanced or metastatic urothelial carcinoma (DANUBE): A randomised, open-label, multicentre, phase 3 trial. Lancet Oncol 2020;21(12):1574-1588. https://doi.org/10.1016/S1470-2045(20)30541-6

14. Graf R.P., Fisher V., Huang R.S.P., et al. Tumor mutational burden as a predictor of first-line immune checkpoint inhibitor versus carboplatin benefit in cisplatin-unfit patients with urothelial carcinoma. JCO Precis Oncol 2022;6:e2200121. https://doi.org/10.1200/PO.22.00121

15. Chandran E.B.A., Iannantuono G.M., Atiq S.O., et al. Mismatch repair deficiency and microsatellite instability in urothelial carcinoma: a systematic review and meta-analysis. BMJ Oncol 2024;3(l):e000335. https://doi.org/10.1136/bmjonc-2024-000335

16. Neuzillet Y., Paoletti X., Ouerhani S., et al. A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer. PLoS ONE 2012;7(12):e48993. https://doi.org/10.1371/journal.pone.0048993

17. Ross J.S., Wang K., Al-Rohil R.N., et al. Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy. Mod Pathol 2014;27(2):271-80. https://doi.org/10.1038/modpathol.2013.135

18. Lin Y., Cheng A., Solanki M., et al. Amplification of CCND1 in urothelial carcinoma. J Assoc Genet Technol 2022;48(l):4-9

19. Jindal T., Zhu X., Bose R., et al. Somatic alterations of TP53 and MDM2 associated with response to enfortumab vedotin in patients with advanced urothelial cancer. Front Oncol 2023;13:1161089. https://doi.org/10.3389/fonc.2023.1161089

20. Faltas B.M., Osman M., Evans M.G., et al. CLONEVO: Preoperative abemaciclib for cisplatin-ineligible muscle-invasive bladder cancer (MIBC) with molecular response assessment. J Clin Oncol 2025;43(l6_suppl):Abstract 4520. https://oi.org/10.1200/JC0.2025.43.16_suppl.4520

21. Hurst C.D., Cheng G., Platt F.M., et al. Stage-stratified molecular profiling of non-muscle-invasive bladder cancer enhances biological, clinical, and therapeutic insight. Cell Rep Med 2021;2(12):100472. https://doi.org/10.1016/).xcrm.2021.100472

22. Gridneva Ya.V., Khmelkova D.N., Volkova M.I., et al. Experience of next-generation sequencing in urothelial carcinoma specimens with panel for 523 genes. Journal of Modern Oncology 2024;26(4):489-494 (In Russ.). https://doi.Org/10.26442/18151434.2024.4.203018

23. Myszka A., Ciesla M., Siekierzynska A., et al. Predictive molecular biomarkers of bladder cancer identified by next-generation sequencing-preliminary data. J Clin Med 2024;13(24):7701. https://doi.org/10.3390/jcml3247701

24. Helsten T., Elkin S., Arthur E., et al. The FGFR landscape in cancer: analysis of 4,853 tumors by next-generation sequencing. Clin Cancer Res 2016;22(l):259-67. https://doi.org/10.1158/1078-0432.CCR-14-3212

25. Loriot Y., Necchi A., Park S.H., et al. Erdafitinib in locally advanced or metastatic urothelial carcinoma. N Engl J Med 2019;381(4):338-48. https://doi.org/10.1056/NEJMoal817323

26. Knowles M.A. FGFR3 - a central player in bladder cancer pathogenesis? Bladder Cancer 2020;6:1-21. https://doi.org/10.3233/BLC-200373

27. Adib E., Klonowska K., Giannikou K., et al. Phase II clinical trial of everolimus in a pan-cancer cohort of patients with mTOR pathway alterations. Clin Cancer Res 2021;27(14):3845-3853. https://doi.org/10.1158/1078-0432.CCR-20-4548

