EBV-negative Hodgkin Lymphoma Research Articles

Evidence for distinct mechanisms of immune suppression in EBV-positive and EBV-negative Hodgkin lymphoma.

Epstein Barr Virus (EBV) has been recognized for its ability to transform B lymphocytes and for its association with different types of cancers including Hodgkin lymphoma. In addition, EBV may also modulate the microenvironment of HL. In this study, we aimed to investigate the prevalence of EBV among HL cases in Ethiopia and to assess the tissue cellular composition of EBV-related and EBV-unrelated cases. We constructed a tissue microarray (TMA) of 126 consecutive cases of classical HL (CHL) and nodular lymphocyte predominant HL (NLPHL) from a tertiary cancer centre, Tikur Anbessa Hospital, Addis Ababa, Ethiopia, and evaluated a panel of immunohistochemical markers. The quantification of immune cells was performed using HALO 2.3, a platform for image analysis from Indica Lab Inc. A total of 77/126 (61.1%) of HL cases expressed LMP1/EBER. Infiltration of CD8+, T-bet+ and FoxP3+ cells was higher in the microenvironment of EBV-related CHL, with P values of <0.001, <0.001 and <0.016, respectively. In contrast, the expression of PD1 was higher in the microenvironment of EBV-unrelated CHL cases (P < 0.001). Unlike in Western countries, the majority of HL cases in Ethiopia were associated with EBV. As FoxP3+ and PD1-expressing cells are thought to participate in down regulation of the immune response by different mechanisms, this finding highlights the previously unrecognized possibility that distinct immunosuppressive mechanisms may be ongoing within EBV positive and negative HL types. This may have important prognostic and therapeutic implications.

EBV Plasma Epstein-Barr Virus (EBV) DNA as a Biomarker for Diagnosis of EBV-positive Hodgkin Lymphoma in Syria.

Epstein Barr Virus - positive Hodgkin lymphoma is defined by the presence of Epstein-Barr virus (EBV) in tumor cells. EBV plays an important role in the development and prognosis of Hodgkin's lymphoma. The standard way to detect EBV in Hodgkin lymphoma is immunohistochemistry stains for latent membrane protein-1 (LMP1) in tumor cells. The present study aimed to evaluate plasma Epstein-Barr virus (EBV) DNA as a noninvasive biomarker for diagnosis of EBV-positive Hodgkin lymphoma. The study included 60 newly diagnosed patients with Hodgkin lymphoma, ranging in age from 4 to 60 years, and 55 sex and age-matched controls. (60) Formalin-fixed paraffin embedded blocks of Hodgkin lymphoma tissue samples were used to investigate the EBV by in immunohistochemistry stains for (LMP1) in tumor cells. Plasma EBV DNA was quantified by real-time quantitative polymerase chain reaction (PCR) for all Hodgkin lymphoma patients prior to therapy and for control. The results showed that (25/60, 41.7%) of Hodgkin lymphoma were positive for histological LMP1, whereas plasma EBV DNA was detectable (range from 1.1×103 to 1.5×104 copies/mL, median: 1.1×104 copies/mL) in all EBV-positive Hodgkin lymphoma samples (25/25). EBV DNA was undetectable in all cases of EBV-negative Hodgkin lymphoma (35/35) and all healthy control (55/55). It is worth mentioning that our results demonstrated that the EBV DNA load was high in the EBV associated Hodgkin lymphoma patients suffering poor prognostic state. Plasma EBV-DNA can be used as a noninvasive biomarker for diagnosis of EBV- positive Hodgkin lymphoma.

Prognostic significance of Epstein-Barr virus (EBV) infection inHodgkin lymphoma patients.

The prognostic significance of Epstein-Barr virus (EBV) infection in Hodgkin lymphoma (HL) is still controversial. In this work we sought to examine the prognostic differences between EBV-positive and -negative HL through a series of 131 cases from Tunisia. The status of EBV was assessed by EBV-encoded RNA (EBER) in situ hybridization. Results were correlated to patients' characteristics and outcome. EBV was detected in the malignant cells in 62 of 131 HL cases (47.3%). EBV-positive HL was associated with extreme age classes (≤15 and>45 years; p=0.0001), male gender (p=0.008), mixed cellularity histologic subtype (p=0.03), and inversely with leukocytosis (white blood cells ≥15000/mm3) (p=0.004) and bulky mediastinum (mediastinal-thoracic ratio≥ 0.35) (p=0.0001). On analysis of all patients, no significant difference between survival rates was found between EBV-positive and EBV-negative HL. In contrast, subgroups analysis revealed a negative effect of EBV infection in terms of overall survival in patients with Ann Arbor stages I/II (p=0.03) and nodular sclerosis HL subgroup (p=0.01), and in terms of event free survival in patients with mediastinal-thoracic ratio less than 0.35 (p=0.03). These results suggest that EBV infection affects the survival of particular subgroups of HL, especially those with early-stage of the disease.

