Discovery metabolomics and genetic analysis reveal lipid pathway alterations associated with malignant phenotype acquisition in pleomorphic adenoma and a novel NTF3::ITPR2 fusion in carcinoma ex pleomorphic adenoma.

Pleomorphic adenoma (PA) is the most common salivary gland tumor. Although it is benign, PA may recur, metastasize, and undergo malignant transformation into carcinoma ex pleomorphic adenoma (CXPA). The mechanisms underlying this transformation are unclear, but it is believed that they involve the accumulation of molecular alterations. This study aimed to analyze the metabolomic profile associated with the acquisition of the malignant phenotype in PA and to identify the metabolic pathways involved in this process. A retrospective analysis was conducted using our institutional Salivary Gland Tumor Registry, which comprises 15 cases each of normal salivary gland (NSG), PA, recurrent PA (RPA), and CXPA. Metabolomic profiling was performed on formalin-fixed, paraffin-embedded tissue samples. Selected CXPA cases underwent genetic sequencing to investigate potential molecular alterations. The analysis revealed changes in carbohydrate, amino acid, and lipid metabolism. Notably, alterations in lipid-related pathways, particularly those involving fatty acids, appeared to play a significanat role in the acquisition of the malignant phenotype. Genetic analysis identified a novel NTF3::ITPR2 fusion in one CXPA case. Additionally, variants were detected in ARID1A, NSD1, XPO1, FOXA1, TP53, GATA2, LZTR1, PIK3CA, IRF4, and CHEK1, with allele frequencies ranging from 10% to 84%, indicating substantial genetic heterogeneity among CXPA cases. This study provides the first comprehensive metabolomic snapshot of malignant phenotype acquisition in PA. Identifying lipid metabolic dysregulation and a novel NTF3::ITPR2 gene fusion highlights potential diagnostic biomarkers and unveils actionable pathways that could be translated into targeted and personalized therapies for salivary gland tumors.