28. Bellmunt J., Maroto P., Bonfill T., et al. Dual mTORl/2 inhibitor sapanisertib (FTH-003/TAK-228) in combination with weekly paclitaxel in patients with previously treated metastatic urothelial carcinoma: a phase II open-label study. Clin Genitourin Cancer 2024;22(5):102123. https://doi.Org/10.1016/j.clgc.2024.102123

29. Janku F., Tsimberidou A.M., Garrido-Laguna I. PIK3CA mutations in patients with advanced cancers treated with PI3K/AKT/mTOR axis inhibitors Mol Cancer Ther 2011;10(3):558-565. https://doi.org/10.1158/1535-7163.MCT-10-0994

30. Durinck S., Moreau Y., Kasprzyk A., et al. BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis. Bioinformatics 2005;2l(l6):3439—3440. https://doi.org/10.1093/bioinformatics/bti525

31. Patel V.G., McBride R.B., Lorduy A.C., et al. Prognostic significance of PIK3CA mutation in patients with muscle-invasive urothelial carcinoma (UC). J Clin Oncol 2016:34(15_suppl):el6002. https://doi.org/10.1200/JCO.2016.34.15_suppl.el6002

32. Loibl S., Majewski I., Guarneri V., et al. PIK3CA mutations are associated with reduced pathological complete response rates in primary HE.R2-positive breast cancer: Pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab. Ann Oncol 2016;27(8):1519-25. https://doi.org/10.1093/annonc/mdwl97

33. Machaalani M., Zarif T.E., Stockhammer P., et al. ERBB2 mutations and association with molecular phenotype in urothelial carcinoma. J Clin Oncol 2024;42(16_suppl):4590. https://doi.org/10.1200/JCO.2024.42.16_suppl.4590

34. Soria F., Moschini M., Haitel A., et al. HER2 overexpression is associated with worse outcomes in patients with upper tract urothelial carcinoma (UTUC) WorldJ Urol 2017;35(2):251-259. https://doi.org/10.1007/s00345-016-1871-x

35. Wysocki P.J., Jung K.H., Oh D.-Y., et al. Efficacy and safety of trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-expressing solid tumors: Results from the bladder cohort of the DESTINY-PanTumor02 (DP-02) study. J Clin Oncol 2024;42(16_suppl):4565. https://doi.org/10.1200/JCO.2024.42.16_suppl.456

36. Hamilton E., Gaisky M.D., Ochsenreither S., et al. Trastuzumab deruxtecan with nivolumab in HER2-expressing metastatic breast or urothelial cancer: analysis of the phase lb DS8201-A-U105 study. Clin Cancer Res 2024;30(24):5548— 5558. https://doi.org/10.1158/1078-0432.CCR-24-1513

37. Zimpfer A., Kdimati S., Mosig M., et al. ERBB2 Amplification as a predictive and prognostic biomarker in upper tract urothelial carcinoma. Cancers (Basel) 2023;15(9):2414. https://doi.org/10.3390/cancersl5092414

38. Rehman H., Chandrashekar D.S., Balabhadrapatruni C., et al. ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors. JCI Insight 2022;7(16):el55899. https://doi.org/10.1172/jci.insight.155899

39. Gui Y., Guo G., Huang Y., et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat Genet 2011;43(9):875-878. https://doi.org/10.1038/ng.907

40. Andricovich J., Perkail S., Kai Y., et al. Loss of KDM6A activates super-enhancers to induce gender-specific squamous-like pancreatic cancer and confers sensitivity to BET inhibitors. Cancer Cell 2018;33(3):512-526.e8. https://doi.org/10.1016/j.ccell.2018.02.003

41. Chen X., Lin X., Pang G., et al. Significance of KDM6A mutation in bladder cancer immune escape. BMC Cancer 2021;21(1):635. https://doi.org/10.1186/sl2885-021-08372-9

42. Qiu H., Makarov V., Bolzenius J.K., et al. KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and drives cell proliferation in bladder cancer. Cancer Res 2023;83(6):814-829. https://doi.org/10.1158/0008-5472.CAN-22-1444.