Epstein-Barr virus latent membrane protein-1 up-regulates cytokines and correlates with older age and poorer prognosis in Hodgkin lymphoma.

Previously, we reported an association between Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL), older age, and poorer prognosis. The aim of this study was to investigate the mechanisms underlying this association. Transfection of HL cell lines with EBV latent membrane protein-1 (LMP1) resulted in up-regulation of many cytokine genes as assessed by the use of oligonucleotide microarrays. The up-regulation of cytokines was validated by using an inflammatory cytokine protein array: macrophage inflammatory protein (MIP)-1α, MIP-1β, and interleukin (IL)-13. Immunostaining of HL samples (n = 104) showed that expression of MIP-1α, MIP-1β and IL-13 correlated with EBV infection and LMP1 expression. Combined expression of these cytokines was more common in patients aged >60 years (P < 0.001), and was associated with a poorer prognosis (P = 0.042). In another cohort, serum levels of MIP-1α, MIP-1β and IL-13 were increased in HL patients (n = 53) and highest in EBV-positive HL patients as compared with healthy controls (n = 40). Xenograft mice injected with EBV-positive HL cells had higher serum levels of MIP-1α, MIP-1β and IL-13 than mice injected with EBV-negative HL cells, although there was no difference in growth. EBV infection appears to promote the release of cytokines in HL patients, and negatively impacts on patient survival. Physiological immunosenescence probably explains the association between EBV infection and older age. Cytokine modulation is a potential therapeutic target for EBV-positive HL patients.

Changing patterns in the Epstein-Barr virus (EBV)and Hodgkin lymphoma association in Tunisia.

We compared the features of the Epstein-Barr virus (EBV) and Hodgkin lymphoma (HL) association in Tunisia in two periods of time, 1991-2001 (111 cases) and 2002-2012 (122 cases). The investigation of the EBV status by EBER in situ hybridization showed a significant decrease in the prevalence of EBV-positive HL from 69.3% for the period 1991-2001 to 40.1% for the 2002-2012 period (p = 0.00001). EBV positivity has decreased in all age groups but was more pronounced among young patients, in the 15-24-year age group (46.1 vs 10.3%, p = 0.003), in the 25-34-year age group (56.2 vs 25%, p = 0.04), and among children (88.4 vs 59.2%, p = 0.01). This decrease in EBV-positive HL over time contrasted with a remarkable increase in EBV-negative HL in young adults aged 15-34years (51.2 vs 83%; p = 0.001), especially among women (59.1 vs 91.2%; p = 0.01). The decrease in EBV-positive HL over time concerns particularly the nodular sclerosis histological subtype (69.2 vs 31.6%, p = 0.000001). These results indicate that the epidemiology of HL and its association with EBV are changing over time, with a trend toward a Western profile, and point toward the emergence of other environmental causative factors, especially among young women, which remain to be identified.

Disruption of Direct 3-Dimensional (3D) Telomere-TRF2 (Telomere Related Factor 2) Interaction Is a Hallmark of Primary Hodgkin (H) and Reed-Sternberg (RS) Cells

