Investigative project for microbiome characterization and immune response in primary and metastatic melanoma and their correlation with survival outcomes

• Grant number: 17/18134-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: April 16, 2018
• End date: December 15, 2018
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Jacks Jorge Junior
• Grantee: Ciro Dantas Soares
• Supervisor: Delphine J. Lee
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil
• Institution abroad: University of California, Los Angeles (UCLA), United States
• Associated to the scholarship: 15/25905-1 - Akt, COX-2, RUNX1 and MMPs expression in metastatic melanomas and Biotechnology prospection of molecules with antineoplastic activity, BP.DR

Abstract

Previous studies described the role of specific microbes in the development of cutaneous melanoma. However, no studies have correlated the skin microbiome with clinical outcomes or measures of immune response in patients with metastatic melanomas. The main goal of this project is to understand how the microbiota and inflammatory infiltrate influence the metastatic process of cutaneous melanomas. Moreover, specifically, we will investigate whether there are any differences in diversity or specific bacterial species between cutaneous primary and metastatic melanomas and the correlation with outcome in these patients. We also will evaluate the correlation of the presence and levels of specific microorganisms with inflammatory infiltrate and COX-2 expression. Other purpose of our investigation will investigate the presence/absence of microorganisms in the skin tumor tissue associated with disease free or overall survival in patients with primary and metastatic melanomas. The clinical records of patients with malignant melanoma will be selected and grouped in relation to the following: (i) diagnosis of primary malignant melanoma with no metastasis [primary nonmetastatic melanomas (PNMM, n =50)]; (ii) diagnosis of primary malignant melanoma with metastasis (PMM, n =50). CD3, CD20, CD4 and CD8 positive cells will be visualized by the immunohistochemistry technique. We will performed the immunohistochemical reactions on 3-¼m-thick sections of paraffin-embedded tissue. For melanin clearance, the sections will be submitted to a bleaching process. We will utilize the following primary antibodies: anti-COX-2, anti-CD3, anti-CD20, anti-CD4 and anti-CD8. Immunohistochemical staining will be performed with Envision. Slides will subsequently expose to diaminobenzidine tetrahydrochloride and will be counterstained with Carazzi hematoxylin. After IHC reactions, the slides will be scanned into high-resolution images using the AperioScanscope CS Slide Scanner. The quantification of positive cells will be performed with Membrane Image Analysis algorithm. The number of CD3, CD20, CD4 and CD8 positive cells will be correlated with the status of patients and with the microbiome. DNA will be extracted from melanoma tissue using FFPE extraction kit from Qiagen. DNA FFPE Tissue kit per manufacturer's instructions. DNA extracted from paraffin embedded melanoma samples will be sequenced by 16S amplicon sequencing. The 16S V4 region will be enriched, amplified and paired-end sequenced for 250 cycles on the MiSeq instrument. The total of paired-end reads will be obtained and their sequencing quality will be assessed by FastQC. Although the concept of microbiome in not completely novel, this is a solid application that will help to define the effect of intratumoral microbiome dysbiosis associated with the role of immune infiltrate on survival outcomes in MM patients. Better characterization of the microbiome and immune cells present in melanoma tissue is an important area of research.