Determination of methylation status of DAPK gene in salivary gland tumour tissues and its association with HPV 16 & 18 positivity

Abstract Salivary gland tumours (SGTs) are heterogeneous neoplasms with diagnostic challenges due to morphological diversity. This study explored methylation status of DAPK gene in SGTs and its association with HPV16/18 and p16 protein expression. A total of 45 formalin-fixed, paraffin-embedded SGT samples were analysed using immunohistochemistry and methylation-specific PCR. Among the cases, 66.67% were benign and 33.34% malignant. DAPK methylation was associated with tumour type but not with HPV markers. Overall, HPV16 L1, HPV16/18 E6, and p16 expression were observed in 68%, 88%, and 77% of cases, respectively, with a higher frequency noted in benign tumours. Patterns observed through hierarchical clustering suggested potential differences in molecular profiles between benign and malignant SGTs; however, these findings remain exploratory, as this distribution deviates from established HPV biology, where E6 expression is typically associated with malignancy, suggesting potential non-specific immunoreactivity or methodological limitations. Overall, DAPK methylation exhibited a heterogeneous pattern across samples, indicating that epigenetic alterations may occur independently of HPV-driven mechanisms in SGTs. While distinct molecular patterns were observed, their biological significance remains uncertain. In conclusion, this study shows an association between DAPK methylation and tumour type in SGTs but does not support a definitive role of HPV in driving these epigenetic changes. The findings also highlight limitations associated with reliance on IHC without molecular HPV confirmation. Further studies incorporating PCR-based HPV typing, functional validation, and larger cohorts are required to clarify these relationships.