Introduction: Numerical and complex structural chromosomal aberrations are regularly identified in primary mononuclear H and multinucleated RS cells and in Hodgkin's lymphoma (HL) derived H cell lines. The transition from H to RS cells is associated with dynamic progressive 3D telomere dysfunction, major changes in the telomere-protecting shelterin complex and formation of giant "zebra" chromosomes. Analogous findings are observed in an in vitro model for post-germinal center B-cell, Epstein-Barr-virus (EBV)-associated HL (Blood. 2015;125 (13):2101-2110). In this experimental system the EBV encoded oncogene LMP1 mediates multinuclearity through downregulation of TRF2. Thus, we hypothesized that the 3D interaction of telomeres and TRF2 is directly associated with the formation of H and RS cells. To this end, we developed a combined quantitative 3D TRF2-telomere immuno FISH protocol (3D TRF2/Telo-Q-FISH) and applied it to monolayers of primary H and RS cells enriched from diagnostic lymph node biopsies allowing 3D analysis of the entire nuclear content (often not achieved using laser microdissection of H and RS cells, given that their nuclei are generally >10 um in diameter and that this technique is performed on 5 um sections).Methods: 3D TRF2/Telo-Q-FISH(Blood. 2015;125 (13):2101-2110; data supplement) was performed on cultured BJ-5ta normal diploid fibroblasts and B-cell rich suspensions (negative selection) of diagnostic lymph nodes, cultured for 12-36 hours. Quality of H and RS cells was assessed by Wright-Giemsa staining. Lymphocytes, H and RS cells of 10 classical HL were analyzed, 3 of them containing LMP1+ tumor cells (EBV-associated HL).Results: Whereas the BJ-5ta normal diploid control fibroblasts and most internal control lymphocytes showed quantitatively and qualitatively intact 1:1, tight physical 3D association of TRF2 with telomeres, with only few very short telomeres (<20%), the H and RS cells of all 10 cases consistently displayed telomere-TRF2 steric disruption, with a significant increase of very short telomeres (49-71%). Interestingly, two opposite disruption patterns of the 3D telomere-TRF2 interaction were identified.The 3 LMP1+ cases were characterized by abundant telomeres without TRF2 interaction (de-protected). TRF2 spots were rare and the quantitative ratio of telomere/TRF2 signals was > 6:1. This loss of TRF2 expression showed gradual progression from H to RS cells.The 7 LMP1- cases showed a different 3D telomere-TRF2 pattern. Three cases with clinically aggressive disease (stages IVA, IVB and IIIB) were characterized by abundant TRF2 signals, most of them not associated with telomeres. In two of them huge multinucleated RS "ghost" cells without any or only few telomeres but numerous TRF2 signals and internuclear DNA-bridges were observed. In one of these two and in the third case bi-nucleated RS cells with grossly different nuclear telomere content of nucleus one and nucleus two were present. Most importantly, these 3 cases showed an inversion of the quantitative ratio of telomere/TRF2 signals <1:3 and the progressive loss of telomere signals from H to RS cells. The remaining 4 cases had a mixed pattern with an overall moderate loss of TRF2 signals including TRF2 de-protected telomeres but also TRF2 signals not associated with telomeres (quantitative, telomere/TRF2 signals ratio of about 1.5-2:1).Conclusions: Disruption of the 3D TRF2-telomere interaction was demonstrated in 100% of primary H and RS cells. However, two opposite pathogenetic mechanisms appear to be operative: in EBV-associated HL telomere de-protection is due to an impressive loss of TRF2 signals physically linked to telomeres, in agreement with the hypothesis of our in vitro model for EBV-associated HL. In clinically aggressive EBV-negative HL massive attrition of telomere signals is associated with an overwhelming increase in TRF2 signals that are not associated with telomeres. In both scenarios, the telomeres are predominantly very short and the crucial 3D interaction is disrupted, thereby qualifying HL as a shelterin-associated disease with the telomere-shelterin complex playing the role of a "plaque tournante" in the pathogenesis of HL. Further molecular dissection of the shelterin-telomere interaction may offer new therapeutic targets for refractory/relapsing disease. DisclosuresNo relevant conflicts of interest to declare.

DNA methylation patterns in EBV-positive and EBV-negative Hodgkin lymphomas.

Hodgkin lymphoma (HL) is characterized by the presence of Hodgkin and Reed-Sternberg cells. Epstein-Barr virus (EBV) infection is thought to play an important role in the development of HL. Although epigenetic alterations, such as aberrant DNA methylation, are known to contribute to the pathogenesis of various malignancies, little is known about such alterations in HL and their putative relationships with EBV infection. We investigated promoter methylation patterns of seven tumor-associated genes in 53 primary HL cases using methylation-specific PCR (MS-PCR). Concomitantly, the EBV infection status was assessed using PCR, in situ hybridization and immunohistochemistry. The gene promoter hypermethylation frequencies observed were 77.3% for P16, 58.5% for RASSF1A, 50.9% for CDH1, 45.3% for DAPK, 43.4% for GSTP1, 37.7% for SHP1 and 24.3% for MGMT. SHP1 gene promoter hypermethylation was more frequently observed in patients at extreme ages (i.e., ≤ 15 and >54years) than in adult patients (p=0.006) and in patients with B symptoms (p=0.03). Interestingly, most of the analyzed gene promoters were more frequently hypermethylated in EBV-negative than in EBV-positive cases, in particular the DAPK gene promoter (58% versus 27%, p=0.04). Furthermore, hypermethylation of multiple gene promoters (≥ 3) was encountered more frequently in females than in males (86% versus 57%, p=0.04), whereas EBV-positive cases were more common among males than females (55% versus 30%, p=0.02). Our results indicate that epigenetic changes frequently occur in both EBV-positive and EBV-negative HL. The rates of these changes were found to vary according to clinico-pathological parameters. These observations probably reflect the multitude of factors involved in HL development and the complexity of their interactions with genetic and/or hormonal factors.

