EBV-associated Hodgkin Lymphoma Research Articles

212 Human ASXL1 Deficiency Causes Epigenetic Dysfunction, Combined Immunodeficiency and EBV–Associated Hodgkin Lymphoma

212 Human ASXL1 Deficiency Causes Epigenetic Dysfunction, Combined Immunodeficiency and EBV–Associated Hodgkin Lymphoma

PD-1 and LAG-3 expression in EBV-associated pediatric Hodgkin lymphoma has influence on survival

In pediatric Hodgkin lymphoma (HL), the inability of the cytotoxic microenvironment induced by EBV presence to eliminate tumor cells could reflect the fact that the virus might be able to induce the expression of exhaustion markers to evade an immune response. Therefore, the expression of exhaustion markers in pediatric EBV–associated HL was evaluated. A balance between cytotoxic GrB and Th1 Tbet markers with regulatory Foxp3 was proved in EBV+ cases. In addition, exclusively in EBV-associated cHL, a correlation between PD-1 and LAG-3 expression was observed. Furthermore, those cases also displayed a trend to worse survival when they expressed LAG-3 and inferior event-free survival when both PD-1 and LAG-3 molecules were present. Therefore, even though a cytotoxic and inflammatory environment was supposed to be triggered by EBV presence in pediatric cHL, it seems that the virus may also induce the synergic effect of inhibitory molecules LAG-3 and PD-1 in this series. These observations may reflect the fact that the permissive and exhausted immune microenvironment succeeds to induce lymphomagenesis.

Lymphotropic Viruses EBV, KSHV and HTLV in Latin America: Epidemiology and Associated Malignancies. A Literature-Based Study by the RIAL-CYTED

The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affect young children, unlike in developed countries, in which adolescents and young adults are the most affected, correlating with an early EBV seroconversion for LA population despite of lack of infectious mononucleosis symptoms. High endemicity of KSHV and HTLV infection was observed among Amerindian populations, with differences between Amazonian and Andean populations.

Risk factors for Epstein Barr virus-associated cancers: a systematic review, critical appraisal, and mapping of the epidemiological evidence.

BackgroundEpstein Barr Virus (EBV) infects 90%-95% of all adults globally and causes ~ 1% of all cancers. Differing proportions of Burkitt’s lymphoma (BL), gastric carcinoma (GC), Hodgkin’s lymphoma (HL) and nasopharyngeal carcinoma (NPC) are associated with EBV. We sought to systematically review the global epidemiological evidence for risk factors that (in addition to EBV) contribute to the development of the EBV-associated forms of these cancers, assess the quality of the evidence, and compare and contrast the cancers.MethodsMEDLINE, Embase and Web of Science were searched for studies of risk factors for EBV-associated BL, GC, HL and NPC without language or temporal restrictions. Studies were excluded if there was no cancer-free comparator group or where analyses of risk factors were inadequately documented. After screening and reference list searching, data were extracted into standardised spreadsheets and quality assessed. Due to heterogeneity, a narrative synthesis was undertaken.Results9916 hits were retrieved. 271 papers were retained: two BL, 24 HL, one GC and 244 NPC. The majority of studies were from China, North America and Western Europe. Risk factors were categorised as dietary, environmental/non-dietary, human genetic, and infection and clinical. Anti-EBV antibody load was associated with EBV-associated GC and BL. Although the evidence could be inconsistent, HLA-A alleles, smoking, infectious mononucleosis and potentially other infections were risk factors for EBV-associated HL. Rancid dairy products; anti-EBV antibody and EBV DNA load; history of chronic ear, nose and/or throat conditions; herbal medicine use; family history; and human genetics were risk factors for NPC. Fresh fruit and vegetable and tea consumption may be protective against NPC.ConclusionsMany epidemiological studies of risk factors in addition to EBV for the EBV-associated forms of BL, GC, HL and NPC have been undertaken, but there is a dearth of evidence for GC and BL. Available evidence is of variable quality. The aetiology of EBV-associated cancers likely results from a complex intersection of genetic, clinical, environmental and dietary factors, which is difficult to assess with observational studies. Large, carefully designed, studies need to be strategically undertaken to harmonise and clarify the evidence.RegistrationPROSPERO CRD42017059806.

