EBV-positive Classical Hodgkin Lymphoma Research Articles

Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways.

Epstein-Barr virus (EBV) is detected in 40% of patients with Hodgkin lymphoma (HL). During latency, EBV induces epigenetic alterations to the host genome and decreases the expression of pro-apoptotic proteins. The present study aimed to evaluate the expression levels of mRNA molecules and the end product of proteins for the JAK/STAT and NF-κB pathways, and their association with clinicopathological and prognostic parameters in patients with EBV-positive and -negative classical Hodgkin lymphoma (CHL). A prospective cohort study was conducted from 2017 to 2022 at the Faculty of Medicine, Zagazig University Hospital (Zagazig, Egypt). Biopsy samples of 64 patients with CHL were divided into EBV-positive and EBV-negative groups. The expression levels of mRNA molecules (JAK2, STAT1, IRF-1, PD-L1, IFN-γ, NF-κB, Bcl-xL, COX-2) and the end product of proteins (PD-L1, Bcl-xL, COX-2) were determined and compared with clinicopathological and prognostic parameters. Data were analyzed using the Chi square test and Kaplan-Meier estimate. EBV-positive CHL patients were significantly associated with positive expression of mRNAs molecules (P<0.001) and the end product of proteins (P<0.001) for the JAK/STAT and NF-κB pathways, B-symptoms (P=0.022), extra-nodal involvement (P=0.017), and advanced stage of CHL (P=0.018). These patients were more susceptible to cancer progression, higher incidence of relapse (P=0.008), poor disease-free survival rate (P=0.013), poor overall survival rate (P=0.028), and higher mortality rate (P=0.015). Through the activation of JAK/STAT and NF-κB signaling pathways, EBV-positive CHL is associated with poor clinicopathological parameters, higher incidence of disease progression, relapse, and poor overall survival. A preprint of this manuscript is available on research square (doi: 10.21203/rs.3.rs-1857436/v1).

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma

Around 30–50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

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.

EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice.

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV’s ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.

Grey zone lymphoma with features intermediate between diffuse large B‐cell lymphoma and classical Hodgkin lymphoma: a clinicopathological study of 14 Epstein–Barr virus‐positive cases

To investigate the clinicopathological features of Epstein-Barr virus (EBV)-positive grey zone lymphoma (GZL) with features intermediate between diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (CHL). We investigated the clinicopathological features of 14 cases of EBV-positive GZL in Japan. The control group included 173 cases of EBV-positive CHL and 64 cases of EBV-positive DLBCL of the elderly (polymorphous type). The patients were 10 men and four women with a median age of 62 years. Twelve patients (86%) had advanced clinical stage, 11 (79%) had B-symptoms, eight (57%) had mediastinal disease, 10 (71%) had elevated serum lactate dehydrogenase (LDH) levels, and five (36%) had thrombocytopenia. All cases had CHL-like morphology but strongly expressed at least one B-cell marker. The neoplastic cells were Hodgkin and Reed-Sternberg-like cells, but with a large number of mononuclear variants. EBV-positive GZL patients were more significantly more likely than EBV-positive CHL patients to have advanced clinical stage (P = 0.023), presence of B-symptoms (P = 0.011), elevated serum LDH levels (P = 0.047), thrombocytopenia (P = 0.042), and mediastinal involvement (P = 0.023). The progression-free survival (PFS) of EBV-positive GZL patients was significantly poorer than that of EBV-positive CHL patients (P = 0.043) but no difference from EBV-positive DLBCL patients was observed (P = 0.367). EBV-positive GZL patients have significantly worse PFS than EBV-positive CHL patients, and are significantly more likely to have adverse clinical parameters such as advanced clinical stage, presence of B-symptoms, and thrombocytopenia. Further studies are needed to better characterize this entity, which may require the development of innovative therapeutic strategies.

Small Bowel Obstruction Caused by Epstein-Barr Virus Positive Extranodal Hodgkinʼs Lymphoma in a Patient with Crohnʼs Disease: A Case Report

Literature in the IBD population has reported increased risks for malignancy including lymphoproliferative disorders associated with biologic and thiopurine medications. A 55 year old male with a history of Crohn's disease presented with multiple symptoms of mid-abdominal pain, nausea, vomiting, constipation, and lack of flatus for three days. He had previous intestinal resections and intermittent therapy including systemic steroids, infliximab, azathioprine, and 5-ASA. The patient was found to have a small bowel obstruction which required surgical intervention and a nonpatent segment of an ileocolonic anastomosis was resected. Histological analysis of the resected anastomotic segment revealed positivity for PAX5, CD30, BCL-2, OCT-2, MUM1 for large cells. Neoplastic cells showed altered B-cell phenotype and decreased expression of PAX5, CD20, CD79a and BOB.1 consistent with Reed Sternberg cells. In-situ hybridization was positive for EBER confirming Epstein-Barr virus. The patient was diagnosed with ileocolonic anastomosis EBV positive classical Hodgkin lymphoma in Crohn's disease.Figure 1Figure 2Figure 3