Human leukocyte antigens and genetic susceptibility to lymphoma.

Familial aggregation, coupled with ethnic variation in incidence, suggests that inherited susceptibility plays a role in the development of lymphoma, and the search for genetic risk factors has highlighted the contribution of the human leukocyte antigen (HLA) complex. In a landmark study published almost 50 years ago, Hodgkin lymphoma (HL) was the first disease to be associated with HLA variation. It is now clear that Epstein-Barr virus (EBV)-positive and -negative HL are strongly associated with specific HLA polymorphisms but these differ by EBV status of the tumours. HLA class I alleles are consistently associated with EBV-positive HL while a polymorphism in HLA class II is the strongest predictor of risk of EBV-negative HL. Recent investigations, particularly genome-wide association studies (GWAS), have also revealed associations between HLA and common types of non-Hodgkin lymphoma (NHL). Follicular lymphoma is strongly associated with two distinct haplotypes in HLA class II whereas diffuse large B-cell lymphoma is most strongly associated with HLA-B*08. Although chronic lymphocytic leukaemia is associated with variation in HLA class II, the strongest signals in GWAS are from non-HLA polymorphisms, suggesting that inherited susceptibility is explained by co-inheritance of multiple low risk variants. Associations between B-cell derived lymphoma and HLA variation suggest that antigen presentation, or lack of, plays an important role in disease pathogenesis but the precise mechanisms have yet to be elucidated.

Free Circulating Epstein Barr Virus DNA Load In Indian Children With Hodgkin Lymphoma As a Biomarker Of EBV-Associated Disease

IntroductionEpstein-Barr virus (EBV) association has been described in a large majority of children with Hodgkin lymphoma (HL) in India. The significance of circulating plasma EBV load and its kinetics during HL therapy is largely unknown. This study aimed at assessing the value of circulating EBV-DNA as a biomarker of EBV-associated pediatric HL and of tumour burden, and the value of serial monitoring during therapy. MethodAll pediatric cases presenting with lymphadenopathy were prospectively recruited between 2007 and 2012. Lymph node biopsy was performed. Children with HL and controls with reactive nodal hyperplasia were enrolled in the study after prior informed consent. Untreated non-lymphoid malignancies and healthy controls were also included in the control group. Plasma EBV real-time quantitative-PCR (RQ-PCR) was assessed with LightCycler2.0, Roche. EBV-associated HL was defined by positive EBV latent membrane protein-1 immunohistochemistry on lymph-node biopsies. Risk-adapted ABVD chemotherapy was given to HL cases, and early treatment response was assessed. RQ-PCR was repeated after the first cycle, at the end of treatment and on follow-up. Kaplan-Meier survival analysis was done, including loss to follow-up as an event. ResultsThirty cases of HL (newly diagnosed–28, relapses–2) and 70 age/ sex-matched controls (benign lymphadenopathy–19, non-lymphoid malignancy–29, Burkitt lymphoma–5, healthy children–17) were included. HL stage distribution was stage I–4, II–9, III–4 and IV–13. EBV immunohistochemistry was positive in 16 (59.3%) out of 27 HL cases analyzed (14/19 MC, 1/6 NS, 0/2 LP, 1 unclassified). RQ-PCR was detectable in 19 (63.3%) out of 30 HL, with 87.5% accuracy (Kappa coefficient=0.69 [0.42-0.97]). All 70 controls were RQ-PCR negative (p<0.0001). RQ-PCR sensitivity and specificity in EBV-associated HL detection were 87.5% and 81.8% respectively. Three out of 4 cases with more than 10,000 EBV copies/mL had advanced stage disease (III-IV) and B symptoms. The highest viral load (430,000 copies/mL) was seen in a boy with stage IV-B disease and end-stage liver involvement. However, viral load was not significantly associated with tumor burden or with survival. All treated Q-PCR positive cases showed EBV clearance after the first cycle. One case, RQ-PCR negative 10 years after primary treatment of stage-I HL, relapsed locally and became RQ-PCR positive. Five-year overall survival (OS) and event-free survival (EFS) were 82.6±8.5% and 77.2±8.3% respectively. OS and EFS were not significantly different in EBV-positive and EBV-negative HL. ConclusionRQ-PCR detection of circulating EBV-DNA is a biomarker of EBV-associated HL, in contrast with pediatric non-lymphoid malignancies, sporadic Burkitt lymphoma and benign lymphadenopathies. EBV-DNA may be used as an early marker of response to therapy in EBV-associated HL and may increase in case of relapse. Disclosures:No relevant conflicts of interest to declare.