Natural Killer cell transcriptome during primary EBV infection and EBV associated Hodgkin Lymphoma in children-A preliminary observation.

Epstein Barr Viral infection is a common childhood infection in India and is also nearly 100 % etiologically associated with pediatric Hodgkin Lymphoma (HL). The main question in EBV immunobiology has been, why only a small subset of infected individuals develop EBV associated malignancies, while the vast majority carry this virus asymptomatically for life. Natural Killer (NK) cells, with a phenotype of CD56dim CD16+ exhibit potent cytotoxicity towards both virus infected cells and transformed cells and hence have been considered to be crucial in preventing the development of symptomatic EBV infection and lymphoma. In order to get an insight into the various possible molecular aspects of NK cells, in the pathogenesis of both these EBV mediated diseases in children we studied the whole transcriptome of MACS sorted CD56dim CD16 + NK cells from four patients from each of the three groups of children viz. Infectious Mononucleosis (IM), HL and age matched controls by using a massively parallel sequencing approach. NK cells from both IM and HL had down-regulated innate immunity and chemokine signaling genes. While down-regulation of genes responsible for polarization of the secretory apparatus, activated NK cell signaling and MAP kinase signaling were exclusive to NK cells in patients with IM, in NK cells of HL, specifically, genes involved in extracellular matrix (ECM) - receptor interaction, cytokine-cytokine receptor interaction, TNF signaling, Toll-like receptor signaling pathway and cytosolic DNA-sensing pathways were significantly down-regulated. Enrichment analysis showed STAT3 to be the most significant transcription factor (TF) for the down-regulated genes in IM, whereas, GATA1 was found to be the most significant TF for the genes down-regulated in HL. Analysis of protein interaction network identified functionally important protein clusters. Top clusters, comprised of down-regulated genes, involved in signaling and ubiquitin-related processes and pathways. These may perhaps be responsible for the hypo-responsiveness of NK cells in both diseases. These possibly point to different deficiencies in NK cell activation, loss of activating receptor signaling and degranulation in IM, versus loss of cytokine and chemokine signaling in HL, in the two EBV associated pathologies investigated. Various suppressed molecules and pathways were novel, which have not been reported earlier and could therefore be potential targets for immunotherapy of NK cell reactivation in both the diseases in future.

Differences in Epstein-Barr Virus Characteristics and Viral-Related Microenvironment Could Be Responsible for Lymphomagenesis in Children.

In Argentina, Epstein-Barr virus (EBV) presence is associated with Hodgkin lymphoma (HL) in patients younger than 10 years, suggesting a relationship between low age of EBV infection and HL. Given that HL is derived from germinal centers (GC), our aim was to compare EBV protein expression and microenvironment markers between pediatric HL patients and EBV+GC in children. Methods: EBV presence and immune cell markers were assessed by in situ hybridization and immunohistochemistry (IHC). Results: Viral latency II pattern was proved in all HL patients and in 81.8% of EBV+ tonsillar GCs. LMP1 and LMP2 co-expression were proved in 45.7% HL cases, but only in 7.7% EBV+ GC in pediatric tonsils. An increase in CD4+, IL10, and CD68+ cells was observed in EBV+ GC. In pediatric HL patients, only the mean of IL10+ cells was statistically higher in EBV+ HL. Conclusions: Our findings point us out to suggest that LMP1 expression may be sufficient to drive neoplastic transformation, that an immune regulatory milieu counteracts cytotoxic environment in EBV-associated Hodgkin lymphoma, and that CD4+ and CD68+ cells may be recruited to act in a local collaborative way to restrict, at least in part, viral-mediated lymphomagenesis in tonsillar GC.

Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency.

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.

The Use of 3D Telomere FISH for the Characterization of the Nuclear Architecture in EBV-Positive Hodgkin's Lymphoma.

