Mutational Signatures and Hypermutation Phenotypes Define Bladder Cancers Arising in Waterpipe and Cigarette Smokers

Bladder cancer is a major malignancy worldwide. Although cigarette smoking is the leading modifiable risk factor, the contribution of waterpipe smoking (WPS), which is increasingly popular globally and often misperceived as safer, remains unclear. In 2019, Lebanese males had the highest bladder cancer incidence in the Arab world (five times the global average), prompting investigation of WPS as a potential carcinogen. We performed a cross-sectional genomic analysis of patients with exclusive WPS and urothelial carcinoma treated at the American University of Beirut Medical Center (n=250 screened; 14 exclusively WPS), integrating targeted sequencing (MSK-IMPACT®) and whole-exome sequencing (TEMPO pipeline). Tumor mutational burden (TMB), microsatellite instability, mutational signatures, and oncogenic alterations were assessed. Previously profiled bladder cancer tumors from cigarette smokers and non-smokers at Memorial Sloan Kettering Cancer Center (n=751) served as comparators. MSK-IMPACT® successfully profiled 13 tumors. WPS-associated tumors exhibited a numerically higher median TMB than cigarette (20 vs. 10 mut/Mb; p=0.10, q=0.2), with 38.5% classified as MSS-hypermutated, a phenotype associated with elevated mutational load despite microsatellite stability. Common mutations included TERT promoter (62%), TP53 and FGFR3 (46%), and PIK3CA (38%). APOBEC mutational signatures predominated across all cohorts, suggesting shared mutagenic processes, while chromatin-modifying gene alterations were less frequent. PD-L1 expression was generally low. Implications: Exclusive WPS induces bladder tumors with overall genomic features resembling cigarette-associated disease, yet with numerically higher TMB and enrichment of MSS-hypermutated phenotypes. These findings provide novel mechanistic evidence of tobacco-related carcinogenesis beyond cigarettes and highlight potential biomarker relevance for immunotherapy in a globally rising exposure group.