Impact of the Epstein–Barr virus positivity on Hodgkin's lymphoma in a large cohort from a single institute in Korea

Epstein-Barr virus (EBV) is considered a prognostic marker in Hodgkin lymphoma (HL) patients, but previous studies have yielded mixed findings because of the confounding effects of factors including age. We examined the prognostic impact of EBV status on 159 patients with HL. The median age at diagnosis was 32 years (range, 4-77 years). The median follow-up time was 5.83 years (range, 0.33-19.69 years). Tumor cell EBV status was positive in 34.5 %. EBV-positive HL was associated with age of ≥ 25 years, male gender, B symptoms, advanced stage, high-risk IPS, nonnodular sclerosis subtype, and treatment with chemotherapy only (P < 0.05). The 5-year disease-specific survival (DSS) rates were 94.1 and 76.4 % for the EBV-negative and EBV-positive HL, respectively, (P < 0.001). On univariate analysis, event-free survival, DSS, and overall survival (OS) were significantly associated with age 40 years or older, B symptoms, and high-risk international prognostic score (≥ 4). On multivariate analysis, EBV positivity was found to be a significant prognostic factor for DSS, particularly in adults 25 years or older. Subgroup analysis showed significant association of EBV-positive HL with poorer DSS and OS in adults 25 years or older with advanced stage disease. In the present series of HL patients, the presence of EBV in tumor cells is associated with adverse prognostic factors. EBV-positive HL is significantly associated with poorer DSS in all age groups.

Hodgkin's lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses.

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.

Correction: Improving T-cell Therapy for Relapsed EBV-Negative Hodgkin Lymphoma by Targeting Upregulated MAGE-A4

Correction: Improving T-cell Therapy for Relapsed EBV-Negative Hodgkin Lymphoma by Targeting Upregulated MAGE-A4

Improving T Cell Therapy for Relapsed EBV-Negative Hodgkin's Lymphoma by Targeting Upregulated MAGE-A4

Abstract 1658Patients with Hodgkin’s Lymphoma (HL) who relapse after hematopoietic stem cell transplant (HSCT) have limited options for long-term cure. We have shown that infusion of cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV+ HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative strategy is to target the cancer/testis antigen MAGE-A4 which is present in EBV antigen-negative Hodgkin’s lymphoma tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. We explored the feasibility of combining adoptive T-cell therapy with epigenetic modification of tumor antigen expression. We first verified whether MAGE-A4-specific CTL could be expanded from autologous and allogeneic donors. Dendritic cells from healthy donors (n=15) or HL patients (n=9) were pulsed with overlapping peptides spanning the MAGE-A4 protein. Healthy donor T-cell lines expanded to sufficient numbers for clinical use and predominantly comprised CD8+ cytotoxic T cells (mean 60%; range 42–81%) and CD4+ T helper cells (mean 20%; range 8–45%). A mean response of 152 interferon gamma (IFNγ) spot forming cells (SFC)/1 × 105 cells (median 78; range 12–635) was observed against MAGE-A4 peptides compared to a mean of 6 IFNγ SFC/1×105 cells (median 4; range 0–30) to irrelevant peptides. Both CD4+ and CD8+ T cell subsets contributed to the interferon response. In T cells expanded from the peripheral blood of patients with HL, a mean of 296 SFC/1×105 cells (range 32–1031) responded to MAGE-A4 peptides compared to a mean of 5 SFC/1×105 cells (range 1–11) responding to irrelevant targets. Five of fifteen CTL lines generated from healthy donors showed specificity for a particular peptide (aa266-285, NPARYEFLWGPRALAETSYV). In contrast, T-cell lines derived from HL patients showed a range of MAGE-A4 epitope specificities. Peripheral blood mononuclear cell and dendritic cell numbers were reduced, and T cells from these patients failed to expand after several stimulations consistent with the profound immune deficiency frequently seen in HL patients. We next evaluated the effect of combining MAGE-CTL with the epigenetic-modifying demethylating agent 2’-deoxy-5’azacytidine. To determine if decitabine would negatively affect the function of the TAA-specific T cells, we examined the effects of the drug on the function of MAGEA4-specific T cells in vitro. Our results showed that treatment of T cells with decitabine – in concentrations that exceed expected levels following drug treatment (1 μM) - did not adversely affect the phenotype and function of MAGE-A4 specific T cells in vitro. To evaluate the in vivo effects of decitabine on tumor antigen specific T cells, patient samples were obtained before and after treatment. In patients who received a decitabine containing regimen (75 mg/m2 daily × 5 days) (n=2), one of whom had clinical responses on PET scan, we observed that MAGE-specific T cells were of greater magnitude and recognized a broader number of MAGE-A4 epitopes after decitabine treatment (see Figure below – a and b are patients with a decitabine containing regimen; c and d are patients receiving no decitabine). In patients (n= 2) who did not receive a decitabine containing regimen, the magnitude of MAGE-specific reactivity was reduced, as measured by IFNγ Elispot. These results suggest that although decitabine may modulate immune function, the effect of other chemotherapies may result in the lack of appreciable endogenous T-cell responses to tumor antigens in these patients. Hence, adoptive transfer of MAGE-A4 specific T cells combined with the administration of epigenetic-modifying drugs to increase expression of the target antigens may improve the efficacy of treatment of relapsed Hodgkin’s Lymphoma. [Display omitted] [Display omitted] Disclosures:Younes:Celgene: Speakers Bureau; Syndax: Research Funding; SBIO: Research Funding; Genentech: Research Funding; Sanofi-Aventis: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau, Travel Expenses; Gilead: Honoraria; Pharmacyclic: Honoraria.