The 3D nuclear architecture is closely related to cellular functions and chromosomes are organized in distinct territories. Quantitative 3D telomere FISH analysis (3D Q-FISH) and 3D super-resolution imaging (3D-SIM) at a resolution up to 80 nm as well as the recently developed combined quantitative 3D TRF2-telomere immune FISH technique (3D TRF2/Telo-Q-FISH) have substantially contributed to elucidate molecular pathogenic mechanisms of hematological diseases. Here we report the methods we applied to uncover major molecular steps involved in the pathogenesis of EBV-associated Hodgkin's lymphoma. These methods allowed us to identify the EBV-encoded oncoprotein LMP1 as a key element in the formation of Hodgkin (H-cell) and multinucleated Reed-Sternberg cells (RS-cell), the diagnostic tumor cell of classical Hodgkin's lymphoma (cHL). LMP1 mediates multinuclearity through downregulation of shelterin proteins, in particular telomere repeat binding factor 2 (TRF2).

Risk Factors and Clinical Significance of Lymphopenia in Survivors of the Fontan Procedure for Single-Ventricle Congenital Cardiac Disease

Congenital cardiac anomalies are associated with immunologic perturbations. Surgical thymectomy, thoracic duct manipulation, and protein- losing enteropathy (PLE), a condition related to stressed Fontan hemodynamics, presumably contribute to low peripheral absolute lymphocyte counts (ALCs) and quantitative immunoglobulins. Clinical significance of lymphopenia and hypogammaglobulinemia in single-ventricle survivors requires additional study. Although immunologic laboratory anomalies are common in this population, we hypothesize that clinically significant immunodeficiency requiring intervention is rarely required. A retrospective chart review of the immunologic parameters of patients enrolled in the Single Ventricle Survivorship Program (SVSP) at the Children's Hospital of Philadelphia was performed. The age range of the 178 SVSP patients was 3 to 26 years, with a median of 10.8 years. Most of the SVSP patients had some degree of lymphopenia. In the non-PLE group, the range of ALCs varied from 530 to 5322 cells/μL, with 17 patients without PLE maintaining an ALC of less than 1000 cells/μL. Among those with PLE, the median ALC and the IgG level were lower (672 cells/μL and 200 mg/dL, respectively) than in those without (1610 cells/μL and 868 mg/dL, respectively). Despite lymphopenia in the majority, few were severely clinically affected: 24% had delayed clearance of cutaneous viral infections, 63% had atopy, and 1 died of EBV-associated Hodgkin lymphoma. Immunoglobulin replacement was clinically indicated for 3 patients, 1 of whom had common variable immunodeficiency. Four patients with normal splenic function were treated with daily antibiotic prophylaxis. Patients with repaired single-ventricle physiology often demonstrate T-cell lymphopenia and hypogammaglobulinemia. A significant portion of patients without PLE also have lymphopenia. The most common clinical manifestation was delayed clearance of cutaneous viral infections, but significant systemic opportunistic infections were not seen despite laboratory abnormalities and lack of antimicrobial prophylaxis.

Pretreatment whole blood Epstein-Barr virus-DNA is a significant prognostic marker in patients with Hodgkin lymphoma.

Epstein-Barr virus (EBV) in the peripheral blood has become a significant predictor of clinical outcomes in EBV-associated Hodgkin lymphoma (HL). However, due to its relative rarity, prevalence and prognostic role of circulating EBV-DNA has not been well established in Asian patients. Seventy patients with newly diagnosed HL were prospectively registered between October 2007 and January 2013, and underwent pretreatment whole blood (WB) EBV-DNA quantitation using real-time polymerase chain reaction (RT-PCR). WB EBV-DNA in baseline and serial RT-PCR within 1 year were investigated. Clinicopathologic parameters of the patients according to pretreatment WB EBV-DNA were also explored. Twelve patients (17.1 %) demonstrated WB EBV-DNA(+), which was significantly associated to older age, advanced stages, frequent involvements of extranodal sites, low serum albumin and hemoglobin levels, and high international prognostic scores ≥2. Three-year event-free survival (EFS) and overall survival (OS) were significantly inferior in patients with pretreatment WB EBV-DNA(+) (53.5 vs 67.0 and 65.6 vs 90.2 %) (p < 0.032 and <0.01). Negatively conversed EBV-DNA within 1 year after chemotherapy also significantly affected favorable EFS (p < 0.01). Taken together, pretreatment WB EBV-DNA(+) may be a significant predictor of inferior EFS and OS over EBV-encoded RNA in situ hybridization (EBER-ISH)(+) in Korean patients with HL. Serial EBV-DNA monitoring following chemotherapy also seems helpful to predict survival outcomes.

