Malignant transformation in pleomorphic adenoma: the impact of DNA methylation profiling and pathogenic mutations.

Salivary gland tumors (SGTs) are a rare and heterogeneous group of lesions with diverse microscopic appearances and variable clinical behavior. The most common SGT subtype, pleomorphic adenoma (PA), can undergo malignant transformation into carcinoma ex pleomorphic adenoma (CXPA). Carcinomatous transformation from PA to CXPA is a highly progressive and multi-step process. Distinguishing a PA with some atypia from a low-grade intracapsular or minimally invasive CXPA is primarily subjective as no mitotic count thresholds exist to help pathologists distinguish between them in difficult cases. In this prospective study, we collected 140 cases encompassing both PA and CXPA to study their molecular signatures. The primary objective was to investigate the use of DNA methylation profiling as a potential molecular tool for differentiating between these two entities. Methylation analysis was performed on 33 PA cases and 33 CXPA cases. We were able to demonstrate that based on their methylation profiles, PA and CXPA could be classified into three distinct clusters that we called benign, intermediate, and malignant. We also revealed that the presence of TP53, HRAS, PTEN and/or TERT pathogenic mutations were exclusively present in CXPA cases; and that chromosomal alteration on chromosomes 5 and 8 are potentially associated with malignant transformation. In conclusion, our study provides a comprehensive molecular framework for PA and CXPA. The presence of a pathogenic mutation in TP53, HRAS, PTEN, or pTERT or HER2 amplification could be integrated into a molecular diagnosis of CXPA for tumors within the PA CXPA spectrum. In the future, we aim to improve our methylomic classification to make it a precision medicine diagnostic tool for the treatment management of tumors within the PA-CXPA spectrum.