Improving T-cell Therapy for Relapsed EBV-Negative Hodgkin Lymphoma by Targeting Upregulated MAGE-A4

Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells. Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo. Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

The molecular pathogenesis of Hodgkin lymphoma

Hodgkin lymphoma (HL) is an unusual malignancy in that the tumour cells, the Hodgkin and Reed-Sternberg (HRS) cells, are a minor component of the tumour mass, the bulk of which is a mixed cellular infiltrate. There is compelling evidence that HRS cells are clonal B cells that have lost their B cell phenotype. Mature B cells lacking B cell receptors would normally die by apoptosis, and therefore HRS cells must have developed mechanisms to facilitate survival. The escape from apoptosis and transcriptional reprogramming of HRS cells are interlinked and appear central to disease pathogenesis. Epstein-Barr virus (EBV) is present in the HRS cells of a proportion of cases and expresses genes with a plausible oncogenic function. It is likely that EBV plays a role in reprogramming and survival through dysregulation of several signalling networks and transcription factors, including nuclear factor (NF)-κB. Activation of NF-κB is a feature of all HRS cells and gene mutations affecting this pathway appear common in EBV-negative HL. The HRS cell furthers its own survival by attracting a supportive microenvironment of immune and stromal cells, and suppressing local immune responsiveness. Although many questions remain unanswered, the last two decades have witnessed a considerable increase in our knowledge of this complex disease.

Comparison of Clinical Characteristics, Plasma Cytokine Signature, Cytokine Gene Polymorphisms and Outcome Between EBV Positive and Negative Hodgkin Lymphoma: a Study of the Groupe Des Lymphomes De l'Adulte (GELA)