Epidemiology of Epstein-Barr virus-associated pediatric lymphomas from Argentina

More than 90% of the population is infected by Epstein-Barr virus (EBV), which has sophisticatedly evolved to survive silently in B cells for the life of infected individuals. However, if the virus-host balance is disturbed, latent EBV infection could be associated with several lymphomas. The age at primary infection varies substantially worldwide, and exposure to EBV is likely to be due to socioeconomic factors. In Argentina, EBV infection is mostly subclinical and 90% of patients are seropositive by the age of 3 years; therefore, its epidemiological characteristics resemble those of an underdeveloped or developing population. EBV-positive Hodgkin lymphoma (HL) in young adults from developed populations has been attributed to delayed primary EBV infection as suggested by the association with recent mononucleosis development. EBV-associated Burkitt lymphoma and Hodgkin lymphoma in children from Argentina display frequencies similar to those observed in developed countries, whereas EBV presence in pediatric diffuse large B-cell lymphoma is slightly increased compared to those populations. However, EBV presence is statistically associated particularly with patients < 10 years of age in all three entities. Therefore, a relationship between low age of EBV seroconversion and B-cell lymphoma development risk could be suggested in children from Argentina.

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.

Plasma Epstein Barr virus (EBV) DNA as a biomarker for EBV associated Hodgkin lymphoma.

To assess plasma Epstein-Barr virus (EBV) DNA as a biomarker of tumour burden at diagnosis and during therapy in children with Hodgkin lymphoma. Case-control study, with prospective follow-up of the Hodgkin lymphoma cohort (2007-2012). Pediatric Hematology Oncology unit of a tertiary care hospital in Delhi. Thirty children with Hodgkin lymphoma and 70 sex and age-matched controls (benign lymphadenopathy 19, non-lym-phoid malignancy 29, Burkitt lymphoma 5, healthy children 17). Positive EBV-staining on immunohistochemistry was defined as EBV-associated Hodgkin lymphoma. Plasma EBV real-time quantitative polymerase chain reaction (PCR) was tested at presentation, after first and last chemotherapy cycles, and on follow-up. Plasma EBV quantitative PCR was compared between cases and controls. Its kinetics was assessed during and after chemotherapy. EBV quantitative PCR was positive in 19 (63%) Hodgkin lymphoma cases (range 500 to 430,000 copies/mL), with 87.5% accuracy (kappa=0.69) as compared with EBV immunohistochemistry. Sensitivity and specificity of the quantitative PCR were 87.5% and 81.8%, respectively. Only boys showed positive EBV immunohistochemistry and,or quantitative PCR positivity. All controls were quantitative PCR negative. All quantitative PCR positive cases with follow up blood sample showed EBV clearance after the first cycle. A quantitative PCR negative case in long-term remission became positive at relapse. EBV status did not influence survival. Plasma EBV-DNA, detectable in EBV-associated Hodgkin lymphoma, becomes undetectable early after initiating therapy. It can be used as a biomarker of treatment response in EBV-associated Hodgkin lymphoma.

The Biology and Clinical Utility of EBV Monitoring in Blood.