An Aggressive Transformation to EBV-Positive Hodgkin Lymphoma after Bendamustine-Containing Chemotherapy for Marginal Zone Lymphoma

EBV-positivelymphoproliferative disorders are well documented phenomenon after fludarabine therapy. Although bendamustine shares common chemical structure with fludarabine, therapy-related malignancies have rarely been reported. A seventy-three-year-old man was presented with splenomegaly and pharyngeal mass. The biopsies of bone marrow and the mucosal mass revealed he had splenic marginal zone lymphoma, stage IV. Six months later, he received one course of R-CHOP and splenectomy. Three months after the surgery, the lymphoma progressed with massive pleural effusion, and six courses of bendamustine plus rituximab, yielding minimal response of the disease. Six months after the completion of bendamustine, however, Pel-Ebstein-like fever and progression of lymphadenopathy ensued. A lymph node biopsy exhibited he had EBV-positive classical Hodgkin lymphoma concomitant with marginal zone lymphoma. The patient achieved complete response after brentuximab vedotin therapy. The Hodgkin lymphoma in the present case might be a result of an aggressive transformation of marginal zone lymphoma, and it could be speculated that the purine analogue-like property of bendamustine be associated with the transformation.

Tumor microenvironment and RIG-I signaling molecules in Epstein Barr virus-positive and -negative classical Hodgkin lymphoma of the elderly.

Classical Hodgkin lymphoma (CHL) is a B-cell neoplasm characterized by Hodgkin and Reed-Sternberg (HRS) cells. Its prevalence exhibits a bimodal pattern of peaking in young adults and the elderly. There is an association with Epstein-Barr virus (EBV) infection in about 50% of cases of CHL of the elderly, and the outcome of these patients is unfavorable. It is not well known how the latent infection of EBV is involved in the pathophysiology of CHL of the elderly. To address this issue, we examined the tumor microenvironment (TME) and the expression of molecules related to EBV infection in HRS cells in 10 EBV-positive CHL and 7 EBV-negative CHL patients older than 50 years. In EBV-positive CHL, we found an increased population of FOXP3(+) cells, while that of granzyme B(+) cells was reduced, compared with those in EBV-negative CHL. The expression of inhibitory chemokine CCL20 was increased in EBV-positive HRS cells compared with that in EBV-negative HRS cells. In addition, despite increased expression of a pattern recognition receptor, RIG-I, in intracellular innate immunity, there was no evidence of interferon regulatory factor 3 activation or interferon-ß induction in EBV-positive HRS cells in CHL of the elderly. The disease recurred frequently (50%) in EBV-positive CHL. The current study thus suggests the possibility that the latent infection of EBV alters the expression of chemokines and the innate immunity response in HRS cells and modulates TME to an immunosuppressive state, which may account for the unfavorable disease course in CHL of the elderly.

BOB.1-positive Classical Hodgkin's Lymphoma Carries Hypermethylation of Its Promoter as Epigenetic Marker of Gene-silencing Memory.

Analysis of archival formalin-fixed, paraffin-embedded (FFPE) pathological specimens of three case of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) and three cases of classical Hodgkin lymphoma (CHL) revealed that hypermethylation of the BOB.1 gene promoter was exclusively observed in CHL. A discrepancy was observed, however, between the methylation status of the BOB.1 gene promoter and its expression in the EBV-positive mixed cellular CHL (MCCHL). Since MCCHL lacks the typical B-cell phenotype even in the presence of abundant BOB.1 transcription factors, functional activity of BOB.1 may be lost or reduced by a mechanism other than epigenetic gene silencing. When some tumor-suppressor gene products have lost their biological function, impact or significance of derepression of such genes may be little. Therefore, when interpreting immunohistochemical results for diagnostic or research purposes, it must be borne in mind that apparent positive immunostaining can merely be the result of chromatin remodeling and that such transient expression often has little functional significance. Any apparent positive immunohistochemical result needs to be interpreted carefully with the help of the hypermethylation status as a molecular marker of gene silencing memory.

