The tumor microenvironment of classical Hodgkin lymphoma (cHL) is clearly responsible for the maintenance of the malignant Hodgkin-Reed-Sternberg (HRS) cells, and Epstein-Barr virus (EBV) has been shown to play a role in this immune evasion. EBV can increase the migration of CD4(+)CD25(+)FOXP3(+) lymphocytes, named regulatory T cells (Tregs). In this study, we assessed the distribution and biological significance of Tregs in patients with cHL. Tissue microarrays were constructed using diagnostic biopsies available in 130 cHL patients and stained with CD4, CD8, CD25, and FOXP3 antibodies. For the present study, only cHL patients whose histology could be confirmed and EBV association established were studied. From the 130 cHL patients selected for this study, 56 were classified as EBV-related and 74 EBV non-related cHL. There were no association between clinical characteristics and the expression of Tregs. However, higher levels of Tregs correlated with EBV presence on HRS cells (p = 0.02), although it did not influence event-free survival (EFS) and overall survival (p = 0.98 and p = 0.59, respectively). This study demonstrates that Tregs expression correlates with EBV presence in HRS cells and has no impact on survival of patients with cHL. Further studies investigating the mechanisms in which EBV recruits Tregs to the tumor microenvironment will contribute not only to our understanding on the pathogenesis of cHL but also to the development of new therapeutic strategies.
4778 Cytotoxic T lymphocyte antigen-4 (CTLA-4) is one of the basic antigens involved in immune responses regulation associated with autoimmune diseases and cancer. Its key role in regulating the immune system has made CTLA-4 an attractive target for cancer. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. Objective: The aim of the study was to evaluate the surface expression of CTLA-4 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods: This is an open study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Eighteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Three patients relapsed, and blood was also drawn at this time. Results: From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 7.36 vs 2.73; P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment and complete response (7.36 vs 4.53; p=0.008), with values similar to healthy controls (4.53 vs 2.73; p=0.07). Interestingly, CD4+CTLA-4+ T lymphocytes on relapse were significantly different from post-treatment values and similar to pre treatment. There was no difference on CD4+CTLA-4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, CD4+CTLA-4+ T lymphocytes strongly correlated with erythrocyte sedimentation rate (ESR) values (r=0.67; p=0.002). Conclusions: We showed that CD4+CTLA-4+ T lymphocytes are increased in Brazilian cHL patients at diagnosis compared with post-treatment values and healthy controls. These results suggest a role of CTLA-4 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. The promising immunotherapy regimen targeting CTLA-4 might be beneficial in classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.
4780 Introduction: Epstein-Barr virus (EBV) can be found latently infecting Reed-Sternberg (RS) malignant cells in approximately 50% of classical Hodgkin lymphoma (cHL) patients in Brazil. EBV signaling leads to a disbalance between effector and regulatory CD4 T lymphocytes in the tumor microenvironment, promoting the immune evasion of RS malignant cells. However, little is known about these lymphocytes subpopulations in the peripheral blood of patients with cHL and how treatment can modify this regulatory/effector ratio. In this study, we analyzed the regulatory and effector CD4+ subpopulations in peripheral blood in patients with EBV related and non-related cHL and the impact of treatment on these cells. Material and Methods: This is an open multicentric study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and after completion of treatment (1 to 4 months). Eighteen healthy blood donors volunteers were recruited as controls. Quantification of regulatory and effector T lymphocytes was done by flow cytometry using CD3, CD8, CD4, CD25, Foxp3, GITR, CD127 and interleukin-17 (IL17) antibodies and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. In this study, only cHL patients whose histology could be confirmed and EBV association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Results: From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+CD25highFoxP3+ and CD4+GITR+ at diagnosis was significantly different from healthy controls (median 1.04 vs 0.26, p=0.02; 4.2 vs 2.2, p=0.003; respectively). CD4+CD127+ T lymphocytes were not different from controls (p=0.3). Additionally, CD4+ T lymphocytes with effector phenotype (CD4+IL17+) were significantly increased in cHL patients compared with controls (0.42 vs 0.13, p<0.001). When we compared pre-treatment values of regulatory and effector CD4+ T lymphocytes with post-treatment values, we did not find any statistical difference. Interestingly, post-treatment values were not statistically different from healthy controls. There was no difference on regulatory and effector CD4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, patients with advanced disease and B symptoms presented with increased CD4+CD25highFoxP3+ and CD4+GITR+ T lymphocytes (p=0.03 and p=0.01, respectively). Conclusions: Our results demonstrate that patients with cHL presented with increased CD4+CD25highFoxP3+, CD4+GITR+ and CD4+IL17+ at diagnosis compared with healthy controls. Also, treatment had no impact on these CD4+ T lymphocytes populations. Probably, the moment blood was drawn after completion of therapy could have influenced our results as we know that immunological reconstitution in patients with cHL may take several months. Further studies investigating these CD4+ T lymphocytes subpopulations together with functional assays will contribute not only to our understanding on the pathogenesis of cHL but also to the development of therapeutic strategies designed to manipulate regulatory activity. Given that the incidence of EBV-related cHL, disease presentation and severity are different in developing countries than in developed ones, we emphasize the importance of this ongoing Brazilian multicentric project. Disclosures: No relevant conflicts of interest to declare.
Introduction CD4+CTLA-4+ T lymphocytes has long been recognized as regulatory T cells, potentially decreasing antitumor immune response. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. CD4+CD127+ T lymphocytes also participate in immune homeostasis and T-cell development. The increased expression of this marker on CD4+ T cells is associated with a effector phenotype. CD127-mediated signaling in human leukemia T-cells that may be of therapeutic value, namely regarding the potential use of PI3K and mTOR pharmacological inhibitors. Increased frequencies of regulatory CD4+ cells, together with decreased effector CD4+ cells in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for the immunosuppression state observed in classical Hodgkin lymphoma (cHL) patients. However, little is known about CD4+ T cells subsets in patients with classical Hodgkin lymphoma (cHL) and how treatment can modify these cells. Objective The aim of the study was to evaluate the surface expression of CTLA-4 and CD127 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods This is an open multicentric study and, so far, we included 54 patients from december 2009 to July 2013. Thirty-four patients have completed therapy until July 2012 and were included in this study. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and CD127 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Nineteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Results From the 34 cHL patients recruited for this study, 17 (50%) were male, 16 (47%) had Epstein-Barr virus (EBV) related cHL, 27 (79%) patients presented with B symptoms and 18 (53%) patients had advanced diseases at diagnosis. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 8.7 (0.8 - 30.3) vs 2.5 (0.7 - 11.2); P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment (8.7 (0.8 - 30.3) vs 3.9 (0.8 - 10.3); p=0.01), with values similar to healthy controls (3.9 vs 2.5; p=0.42). By contrast, CD4+CD127+ T lymphocytes were decreased at diagnosis, with values increasing after therapy (41.2 (3.3 – 75.7) vs 54.9 (17.1 – 81.3); p=0.002), similar to healthy controls (54.9 (17.1 – 81.3) vs 58.2 (41.2 – 89.8); p=0.21). The expression of CD127 on CD4+ T cells negative correlated with the expression of CTLA-4 (p<0.001). In this study, these CD4+ T cells subpopulations were neither associated with treatment response nor relapse. The frequencies of these cells were not correlated with age, gender, disease stage, erythrocyte sedimentation rate (ESR), albumin levels and EBV status. Conclusions In this study we showed a negative correlation between CTLA-4 expression on CD4+ T cells with the expression of CD127 at diagnosis of patients with cHL. These results suggest a role of CTLA-4 and CD127 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. Further studies investigating these CD4+ T lymphocytes subpopulations with functional assays are warranted. The promising immunotherapy regimen targeting CTLA-4 and the use of drugs that alter CD127 signaling might be beneficial in classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.
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.
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