AbstractAbstract 3884 Background:Hodgkin Lymphoma (HL) has a remarkable pathogenesis with rare neoplastic cells surrounded by numerous reactive cells that interact with cytokines, chemokines and soluble receptors. EBV is found in about 40% of HL. Recent clinical series showed that prognostic between EBV-positive and negative could be different especially in patients older than 50 years old, with a worse prognosis for patients with EBV-positive tumors (Diepstra et al. JCO 2009). Gene expression profile analysis of HL tumors are also in favor of different pathogenesis between EBV-positive and negative tumors. In this context, we assessed whether plasma cytokine pattern and cytokine gene polymorphisms could be different regarding EBV status and compared the clinical characteristics and outcome of patients according to EBV status in a series of 242 HL patients. Patients and Methods:the GELA conducted between 1998 and 2002, a prospective study to assess the prognostic role at diagnosis of plasma levels of TNFA, its soluble receptors TNF-R1, TNF-R2, and IL-10, IL-6, IL-1B, IL-1Ra, sCD30 (Casasnovas et al. JCO 2007). This study was also designed to investigate the role of polymorphisms (SNP) in cytokine genes of IL10 (Four SNPs), TNFA, IL6, IL1B, IL1RN, INFG, CCL17 and IL12. EBV status of classical HL samples could be obtained retrospectively for 242 patients. Results:EBV status was obtained according to the immunohistochemistry detection of latent membrane protein-1 (LMP-1) in 186 patients (77%), to EBER in situ hybridation results in 48 patients (20%) or others techniques in the 8 remaining patients (3%). EBV status was considered as positive and negative for 53 (21%) and 189 (79%) patients, respectively. Comparison of clinical characteristics between EBV-negative and positive HL showed some differences in term of sex-ratio and histological subtypes with a higher proportion of male in EBV-positive cohort (81% vs. 54%, P < .01) and less nodular sclerosis subtypes (74% vs. 85% P = .05). Erythrocyte sedimentation rate and white-cell count were more frequently increased in EBV-negative population but the distribution of prognostic categories according to the Hasenclever score was not different between the two cohorts. The TNFA and TNF-R2 plasma levels were significantly higher for EBV-positive compared to EBV-negative HL (P = .03) but no difference was observed for others cytokines (TNF-R1, IL-10, IL-6, IL-1Ra, sCD30). For the eleven studied SNPs, no difference in term of genotype distribution was observed between EBV-positive and negative tumors. The 6-year overall survival (OS) (91% vs. 88% P = .3) and progression free survival (PFS) (81% vs. 79% P = .3) were not statistically different between the EBV-negative and positive cohorts. Analysis of outcome by age groups, showed a non-significant trend for a worse 6-year OS (72.1% vs. 58.3% P = .14) and 6-year PFS (57.2% vs. 48% P = .42) for EBV-positive HL after 50 years old with a similar proportion of EBV-positive and negative HL tumors before and after 50 years old. Conclusions:In this study, we confirm difference of histological subtype distribution between EBV-positive and negative HL. A difference of plasma levels of TNFA and TNF-R2 was observed suggesting a different inflammatory profile between EBV-positive and negative EBV HL but genotype distribution of germline cytokine gene polymorphisms was not different. In this cohort of HL with a global favorable outcome, a trend for a worse prognosis became apparent for older patients with EBV-positive HL. Disclosures:Salles:Roche: Consultancy, Honoraria.

Dominant expression of survival signals of endoplasmic reticulum stress response in Hodgkin lymphoma

The accumulation of viral proteins in endoplasmic reticulum (ER) may cause ER stress responses and lead to either apoptosis or survival depending on the driving signals. The strong expression of latent membrane protein-1 (LMP1) in Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL) cells raises the question whether LMP1-induced ER stress response is associated with the characteristic tumor biology in HL. In this study, we investigated the expression of ER stress signals (glucose-regulated protein 78 [GRP78], X-box binding protein 1 [XBP1], activating transcription factor 6 [ATF6], CCAAT enhance-binding protein homologous protein [CHOP] and phospho-apoptosis signal-regulating kinase 1 [pASK1]) on 156 cases of HL. Furthermore, LMP1 transfection on EBV-negative HL cell lines was used to explore the regulation of ER stress signals by EBV-LMP1. Interestingly, we demonstrated that the survival signals of ER stress response (GRP78, 62%; XBP1u [unspliced], 55%; XBP1s [spliced], 38%; ATF6, 91%) were dominantly expressed over the ER death signals (CHOP, 10%; pASK1, 7%) in all histological subtypes of HL with a similar level in both EBV-positive and EBV-negative cases. However, expression of ER signals did not bear prognostic significance. In vitro, LMP1 transfection increased the expression of GRP78 and XBP1, but attenuated the expression of death signals, CHOP and pASK1. These data indicate that EBV-LMP1 may play a role in shifting EBV-infected cells towards the survival pathway in the presence of ER stress in EBV-positive HL cases.

