Proteomic analysis based on mass spectrometry in the malignant transformation of Pleomorphic Adenoma

Author(s)

Reydson Alcides de Lima Souza

• Document type: Master's Dissertation
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2020-07-27

Examining board members

Fernanda Viviane Mariano; Adriana Franco Paes Leme; Claúdia Malheiros Coutinho Camillo

• Advisor: Fernanda Viviane Mariano

Abstract

Pleomorphic adenoma (PA) represents the most common neoplasm of salivary glands. Although being a benign lesion, PA can present recurrences, metastases and malignant transformation. About 6% of cases transform into a carcinoma ex-pleomorphic adenoma (CXPA). For being an unusual neoplasm, CXPA etiopathogenesis is still poorly elucidated, however, it is etiology is believed to be due to the accumulation of genetic and epigenetic alterations in PA. In the context of pathologies, proteomic analysis helps to better understand the events associated with carcinogenesis. Based on these aspects, the aim of this study was to verify and analyze the abundance of proteins, by means mass spectrometry, in cases of PA and CXPA and correlate with the malignant transformation of the PA. Thirty cases of AP and CXAP fixed in formaldehyde and embedded in paraffin were selected. Immunohistochemical stains and hematoxylin and eosin were performed as well as cuts in specific membrane slides for later laser capture microdissection (LCM). Microdissection was performed in all selected cases and only neoplastic cells were microdissected, collected and stored at -80 °C. Subsequently, only 10 PA samples, 16 CXPA samples and 4 residual PA (RPA) samples followed for protein extraction and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The proteomic analysis identified and quantified 240 proteins in total. A heat map, grouping and analysis of the main components was performed. Thirty-nine exclusive proteins were identified in the PA, 4 in the APR and 17 in the CXAP, and the proteins CA2, AFDN and ATP3A2 were proposed as protein signature of the lesions respectively. Regarding the shared proteins, 6 subgroups were created to evaluate the protein abundance among the lesions. Through the analyses with and without imputation, proteins that showed statistically significant differences were selected. After analyzing the networks of associations that the proteins in each subgroup presented, the three highest values of Log2-ratio > 2.5 (for positive regulation) or > -2.5 (for negative regulation) in the mean intensity of label-free protein quantification (LFQ). In conclusion, we demonstrate the proteomic profile of PA along its malignant transformation based on MS. Additionally, 15 proteins (AFDN, AP1M1, APOA1, ATP2A3, CA2, DCD, FABP5, FLG, HBB, HP, IgJ, KRT16, MYH9, SLC4A1 and SYCP1) were proposed as potential markers, some of which may be associated with the progression or suppression of malignant transformation of PA (AU)

• FAPESP's process: 19/06809-2 - Analysis of mass spectrometry in the malignant transformation of the pleomorphic adenoma. correlation of data with gene expression and exome
• Grantee: Reydson Alcides de Lima Souza
• Support Opportunities: Scholarships in Brazil - Master