Cytokine gene polymorphisms and progression-free survival in classical Hodgkin lymphoma by EBV status: Results from two independent cohorts

BackgroundCytokines are important immune mediators of classical Hodgkin lymphoma (CHL) pathogenesis, and circulating levels at diagnosis may help predict prognosis. Germline single nucleotide polymorphisms (SNPs) in immune genes have been correlated with cytokine production and function. MethodsWe investigated whether selected germline SNPs in IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629), IL6 (rs1800795), ILRN (rs419598), INFG (rs2430561) and CCL17 (rs223828) were associated with circulating levels of related cytokines at diagnosis and progression-free survival (PFS) in CHL. Patients were from France (GELA, N=464; median age=32years) and the United States (Iowa/Mayo Specialized Program Of Research Excellence [SPORE], N=239; median age=38years); 22% of 346 CHL cases with EBV tumor status were positive. ResultsThere was no association with any of the SNPs with cytokine levels. Overall, there was no association of any of the SNPs with PFS. In exploratory analyses by EBV status, TNFA rs1800629 (HRAA/AG=2.41; 95%CI, 1.17–4.94) was associated with PFS in EBV-negative GELA patients, with similar trends in the SPORE patients (HRAA/AG=1.63; 95%CI, 0.61–4.40). In a meta-analysis of the two studies, TNFA (HRAA/AG=2.11; 95%CI, 1.18–3.77; P=0.01) was statistically significant, and further adjustment for the international prognostic system did not alter this result. ConclusionsThis study showed that germline variation in TNFA was associated with CHL prognosis for EBV-negative patients, which will require confirmation. These results support broader studies on the differential impact of genetic variation in immune genes on EBV-positive vs. EBV-negative CHL pathogenesis.

A malignant lymphoma with histological features and immunophenotypic profile intermediate between EBV-positive diffuse large B-cell lymphoma and EBV-positive classical Hodgkin lymphoma in a 67-year-old female: A “gray zone” lymphoma associated with Epstein-Barr virus in the elderly

Epstein–Barr virus (EBV) can be associated with both classical Hodgkin lymphoma (cHL) and non-Hodgkin lymphoma of the B-cell type, particularly in immunodeficient patients or elderly individuals. While polymorphic variants of EBV-positive large B-cell lymphoma (EBV+ DLBCL) frequently resemble cHL in morphology, and thereby may cause diagnostic difficulty, a true gray zone lymphoma with overlapping morphological and immunophenotypical features of EBV+ DLBCL and EBV+ cHL has not been reported in the literature. We describe a unique case of an EBV+ malignant lymphoma of B-cell origin with hybrid features of EBV+ DLBCL and EBV+ cHL in a 67-year-old female without an identifiable etiology for immunodeficiency. The biopsy of an enlarged lymph node showed a polymorphic infiltrate containing Reed–Sternberg-like pleomorphic large cells, which were positive for CD30 and CD15. Although CD20 was negative and PAX5 and CD45 were down-regulated, the pleomorphic large cells expressed multiple other B-cell antigens which are characteristically absent in cHL. EBV-encoded RNA hybridization (EBER) studies demonstrated nuclear reactivity in the large cells as well as in the smaller bystander cells. A clonal rearrangement of the immunoglobulin heavy chain gene was also detected by PCR. Although the results of the EBV and genotypic studies suggest this case may be an example of EBV+ DLBCL of the elderly instead of EBV+ cHL, the immunophenotype is strikingly ambiguous. Thus, this case may represent an interface between EBV+ DLBCL and EBV+ cHL.

HLA-A*02:07 Is a Protective Allele for EBV Negative and a Susceptibility Allele for EBV Positive Classical Hodgkin Lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10−5). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.

Age-Related EBV-Associated B-Cell Lymphoproliferative Disorders: Comparison with EBV-Positive Classical Hodgkin Lymphoma in Elderly Patients

Age-related Epstein-Barr virus-associated (EBV+) B-cell lymphoproliferative disorder (LPD) is a disease group of EBV+ large B-cell lymphoma characteristically seen in elderly patients without predisposing immunodeficiency. This disease is now going to be listed in the up-coming WHO classification the 4th version. Age-related EBV+ LPD, especially of a polymorphous subtype, which occupied about one-third of cases, is often featured by a rich infiltration of reactive cells in the background, posing the differential diagnostic issue with classical Hodgkin lymphoma (cHL) associated with EBV. However, the clinicopathological differentiation between these two EBV-positive diseases remains to be elucidated. The aim of this study is to clarify the clinicopathological differences between the polymorphic subtype of age-related EBV+ LPD (n=34) and EBV+ cHL (n=108) of patients of 50 years and more of age. Age-related LPD was more closely associated with the following clinical parameters than cHL: a higher age at onset (71 vs. 63 years), lower male predominance (male:female ratio, 1.4 vs. 3.3), and a higher ratio of involvement of the skin (18% vs. 2%), the gastrointestinal tract (15% vs. 4%) and the lung (12% vs. 2%). Furthermore, age-related LPD was histopathologically characterized by a higher ratio of geographical necrosis, a greater increase (>30%) of cytotoxic T cells in the background lymphocytes, a higher positive rate for CD20 and EBNA2 together with an absence of CD15 expression than EBV+ cHL. As predicted by the clinical profile, age-related LPD had a significantly poorer prognosis than EBV+ cHL (P = 0.0001). This analysis has clearly demonstrated that the polymorphous subtype of age-related LPD constitutes an aggressive group with an immune response distinct from those of EBV+ cHL.

