Background







Waterpipe smoking is increasingly prevalent in the Middle East and is expanding globally, often driven by misconceptions regarding reduced harm. Evidence indicates that waterpipe smoke delivers substantial levels of nicotine, carbon monoxide, and carcinogens, yet molecular data describing its biological impact remain limited. Epigenetic modifications, particularly DNA methylation, serve as sensitive indicators of tobacco exposure and are implicated in carcinogenesis. Previous IARC-led work in Lebanon demonstrated that blood DNA methylation signatures can distinguish waterpipe from cigarette smoking and accurately classify smoking type using machine learning. Comparable molecular data are lacking for Saudi Arabia, despite the high regional relevance of waterpipe use. This KAIMRC–IARC collaboration aims to address this gap while supporting technology transfer and harmonization of epigenetic and digital research workflows.







Objectives







The primary objectives are to identify DNA methylation biomarkers associated with waterpipe and cigarette smoking among adults in Saudi Arabia and to estimate the incidence of waterpipe exposure using Saudi Biobank data. Additional objectives include comparison of identified molecular signatures with international IARC datasets, introduction of IARC epigenetic laboratory and digital protocols into Saudi research settings, and generation of early molecular evidence relevant to tobacco control policy. Secondary objectives include exploratory analyses of smoking-related biological pathways.







Methods







This pilot cross-sectional study is initiated in 2026 through a formal collaboration between KAIMRC and the International Agency for Research on Cancer. Blood samples will be obtained from the Saudi Biobank, with supplementary recruitment from MNGHA clinics if required. Adults aged 18 years or older will be classified into waterpipe smokers, cigarette smokers, and non-smoker controls based on documented exposure history. DNA will be extracted using standardized protocols and profiled using the Infinium MethylationEPIC v2.0 array. Laboratory procedures, quality control, normalization, and data processing will follow IARC-developed epigenetic and digital workflows transferred to KAIMRC as part of the collaboration. Differential methylation analyses will compare exposure groups, supported by surrogate variable analysis and dimension reduction techniques. Machine learning models will be developed to evaluate the ability of methylation markers to classify tobacco exposure type. Candidate biomarkers will be prioritized for validation using targeted assays. No outcome data have yet been generated.







Results







At the time of submission, the study is in its initiation phase in 2026. Collaborative activities have focused on protocol harmonization, training, and implementation of IARC quality-assured laboratory and analytical pipelines at KAIMRC. Data generation and downstream analyses are ongoing. Expected outputs include differentially methylated CpGs and regions associated with waterpipe and cigarette exposure, exposure classification model performance metrics, and pathway-level insights. Validation and replication phases are planned within the collaborative framework.







Conclusions and Implications







This KAIMRC–IARC collaborative study will generate the first molecular dataset describing epigenetic biomarkers of waterpipe and cigarette smoking in Saudi Arabia. By integrating epidemiologic data with standardized IARC laboratory and analytical workflows, the project aims to strengthen national research capacity and improve understanding of tobacco-related epigenetic alterations relevant to cancer risk. The findings are expected to support future cessation strategies and contribute evidence to regional and international tobacco control policy discussions.