Plasma EBV-DNA monitoring in Epstein-Barr virus-positive Hodgkin lymphoma patients

Epstein-Barr virus (EBV) is associated with approximately one-third of Hodgkin lymphoma (HL) cases. EBV-DNA is often present in the plasma and whole blood of EBV-associated HL patients. However, the significance of EBV-DNA monitoring is debated. In a cohort of 165 adult HL patients, EBV-DNA viral load was prospectively monitored both in the plasma and whole blood. Diagnostic tissue samples of all patients were histologically reviewed; in 72% nodular sclerosis was detected, 24% presented with mixed cellularity (MC), and 5% had other type of HL. Tissues from 150 patients were also analyzed for the presence of latent EBV infection using in situ hybridization for EBV-encoded RNA (EBER) and immunohistochemistry for latent membrane protein (LMP1). Using these methods, 29 (19%) patients were classified as EBV positive. Using real-time quantitative PCR, 22 (76%) of EBV-positive HL patients had detectable EBV-DNA in the plasma and 19 (66%) patients in whole blood prior to therapy. In the group of EBV-negative HL cases, three (2%) patients had detectable plasma EBV-DNA and 30 (25%) patients whole blood EBV-DNA before treatment. EBV-positive HL was significantly associated with EBV-DNA positivity both in the plasma and whole blood in pretreatment samples, increasing age and MC subtype. Serial analysis of plasma EBV-DNA showed that response to therapy was associated with decline in viral load. Moreover, significantly increased plasma EBV-DNA level recurred before disease relapse in one patient. Our results further suggest that the assessment of plasma EBV-DNA viral load might be of value for estimation of prognosis and follow-up of patients with EBV-positive HL.

3D structural and functional characterization of the transition from Hodgkin to Reed-Sternberg cells

Recent research using an innovative 3D quantitative FISH approach of nuclear remodelling associated with the transition from mononuclear Hodgkin to diagnostic multinuclear Reed-Sternberg cells revealed profound changes in the 3D nuclear organization of telomeres. Analogous 3D telomere dynamics were identified in Hodgkin's lymphoma derived cell-lines and diagnostic patient biopsies. These changes were observed in both, EBV positive and EBV-negative Hodgkin's lymphoma and independent of the age of the patients at presentation. Compared to mononuclear Hodgkin cells, multinuclear Reed-Sternberg cells are characterized by a highly significant increase of telomere aggregates, often composed of very short telomeres, telomere shortening and loss. RS-cells with telomere free "ghost" nuclei are regularly observed. The telomere protecting shelterin complex appears to be disrupted and deregulation of DNA-repair mechanisms is observed. Our findings are consistent with the hypothesis that distinct 3D telomere changes and shelterin disruption represent a common pathogenetic denominator in the generation of Reed-Sternberg cells.

Risk of EBV-Positive Hodgkin Lymphoma Varies Over 30-Fold by HLA Class I Genotype and History of Infectious Mononucleosis.

Abstract 269 Introduction:Markers in the human leukocyte antigen (HLA) class I locus and infectious mononucleosis (IM) have both been associated with Epstein-Barr (EBV)-positive Hodgkin lymphoma (HL). IM is caused by late infection with EBV. To further explore associations between HLA class I genes and EBV-positive HL and to investigate potential interactions between genetic (HLA) and environmental risk factors in the development of HL, we performed a case-only study of 934 HL cases. Methods:HLA-A and B typing were performed by PCR sequence specific oligonucleotide assay. Logistic regression was used to estimate odds ratios (ORs) of EBV-positive HL relative to EBV-negative HL for HLA-A and B genotypes and other known risk factors. Results:Increasing age, male sex, HLA-A*01 and a previous history of IM were all associated with a statistically significant increased odds of EBV-positive HL while HLA-A*02 and HLA-B*07 were associated with a protective effect. Significant interactions were detected between HLA-A*01 and HLA-B*07 and between prior IM and HLA-A*02, such that HLA-B*07 abolished the increased odds associated with HLA-A*01 and HLA-A*02 abrogated the increased odds associated with prior IM. In our final model of EBV-positive HL, HLA-B*07 was associated with a dominant protective effect (OR=0.57, 95% confidence interval (CI): 0.34–0.94); HLA-A*02 was associated with a protective effect which was additive (for individuals with no prior IM, ORper allele =0.69, 95% CI: 0.50–0.97); and HLA-A*01 was associated with an increased odds in patients who were HLA-B*07-negative (ORper allele=2.15, 95% CI: 1.54–3.01). Prior IM was independently associated with EBV-positive HL (OR=3.35, 95% CI: 1.67–6.73). This resulted in a >32-fold higher odds of EBV-positive HL in HLA-A*01 homozygotes with IM compared to HLA-A*02 homozygotes without IM. Conclusions:We have identified strong associations between both genetic and environmental risk factors and EBV-positive HL. Our findings suggest that HLA class I-restricted, cell-mediated responses to EBV and a prior history of IM play critical roles in the pathogenesis of EBV-positive HL. Disclosures:No relevant conflicts of interest to declare.