Epstein-Barr virus (EBV) DNA in blood can be quantified in peripheral blood mononuclear cells, in circulating cell-free (CCF) DNA specimens, or in whole blood. CCFviral DNA may be activelyreleasedor extruded from viable cells, packaged in virions or passivelyshed from cellsduring apoptosis or necrosis. In infectious mononucleosis, viral DNA is detected in each of these kinds of specimens, although it is only transiently detected in CCF specimens. In nasopharyngeal carcinoma, CCF EBV DNA is an established tumor marker. In EBV-associated Hodgkin lymphoma and in EBV-associated extranodal NK-/T-cell lymphoma, there is growing evidence for the utility of CCF DNA as a tumor marker.

Pediatric Hodgkin Lymphoma in a South Indian Regional Cancer Center: Its Immunomorphology, Tumor-Associated Macrophages, and Association with Epstein–Barr Virus

Pediatric Hodgkin lymphoma (HL) comprises approximately a fifth of all patients with HL in India. Seventy-four cases of pediatric classical Hodgkin Lymphoma (cHL) from a regional cancer center in southern India were analyzed on a tissue microarray (TMA) for the stage of B-cell differentiation of the Hodgkin/Reed Sternberg (HRS) cell by immunohistochemistry (IHC) using CD10, bcl6, MUM1/IRF4, and CD 138. Fifty-two of seventy-four (70.3%) cases were of late germinal center/early post-germinal center phenotype (CD10−/bcl6−/MUM1+/CD138−). Epstein–Barr virus (EBV) association using Epstein-Barr virus encoded RNA (EBER) RISH and EBV-LMP1 immunohistochemistry (IHC) revealed an EBV association of 93%. Tumor-associated macrophages (TAM) in the microenvironment were also assessed on the TMA by CD68 IHC, and most cases (59.7%) showed >25% TAMs, with no case showing ≤5%. These findings indicate that pediatric cHL in India is a tumor, predominantly, of late germinal center/early post-germinal center B cells, is almost invariably EBV associated, and with a high number of TAMs in the microenvironment. This latter finding suggests that criteria other than TAM scores need to be developed for risk stratification of pediatric EBV-associated HL especially in developing countries.

Epstein-Barr virus, the germinal centre and the development of Hodgkin's lymphoma.

The relationship between Epstein-Barr virus (EBV) and the germinal centre (GC) of the asymptomatic host remains an enigma. The occasional appearance of EBV-positive germinal centres in some patients, particularly those with a history of immunosuppression, suggests that EBV numbers in the GC are subject to immune control. The relationship, if any, between lymphoid hyperplasia with EBV-positive germinal centres and subsequent or concurrent lymphomagenesis remains to be clarified. As far as the development of EBV-associated Hodgkin's lymphoma is concerned, the suppression of virus replication, mediated by LMP1 on the one hand, and the loss of B-cell receptor signalling on the other, appears to be an important pathogenic mechanism. A further important emerging concept is that alterations in the microenvironment of the EBV-infected B-cell may be important for lymphomagenesis.

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.

Characterizing The CpG Methylation Of Epstein-Barr Virus DNA In The Plasma Of Patients With Hodgkin Lymphoma and HIV-Associated Burkitt Lymphoma

