In this study, we evaluated the prognostic significance of APOBEC3B expression in metastatic bladder cancer. In a previous comparative study of 14 cancer types, APOBEC-mediated mutations in bladder cancer were observed at a higher rate than in other cancers . APOBEC mutation has been report to correlate with APOBEC mRNA expression and APOBEC1, APOBEC3A, APOBEC3B, APOBEC3F, and APOBEC3G among of the APOBEC family members are statistically confirmed to correlate . APOBEC3B presented the strongest correlation (Spearman = 0.30, p < 0.001), and the expression level of APOBEC3B mRNA in bladder cancer was significantly higher than the median value of all samples of 14 cancer types evaluated in the previous study . In addition, only APOBEC3B of the APOBEC family showed significantly higher mRNA expression in bladder cancer than in the normal tissue (p < 0.001) . Therefore, we selected APOBEC3B from several APOBEC enzymes as the subject of this study. The survival curves were clearly grouped depending on the APOBEC3B expression status. Previous studies support our current results, although there are no studies on APOBEC3B specifically [18, 19].
APOBEC3 is one of the cytidine deaminases involved in C to U editing . It was first discovered in relation to a restriction factor against viral infection and adaptive immunity through antibody diversification [10, 11]. Subsequently, APOBEC mutation of the tCw motif was identified, and it was found to be highly specific in single-stranded DNA and responsible for somatic mutations that may induce carcinogenesis and cancer evolution in various type of human solid cancers [10, 11]. Gene mutations in bladder cancer resulting in a high expression of APOBEC are frequently observed in genes related to DNA damage response, chromatin modification, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), and tumor protein p53 (TP53) . In contrast, specimens with a low expression of APOBEC are reported to have mutations in KRAS and fibroblast growth factor receptor 3 (FGFR3) . These distinct mutational characteristics suggest that the tumor biology of bladder cancer may be different between those with high and low expression of APOBEC3B and that the APOBEC3B activity may be associated with DNA damage response. A previous study in a breast cancer cell line reported that the loss of tumor suppressor genes, such as TP53, and DNA replication stress induce increased transcription of APOBEC3B . The replication stress increases replication fork stalling, and exposure of single-stranded DNA may favored APOBEC3B function [11, 21]. In addition, in APOBEC3A-expressing cells, inhibition of ataxia telangiectasia and Rad3-related protein (ATR), a protein involved in DNA damage response, results in an increase in apurinic/apyrimidinic (abasic) sites and the accumulation of single-stranded DNA, which drive cells into a replication catastrophe, including massive DNA breakage and cell death . Therefore, we thought that increased APOBEC3B activity may accelerate the production of various neoantigens in bladder cancer cells. The difference in antigenicity may ultimately result in tumor immunity in APOBEC3B-expressing bladder cancer. However, the association between tumor mutation burden and TILs, which can reflect tumor immunity, is still unclear, and further study is needed in this regard .
We assessed intra-tumoral and peri-tumoral TILs, but not tumor mutation burden, in relation to APOBEC3B expression. A previous study reported that a high density of intra-tumoral CD8 + T cells is associated with longer OS (p = 0.02) . Consistent with this finding, intra-tumoral and stromal TILs in breast cancer influence treatment outcomes . In an analysis of TCGA RNA-sequencing data, the expression of the APOBEC3 family members correlated with T-cell markers in various types of solid cancers and APOBEC3G exhibited a strong correlation with activated cytotoxic T-cell markers, such as granzyme B (GZMB) and perforin (PFR1) in high-grade ovarian cancer (r = 0.6591, p < 0.0001 and r = 0.6422, p < 0.0001, respectively) . With respect to tumor mutation burden, APOBEC3B expression is strongly correlated with the total mutation burden in bladder cancer (r = 0.3308, p < 0.001) . These previous study results suggest that in metastatic bladder cancer, the differences in survival curves depending on APOBEC3B expression may be associated with APOBEC-mediated mutations and TILs. Consistent with this, our study showed that there were distinct high-expressing and low-expressing APOBEC3B groups in terms of TILs and survival curves.
Our study results suggested that APOBEC3B was an independent prognostic factor for metastatic urothelial carcinoma and appeared to be associated with TILs, which can represent tumor immunity. However, there were limitations to our study. First, the immune response against cancer cells is substantially complicated and cannot be simply explained by only infiltrating cytotoxic T-cells . Many factors associated with immune suppressive activity are present in the tumor microenvironment, such as TGFβ, and tumor cells continue to evolve through mechanisms such as epithelial–mesenchymal transition, angiogenesis, and immune inhibitory molecule production, which help in promoting immune evasion . We did not evaluate these various factors of the tumor microenvironment in the present study. Second, there is considerable evidence from previous studies regarding correlations between total non-synonymous mutation of cancer-associated genes and mRNA expression of APOBEC3B [11, 12]. However, these studies were limited in relation to the association of mRNA expression with protein expression, especially in metastatic urothelial cancer. Therefore, based on the previous mRNA studies, we assumed that the high expression of APOBEC3B protein may lead to cancer-associated mutagenesis and the first quartile of the APOBEC3B H-score was determined as a cut-off value, because the proportion of APOBEC3B mRNA expression was approximately 80%. Finally, our study was a retrospective study and inevitably was limited in the ability to adjust for various confounding factors. Given the total number of patients and cut-off value, there were fewer patients in the low-expression group compare with those in the high-expression group. A future well-designed prospective study is required to overcome these limitations and to verify our findings.
There are some interesting questions that our study raises. Recognition of tumor antigen is important for infiltrating cytotoxic T-cells to operate in the immune system . For this purpose, immunogenic cell death is needed to expose antigens in identifiable states to appropriate immune cells . Platinum agents are well known to be involved in immunogenic cell death . Perhaps this is related to immune surveillance and is a factor leading to a better prognosis of patients with high APOBEC expression in bladder cancer undergoing cytotoxic chemotherapy than patients with low APOBEC expression . On the contrary, chemotherapy could induce replication stress and higher APOBEC3B expression, which may ultimately lead to the development of and increase in clones during chemotherapy resistant to cytotoxic drugs . Taken together, APOBEC3B may have lasting effects of the initial cytotoxic chemotherapy response to acquired resistance.
Another question is whether APOBEC3B expression influences the effect of ICIs. In bladder cancer, anti PD1 or PD-L1 monoclonal antibodies are among the therapeutic choices for treating patients with metastatic urothelial carcinoma . However, in the present study, we did not evaluate the PD-L1 status, which is often used as a predictive marker for anti PD1 or PD-L1 antibodies. A previous study reported that APOBEC3B expression was marginally associated with PD-L1 expression on immune cells in metastatic urothelial carcinoma (p = 0.05) . Another study suggested that APOBEC-induced mutagenesis affects immune evasion of cancer cells through HLA mutations in urothelial carcinoma . Contrary to a good response expected due to increased tumor mutation burden, these results support the possibility of resistance to immune check point inhibitors in metastatic urothelial carcinoma by harboring APOBEC3B-mediated mutations. Therefore, besides cytotoxic chemotherapy, further studies are needed to evaluate the influence of APOBEC3B mutation with immune check point inhibitors in urothelial carcinoma.