Genomic profile of different stages of carcinoma ex pleomorphic adenoma development: a comprehensive analysis using whole exome sequencing and array-based comparative genomic hybridization

Author(s)

João Figueira Scarini

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2024-02-02

Examining board members

Fernanda Viviane Mariano; Rogério de Oliveira Gondak; Erika Said Abu Egal; Rogério Moraes de Castilho; Pablo Agustin Vargas

• Advisor: Fernanda Viviane Mariano

Abstract

Pleomorphic adenoma (PA) is susceptible to genetic alterations that can result in its malignant counterpart, carcinoma ex-pleomorphic adenoma (CXPA), which is a rare and aggressive tumor with distinct behavior and prognosis. We used whole exome sequencing (WES) to analyze the DNA of 7 patients with PA, 13 with CXPA, and 5 with residual PA. All samples were analyzed for single nucleotide polymorphisms (SNPs) and filtered based on a population disease allele frequency (AF) ?0.2. The results were validated using immunofluorescence. A total of 823 genes with non-synonymous mutations were identified, with 30.5% exclusive to PA, 28.7% to residual PA, and 18.3% to CXPA. Notably, 12.6% of these genes showed persistent mutations from PA to CXPA, while 7.9% showed mutations along the adenoma-carcinoma sequence. The study focused on various genes, including extracellular matrix (ECM)- related genes such as TNXB, tumor suppressor genes like PCDH9, LRMP, KDM5A, RB1, TP53, and key oncogenes such as USP4, MUC4, BRAF, GNAS. These genes are involved in important cancer pathways, including the MYB gene point mutation in all three groups analyzed. Immunofluorescence analysis showed c-Myb expression in all groups, with a decrease along the adenoma- carcinoma sequence, reflecting the impact of copy number alterations (CNAs) in the loss of this gene along the adenoma-carcinoma sequence. These findings were confirmed by analyzing 27 CXPA cases and 14 matched residual PA samples using array-based comparative genomic hybridization (aCGH). Additionally, our aCGH results demonstrate the persistence of PLAG1 chromosomal gain in both residual PA and CXPA. Amplification of HMGA2 and RPSAP52 was prevalent, while GRB7 amplification may represent an initial step in malignant transformation from PA to CXPA. The genes that were amplified in the tumor-transformed areas were enriched for biological processes related to immune signaling, such as INF-? family genes. In the context of the malignant transformation of PA, we observed alterations in FASN and GLUT-1 in malignant tumors, as evidenced by different methodologies. Additionally, the myoepithelial cell appears to play a prominent role in CXPA development through various processes and interactions, although its role is unclear in the literature. Our WES analysis indicates that mutations in different genes and pathways may be linked to myoepithelial cells, especially in the context of changes in the tumor microenvironment with reference to the ECM. It is important to note that the genetic alterations identified in CXPA were already present in the adjacent benign residual area adjacent to the transformation area. In summary, this study illuminates the complex interplay between point mutations and chromosomal alterations that occur concurrently in oncogenes and tumor suppressor genes to facilitate the transformation from PA to CXPA. Our results also highlight the early presence of crucial genetic alterations in the residual benign area, which, although displaying morphological characteristics of a conventional PA, exhibits a pre-malignant molecular signature (AU)

• FAPESP's process: 19/09419-0 - Genomic alterations in the malignant transformation of the Pleomorphic Adenoma
• Grantee: João Figueira Scarini
• Support Opportunities: Scholarships in Brazil - Doctorate