BackgroundEpstein-Barr virus (EBV) is harbored as latent virus within the tumor cells of a subset of classical Hodgkin lymphoma (HL) and HIV-associated Burkitt lymphoma (BL). In HL, we and others have previously shown that elevated EBV DNA copies in plasma accurately distinguish EBV(+) tumors from EBV(-) tumors. However, EBV DNA may be present in plasma due to apoptosis of latently infected EBV(+) tumor cells (or latent EBV(+) benign lymphocytes), as a result of replicative lytic infection (particularly in immunocompromised hosts), or both. Thus, detecting elevated EBV DNA copies in plasma by does not always signify the presence of an EBV(+) tumor. However, a distinction is that the EBV DNA derived from latently infected cells is replicated by cellular DNA polymerase and is usually CpG methylated, whereas EBV DNA in lytic infection is replicated by the viral DNA polymerase and is never CpG methylated. MethodsTo improve upon plasma EBV DNA as a tumor-specific marker in EBV(+) lymphomas, we assessed the CpG methylation of EBV DNA in a subset of plasma specimens from immunocompetent patients with HL (enrolled in the Eastern Cooperative Oncology Group E2496 trial) and a subset from patients with HIV-associated BL (enrolled in the AIDS Malignancy Consortium 048 trial). The EBV tumor status was determined by in situ hybridization (ISH) on tissue for EBV RNAs (EBER). For the CpG methylation assay, methyl binding domain paramagnetic beads were used to first fractionate CpG methylated DNA from CpG unmethylated DNA in plasma. EBV DNA in each fraction were quantified by real time polymerase chain reaction (qPCR) to determine the percentage of methylated EBV DNA. Specimens with > 100 EBV DNA copies/mL plasma were analyzed. ResultsAmong patients with EBER(+) HL and elevated EBV DNA in plasma prior to therapy (n=8), EBV DNA was largely CpG methylated (median 95%, range 54%-99%). By contrast, in the rare cases where patients with EBER(-) HL tumors had high EBV DNA copy number in plasma prior to therapy (4 of 92, 4%), only small percentages of EBV DNA were methylated in 3 patients (5%, 9%, and 12%). In the fourth patient, the plasma EBV DNA was 99% CpG methylated. We suspect that patient 4 had an EBV-associated HL with either a technical failure of EBER-ISH or an instance of a tumor that harbors viral DNA but does not express EBERs. In patients with HIV-associated BL, plasma EBV DNA copies were elevated in 100% of patients with EBER(+) BL (n=4), but also in 4 of 9 (56%) patients with EBER(-) BL. This propensity to detect EBV DNA in the plasma of patients with HIV but without EBV(+) BL likely reflects replicative lytic infection in immunocompromised hosts, making plasma EBV DNA less reliable as a marker of EBV(+) lymphoma in this setting. Thus, we applied the methylation assay to specimens from patients with HIV and BL. Patients with HIV and EBER(+) BL tumors (n=4), EBV DNA was 28%, 46%, 47%, and 60% methylated, whereas a patient with HIV and EBER(-) BL had lower methylation at 8%. To better elucidate the meaning of plasma EBV DNA elevations during lymphoma treatment, we applied the methylation assay to specimens from patients with EBV(+) HL and elevated EBV DNA in plasma at month 6 of therapy (n=4). Dense CpG methylation (96%) of plasma EBV DNA was detected in 1 patient who went on to relapse at Month 15, while the plasma EBV DNA was largely unmethylated in 3 patients who remain in remission each with over 5 years of follow-up. ConclusionIn settings where EBV DNA is detected in plasma, the CpG methylation assay helps differentiate patients with EBV(+) HL or BL from those with EBV(-) tumors. In patients with HIV where lytic EBV replication can contribute greatly to elevations of EBV DNA in plasma, the fractionated quantification of plasma EBV DNA by the methylation assay aids in distinguishing EBV DNA that is tumor-derived from that which is the result of lytic replication. With growing interest in plasma EBV DNA as a serial marker of disease activity in EBV(+) lymphomas, the methylation assay is promising in its ability to differentiate patients with elevated EBV DNA in plasma but ongoing remission from those in whom plasma EBV DNA is tumor-derived and an early harbinger of relapse. Disclosures:No relevant conflicts of interest to declare.

Interleukin 15 Provides Relief to CTLs from Regulatory T Cell–Mediated Inhibition: Implications for Adoptive T Cell–Based Therapies for Lymphoma

Systemic administration of recombinant interleukin (IL)-2 is used to support the expansion and persistence of adoptively transferred antigen-specific CTLs in patients with cancer. However, IL-2 also expands regulatory T cells (Treg) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein-Barr virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin lymphoma, and Tregs are frequently abundant in these patients. Tregs were isolated from the peripheral blood of healthy donors and patients with Hodgkin lymphoma or from Hodgkin lymphoma tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function, and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs but instead preferentially supported the proliferation of CTLs and effector T cells as compared with Tregs. IL-15 selectively favors the survival, proliferation, and effector function of antigen-specific CTLs in the presence of Tregs, and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in patients with cancer, including those infused with EBV-CTLs for treatment of EBV-associated malignancies.