Methylation heterogeneity of the AQP1 promoter as a candidate prognostic biomarker in cholangiocarcinoma

Cholangiocarcinoma (CC) is an aggressive cancer with limited prognostic biomarkers. Aquaporin 1 (AQP1), a water channel protein, is implicated in cancer progression. Moreover, DNA methylation plays a key role in epigenetic gene regulation. Therefore, the present study aimed to investigate the prognostic significance of AQP1 promoter methylation haplotypes in CC using high-resolution bisulfite amplicon sequencing. AQP1 mRNA expression in 102 patients was evaluated first to examine its prognostic impact. Subsequently, methylation haplotypes in non-neoplastic and neoplastic tissues from 96 patients were analyzed. Methylation heterogeneity was assessed using t-distributed stochastic neighbor embedding (t-SNE) and k-means clustering. Although AQP1 mRNA expression alone was not a robust independent prognostic factor, a t-SNE-based classification of the methylation signature identified two subgroups with distinct overall survival in both non-neoplastic and neoplastic tissues. Multivariate analysis using Firth's penalized Cox regression indicated that this methylation signature was independently associated with prognosis (hazard ratio 0.322, p = 0.001). Similar haplotype alterations were observed in non-neoplastic and neoplastic tissues, consistent with the possibility of an epigenetic field effect. These findings suggest that evaluating the methylation signature of the AQP1 promoter may support future less-invasive prognostic assessment and risk stratification, suggesting its potential utility as a candidate biomarker in CC.