Regulatory T Lymphocytes and Tumor-Associated Macrophages Have No Impact On Treatment Response and Survival in Brazilian Patients with Classical Hodgkin Lymphoma
Authors: Assis Mariane Cristina Gennari , Campos Antonio H. F. M. , de Oliveira José Salvador Rodrigues , Soares Fernando A , Silva Joyce M. K. , Silva Priscilla B , Penna Adriana M. Damasco , Souza Eni Maria , Baiocchi Otávio Cesar Carvalho Guimarães 
Published: October 2019

Abstract
Abstract 4777
Understanding the mechanisms of how tumor microenvironment of classical Hodgkin lymphoma (cHL) fosters immune privilege and survival of Hodgkin-Reed-Sternberg (HRS) cells is crucial for the development of new biomarkers and therapy strategies. Recently, infiltrating regulatory T CD4+CD25+FOXP3+ lymphocytes (Tregs) and tumor-associated macrophages CD68+ (TAMs) have been shown to play a role in HRS immune evasion, disease progression and survival. However, data arising from studies of different populations of cHL patients are conflicting.

Purpose:
In this study, we evaluated the importance of infiltrating Tregs and TAMs in a subset of 130 cHL patients treated in public hospitals in southeast Brazil and correlated these findings with Epstein-Barr virus (EBV) presence in HRS cells.

Material and Methods:
Tissue microarrays were constructed using diagnostic biopsies available in 130 patients and stained with CD4, CD8, CD25, FOXP3, CD15, CD30, CD68 e LMP1. Quantification of TAMs and Tregs was performed using automated slide scanning and image analysis (Aperio ScanScope XT Slide Scanner and Aperio ImageScope Software with Aperio Positive Pixel Count Sample Macro algorithm). Immunohistochemical scoring ranged from 1 to 4 for the antibodies tested, with higher scores indicating a greater proportion of positive cells. For Tregs and TAMs quantification, score 1 was considered negative (≤ 25 % of Tregs or TAMs) and scores 2, 3, and 4 (more than 25 % of positive cells) were considered positive. All patients underwent similar chemotherapy protocols. For the present study, only cHL patients whose histology could be confirmed and EBV-association established were studied.

Results:
From the 130 cHL patients selected for this study, 56 (43%) were classified as EBV related and 74 (57%) EBV non-related cHL. The expression of Tregs (CD4/CD25/FOXP3) was more common in the EBV related cHL group (p=0.02). TAMs did not correlate with EBV presence in HRS cells. Response to treatment, either complete response or partial response, and relapse rate were independent of Tregs and TAMs quantification and EBV status. Increased Tregs and TAMs in the tumor microenvironment did not influence event-free survival (EFS) and overall survival (OS). For further analysis, we stratified our patients into 4 groups, according to Tregs and TAMs quantification and EBV status and we still did not find any difference on EFS and OS. Additionally, stratified survival analysis according to age, stage and IPS-risk group did not identify any impact of Tregs and TAMs quantification on EFS and OS.

Conclusion:
This study demonstrates that increased Tregs and TAMs in the tumor microenvironment of cHL patients neither correlate with treatment response nor survival. Additionally, increased Tregs correlated with EBV presence in HRS cells. It is well known that the incidence of EBV-related cHL in developing countries is different from that in developed ones, as well as the severity of the disease at presentation, with advanced disease being more common at diagnosis. Our results, although different from those recently published, probably reflect the reality of the Brazilian population enrolled in the public health system, highlighting the importance of studying the same disease and their potential biomarkers within different populations.

Disclosures:
No relevant conflicts of interest to declare.