Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer

Abstract

In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self-renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations (TERTp^Mut ) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERTp^Mut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort (n = 237). We verified that TERTp^Mut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher-risk disease in nonmuscle invasive bladder cancers (NMIBC). TERTp^Mut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THOR^high /TERTp^Mut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERTp^wt and TERTp^Mut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERTp^Mut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer.

Keywords: TERT promoter methylation; TERT promoter mutations; progression; recurrence; telomerase; urothelial bladder cancer.

Figure 1

THOR methylation in urothelial bladder cancer. (a) THOR methylation status in normal urothelium and tumor tissue in the entire cohort (****, p < 0.0001) (b) The ratio of tumor/normal tissue in paired samples from the same patient (n = 38). Note a mean of twofold increase in THOR methylation status in the malignant tissue. (c) THOR methylation status in different tumor stages. (d) THOR methylation status within tumor grades, low grade has significantly higher THOR methylation than normal urothelium (****, p < 0.0001). NS = nonsignificant. Error bars represent median and Interquartile range (IQR). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Survival estimates for patients with NMIBC stratified by TERT promoter alterations. (a) Disease free and (b) progression free survival for patients with NMIBC stratified by TERT promoter mutation status. (c) Disease free and (d) progression free survival for patients with NMIBC stratified by TERT promoter methylation status. THOR^low = THOR hypomethylated, THOR^high = THOR hypermethylated. TERTp^Wt = TERT promoter wild type (for the two studied mutations); TERTp^Mut = TERT promoter mutant. Yellow = TERTp^Wt and THOR^low; Blue = TERTp^Mut and THOR^high.

Figure 3

Survival analysis of combined TERT promoter alterations in patients with NMIBC. Kaplan–Meier analysis for (a) disease free and (b) progression free survival stratified by combined TERT promoter mutations and THOR methylation status in nonmuscle invasive bladder cancer patients. THOR^low/TERTp^Wt = blue; THOR^low/TERTp^Mut = light red; THOR^high/TERTp^Wt = green; THOR^high/TERTp^Mut = dark red.

Figure 4

Estimated probability of disease progression based on THOR methylation levels. Risk of progression is estimated by any TERT promoter mutation status (wild type or mutant) as a result of increased THOR methylation. Red‐TERT promoter mutant NMIBC, Black‐TERTp^Wt NMIBC.