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.

Bioptic Laboratory Ltd Pilsen Czech Republic

Biorepository and Molecular Pathology Huntsman Cancer Institute University of Utah Salt Lake City UT USA

Department of Head and Neck Surgery and Otorhinolaryngology A C Camargo Cancer Center São Paulo Brazil

Department of Oral Diagnosis Piracicaba Dental School Universidade Estadual de Campinas Piracicaba São Paulo Brazil

Department of Pathology Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Department of Pathology School of Medical Sciences Universidade Estadual de Campinas Tessália Vieira de Camargo 126 Barão Geraldo Cidade Universitária 13414 903 Campinas SP 13083 887 Brazil

Head and Neck Surgery Department University of São Paulo Medical School São Paulo Brazil

Laboratory of Analysis of Biomolecules Tiselius Universidade Estadual de Campinas Campinas São Paulo Brazil

National Institute of Science and Technology in Bioanalytics Campinas São Paulo Brazil

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Integrative diagnostic approach to salivary gland tumors: from clinical evaluation to molecularly targeted therapy