Analysis of Several Common APOBEC-type Mutations in Bladder Tumors Suggests Links to Viral Infection

Abstract

FGFR3 and PIK3CA are among the most frequently mutated genes in bladder tumors. We hypothesized that recurrent mutations in these genes might be caused by common carcinogenic exposures such as smoking and other factors. We analyzed 2,816 bladder tumors with available data on FGFR3 and/or PIK3CA mutations, focusing on the most recurrent mutations detected in ≥10% of tumors. Compared to tumors with other FGFR3/PIK3CA mutations, FGFR3-Y375C was more common in tumors from smokers than never-smokers (P = 0.009), while several APOBEC-type driver mutations were enriched in never-smokers: FGFR3-S249C (P = 0.013) and PIK3CA-E542K/PIK3CA-E545K (P = 0.009). To explore possible causes of these APOBEC-type mutations, we analyzed RNA sequencing (RNA-seq) data from 798 bladder tumors and detected several viruses, with BK polyomavirus (BKPyV) being the most common. We then performed IHC staining for polyomavirus (PyV) Large T-antigen (LTAg) in an independent set of 211 bladder tumors. Overall, by RNA-seq or IHC-LTAg, we detected PyV in 26 out of 1,010 bladder tumors with significantly higher detection (P = 4.4 × 10-5), 25 of 554 (4.5%) in non-muscle-invasive bladder cancers (NMIBC) versus 1 of 456 (0.2%) of muscle-invasive bladder cancers (MIBC). In the NMIBC subset, the FGFR3/PIK3CA APOBEC-type driver mutations were detected in 94.7% (18/19) of PyV-positive versus 68.3% (259/379) of PyV-negative tumors (P = 0.011). BKPyV tumor positivity in the NMIBC subset with FGFR3- or PIK3CA-mutated tumors was also associated with a higher risk of progression to MIBC (P = 0.019). In conclusion, our results support smoking and BKPyV infection as risk factors contributing to bladder tumorigenesis in the general patient population through distinct molecular mechanisms.

Prevention relevance: Tobacco smoking likely causes one of the most common mutations in bladder tumors (FGFR3-Y375C), while viral infections might contribute to three others (FGFR3-S249C, PIK3CA-E542K, and PIK3CA-E545K). Understanding the causes of these mutations may lead to new prevention and treatment strategies, such as viral screening and vaccination.

Figure 1.. BKPyV infection as a possible cause of APOBEC-mediated mutagenesis in NMIBC.

A) Several viruses were detected by RNA-seq analysis of 376 UROMOL-NMIBC tumors: BK Polyomavirus (BKPyV), Herpes Simplex Virus 2 (HSV2), Cytomegalovirus (CMV), Human papillomavirus (HPV), Herpes Simplex Virus 1 (HSV1), Human Betaherpesvirus 7 (HHV7), and Epstein-Barr Virus (EBV). Within the subset of 279 tumors with FGFR3/PIK3CA mutations, APOBEC-type driver mutations in these two genes were most common in the BKPyV-positive tumors. P-values are for Fisher’s exact test. B) Time to progression to MIBC in NMIBC patients with FGFR3 or PIK3CA mutations from the UROMOL study. Progression-free survival (PFS) was evaluated by Kaplan-Meier analysis in 279 patients with FGFR3 or PIK3CA mutations according to BKPyV status (yes/no) by RNA-seq. C) Representative IHC images demonstrating H&E and Large T antigen (LTAg) staining in one of the 5 positive NMIBC tumors; images from additional tumors are shown in Figure S1A. Marked areas are shown at a higher magnification. Arrows point to cells positive for LTAg staining (brown dots). NMIBC patient 1 is a 71-yr old never-smoking male with stage Ta, grade 1 bladder tumor with the APOBEC-type FGFR3-S249C mutation. Positive control for LTAg antibody: HeLa cells transfected with an expression construct for truncated T antigen. The table shows results for NMIBC tumors with FGFR3/PIK3CA mutations based on BKPyV status, as determined by RNA-seq or IHC-LTAg. Tumors positive for BKPyV were more likely to harbor an APOBEC-type FGFR3/PIK3CA mutation than BKPyV-negative tumors. P-values are for Fisher’s exact test. D) Variance stabilized read counts for APOBEC3A (A3A) and APOBEC3B (A3B) RNA expression corresponding to mock-infection and 1, 3, 5, and 7 days post-BKPyV infection. E) Representative coverage plots for BKPyV RNA-seq on different days post-infection in HBLAK cells (n=3) (blue), NMIBC tumors from UROMOL (green) and the only BKPyV-positive MIBC tumor (TCGA-BLCA), in which virus is integrated (red). The y-axis shows the read depth per million human reads. Positions and open reading frames within the 5-Kb BKPyV genome are shown under the X-axis. Agnoprotein – regulates viral proliferation; VP1 and VP2 - encode structural proteins for virion capsids during viral proliferation; LTAg and st - encode large and small tumor (T) antigens involved in the initiation of viral replication that causes an oncogenic transformation of the host genome.