Quantification of Regulatory/Effector CD4+ T Lymphocytes Pre and Post-Treatment in Patients with Classical Hodgkin Lymphoma in Brazil

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4780-4780
Author(s):  
Joyce M. K. Silva ◽  
Adriana M. Damasco Penna ◽  
Maria Mirtes Sales ◽  
Elma Maria Chaves ◽  
Priscilla Brito Silva ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Post Treatment ◽  
Interleukin 17 ◽  
Healthy Controls ◽  
Malignant Cells ◽  
Classical Hodgkin Lymphoma ◽  
Multicentric Study ◽  
The Impact

Abstract Abstract 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.

CD4+ctla-4+ t Lymphocytes Inversely Correlate With CD4+CD127+ T Cells In Brazilian Patients With Classical Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4240-4240
Author(s):  
Joyce M. K. Silva ◽  
Adriana M. Damasco Penna ◽  
Maria Mirtes Sales ◽  
Elma Maria Chaves ◽  
Priscilla Brito Silva ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Lymphocytes ◽  
Hodgkin Lymphoma ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
Post Treatment ◽  
Healthy Controls ◽  
Classical Hodgkin Lymphoma ◽  
Epstein Barr

Abstract 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.

Impact of Treatment On CD4+CTLA-4+ T Cell Subset in Brazilian Patients with Classical Hodgkin Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4778-4778
Author(s):  
Joyce M. K. Silva ◽  
Maria Mirtes Sales ◽  
Adriana M. Damasco Penna ◽  
Elma Maria Chaves Maria Chaves ◽  
Priscilla B Silva ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Hodgkin Lymphoma ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
Post Treatment ◽  
Healthy Controls ◽  
Classical Hodgkin Lymphoma

Abstract Abstract 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.

Impact Of Treatment On Regulatory CD4+ T Lymphocytes, TH17 and Cytokines In Patients With Classical Hodgkin’s Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1756-1756
Author(s):  
Adriana M. Damasco Penna ◽  
Priscilla Brito Silva ◽  
Joyce M. K. Silva ◽  
Maria Mirtes Sales ◽  
Elma Maria Chaves ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Treatment Strategies ◽  
Interleukin 17 ◽  
Barr Virus ◽  
Epstein Barr ◽  
The Impact

Abstract Introduction Although immunosuppression has long been recognized in classical Hodgkin lymphoma (cHL), the underlying basis for the lack of an effective immune response against the tumor remains unclear. Increased frequencies of regulatory CD4+ T lymphocytes in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for this anergic state. However, little is known about the disbalance between regulatory and effector CD4+ subpopulations and cytokines in the peripheral blood of cHL patients and how treatment can modify this regulatory/effector ratio. In this study, we analyzed the regulatory and effector CD4+ subpopulations together with pro and anti-inflammatory cytokines in peripheral blood of cHL patients and the impact of treatment on these cells and cytokines. Material and Methods This is an open multicenter study and, so far, we included 54 patients from December 2009 to July 2013. Thirty-four patients have completed therapy on July 2012 and were included in this study. Blood was drawn at diagnosis and after completion of treatment (1 to 4 months). Nineteen 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, CTLA4, GITR and interleukin-17 (IL17) antibodies. Ten cytokines were studied: IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, sIL-2Rα, TNF-α, IFN-γ, and VEGF. Cytokine levels were determined by multiplexed immunoassay system. All these parameters were 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 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. Results of subsets of CD4+ T cells and cytokines are summarized in the following table: After treatment, the percentage of regulatory CD4+CD25highFoxP3+ and effector CD4+IL17+ T lymphocytes were not different from diagnosis (0.9 vs 0.4, p=0.45; 0.6 vs 0.9, p=0.52; respectively) and from controls (0.9 vs 0.3, p=0.22; 0.6 vs 0.7, p=0.84; respectively). Interestingly, increased CD4+CD25highFoxP3+ T lymphocytes were correlated with advanced disease at diagnosis (p=0.03) and an erythrocyte sedimentation rate (ESR) > 30 mmHg (p=0.01). Additionally, we found a negative correlation between soluble IL-2Rα and CD4+GITR+ (p=0.02) and CD4+FOXP3+ (p=0.02). Conclusions In this study, we showed that, after treatment, there was a decrease of some subsets of CD4+ T cells with regulatory phenotype, together with a decrease of IL-6, IL-10 and sIL-2Rα. Understanding cHL associated immunosuppression and the immune reconstitution after treatment maybe the key to develop new treatment strategies, designed to manipulate regulatory activity. Further studies investigating these CD4+ T lymphocytes subpopulations with functional assays are warranted. 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 multicenter project. Disclosures: No relevant conflicts of interest to declare.

Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3326-3334 ◽  
Author(s):  
Alexey Ushmorov ◽  
Olga Ritz ◽  
Michael Hummel ◽  
Frank Leithäuser ◽  
Peter Möller ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Epigenetic Silencing ◽  
Chain Gene ◽  
Classical Hodgkin Lymphoma ◽  
Regulatory Regions ◽  
The Impact

Abstract Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of “crippling” mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells. (Blood. 2004;104:3326-3334)

scholarly journals Detection of classical Hodgkin lymphoma specific sequence in peripheral blood using a next-generation sequencing approach

2015 ◽  
Vol 169 (5) ◽  
pp. 689-693 ◽  
Author(s):  
Yasuhiro Oki ◽  
Sattva S. Neelapu ◽  
Michelle Fanale ◽  
Larry W. Kwak ◽  
Luis Fayad ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Classical Hodgkin Lymphoma ◽  
Next Generation ◽  
Specific Sequence ◽  
Generation Sequencing

Abstract 2716: A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma

2017 ◽  
Author(s):  
Lance U'Ren ◽  
David Wu ◽  
Jonathan R. Fromm ◽  
Jackie Stilwell ◽  
Eric Kaldjian
Keyword(s):  
Hodgkin Lymphoma ◽  
Malignant Cells ◽  
Classical Hodgkin Lymphoma

The PD1/PD-L1 Axis in the Classical Hodgkin Lymphoma Microenvironment: PD-1 Is Rarely Expressed but Identifies Patients with High-Risk Disease

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1560-1560
Author(s):  
Paul Greaves ◽  
Andrew James Clear ◽  
Andrew Owen ◽  
Andrew Wilson ◽  
Janet Matthews ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Outcome ◽  
Hodgkin Lymphoma ◽  
Adverse Outcome ◽  
Germinal Centre ◽  
Automated Image Analysis ◽  
Image Analysis System ◽  
Malignant Cells ◽  
Classical Hodgkin Lymphoma ◽  
Median Expression

Abstract Abstract 1560 Introduction: Classical Hodgkin lymphoma (CHL) is histologically unique: the malignant Hodgkin Reed-Sternberg cell (HRS) represents a fraction of tumour bulk, the majority being inflammatory cells: a combination of inadequate immune response and tumor-supportive microenvironment. PD-1 is a T-cell expressed CD28-analogue that inhibits activity of the expressing cell by interactions with ligands including the B7 analogue PD-L1. PD-L1 is upregulated in a range of immune and non-immune cells including malignant cells. Immunosuppression through this axis may contribute to failed immune response and adverse outcome in some tumors (Keir et al. Annu Rev Immunol, 2008; 26: 677–704) and there are reports regarding expression levels and pathway activity (Chemnitz et al. Blood, 2007; 110: 3226–33) as well as biological importance in CHL (Yamamoto et al. Blood, 2008; 111: 3220–3224). PD-L1 is overexpressed by HRS cells and encoded by a chromosomal region showing recurrent copy number gains in CHL (Steidl et al. Blood, 2010; 116: 418- 427). PD1/PD-L1 interactions are also essential to the survival of germinal centre B cells (Good-Jacobson et al. Nature Imm; 2010; 11: 535–42) the normal counterparts of the HRS, implying a potential direct supportive role for this pathway independent of the immune response. While the mechanistic importance of this axis in CHL is widely stated, little published evidence exists to substantiate it. This immunohistochemical (IHC) study set out to determine levels and variability of expression of PD-1 and PD-L1 at diagnosis in CHL and determine prognostic importance. Methods: IHC analysis was performed on tissue microarrays (TMAs) from 122 previously untreated CHL patients with known clinical outcome: median age 30 (range 18–80), 35% female, 71% advanced stage, median follow up 16.5 (range 2–40) years. Triplicate cores were made from areas of high cellularity avoiding fibrotic or acellular portions, arrayed and stained for PD-1 or PD-L1. Positive cells were counted across all cores using an automated image analysis system (Ariol), output confirmed by expert histopathologists, and means calculated corrected to % infiltration per 1mm2. 105 cases (86%) had tissue of sufficient quality for full IHC assessment. Groups were determined based on population distribution of infiltration levels and confirmed using the test/validation set method. Outcomes of freedom from first-line treatment failure (FFTF), disease-specific survival (DSS) and overall survival (OS) were assessed using the Kaplan Meier method with statistically significant differences between groups calculated using the log-rank test. Results: PD-1 expression was strikingly low or absent in the microenvironment of the majority of cases: <1% of cells in 40% of cases and <5% of cells in 80%. Median expression was 0.14% of all nucleated cells (range 0–8.9%). When present, this was predominantly as a low-level background infiltrate with frequent clusters/nodules of PD-1 positive lymphoid cells aggregating around HRS cells. Notably, the rare patients with high expression of PD-1 had adverse outcome. The 20% of patients with infiltration >0.5% cells had DSS 61% vs 89% at both 5 and 10 years (p=0.03). In contrast, PD-L1 was expressed at a high level in both HRS and the microenvironment in the majority of cases: >10% cells in 38% of cases, and <1% in only 5%. Median expression was 6.1% (range 0.2%–40%). Level of PD-L1 expression in the microenvironment was not associated with any measure of clinical outcome. Conclusions: PD-1 may have biological significance, as measured by impact on prognosis in a minority of patients with CHL, where overexpression is associated with an increased chance of CHL-related death. This may relate to immunosuppression, but the very low overall levels of expression and tendency to cluster around the malignant cells (a pattern seen more often in nodular lymphocyte predominant HL) imply a more localised HRS interactive role perhaps equivalent to the pathway's activity in a normal germinal centre. Its ligand PD-L1 is highly expressed in the microenvironment as well as in the HRS, which may relate to an activated rather than suppressive microenvironment, but without association with prognosis. We conclude that for the majority of patients with CHL, the definitive role for this pathway remains to be demonstrated conclusively. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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