Racial/ethnic variation in EBV‐positive classical Hodgkin lymphoma in California populations

Epstein-Barr virus (EBV) is detected in the tumor cells of some but not all Hodgkin lymphoma (HL) patients, and evidence indicates that EBV-positive and -negative HL are distinct entities. Racial/ethnic variation in EBV-positive HL in international comparisons suggests etiologic roles for environmental and genetic factors, but these studies used clinical series and evaluated EBV presence by differing protocols. Therefore, we evaluated EBV presence in the tumors of a large (n = 1,032), racially and sociodemographically diverse series of California incident classical HL cases with uniform pathology re-review and EBV detection methods. Tumor EBV-positivity was associated with Hispanic and Asian/Pacific Islander (API) but not black race/ethnicity, irrespective of demographic and clinical factors. Complex race-specific associations were observed between EBV-positive HL and age, sex, histology, stage, neighborhood socioeconomic status (SES), and birth place. In Hispanics, EBV-positive HL was associated not only with young and older age, male sex, and mixed cellularity histology, but also with foreign birth and lower SES in females, suggesting immune function responses to correlates of early childhood experience and later environmental exposures, respectively, as well as of pregnancy. For APIs, a lack of association with birth place may reflect the higher SES of API than Hispanic immigrants. In blacks, EBV-positive HL was associated with later-stage disease, consistent with racial/ethnic variation in certain cytokine polymorphisms. The racial/ethnic variation in our findings suggests that EBV-positive HL results from an intricate interplay of early- and later-life environmental, hormonal, and genetic factors leading to depressed immune function and poorly controlled EBV infection.

Racial/ethnic variation in EBV-positive classical Hodgkin lymphoma in California populations

Racial/ethnic variation in EBV-positive classical Hodgkin lymphoma in California populations

Polymorphisms and Lack of or Aberrant Expression of HLA Class I and II May Influence Antigen Presentation in Classical Hodgkin Lymphoma.

In a previously performed population-based genetic screening analysis we found that polymorphisms in the HLA class I region are specifically associated with EBV-positive classical Hodgkin lymphoma(cHL)(Diepstra et al., Lancet 2005; 365:2216). We now studied the expression of HLA class I and HLA class II in this population (n=450) by immunohistochemistry. HLA-B/C, HLA-G, beta-2-microglobulin and HLA-DP/DQ/DR staining was performed on formalin fixed paraffin embedded tissue sections, and HLA-DM and HLA-DO staining on frozen sections. Hodgkin-Reed Sternberg (HRS) cells showed cell surface expression of HLA class I in 37.5% (159/424) of cHL tumors and expression was observed predominantly in EBV-positive cases (73%; 103/141). Expression of HLA-G, a molecule known to enable evasion from natural killer cell responses, was found in 67% of the cases and was associated with absence of MHC class I and EBV. Only 58% of cHL cases showed HLA class II positive staining in more than 50% of the HRS cells (224/387). The non-classical HLA class II protein HLA-DM, essential for the loading of HLA class II with antigenic peptides was reduced or absent in HRS cells in 7 out of 10 HLA class II positive cases, whereas HLA-DO, the natural repressor of HLA-DM, was not or minimally expressed. Impaired expression of HLA-DM may prevent presentation of antigens at the level of individual HRS cells. Reanalysis of the genotyping data revealed an association of HLA class I positive cases with the known susceptibility polymorphisms in the HLA class I region. However, this association was stronger for the EBV-positive cases than for the HLA class I positive cases. Within the group of EBV-positive cHL patients, the same predisposing alleles were present in both HLA class I negative and positive cases, suggesting that the negative cases probably did express HLA class I during early pathogenesis. Finally, in cases with HLA class II expression on the HRS cells, a genetic association with the HLA class II region was found. These changes may influence (EBV) antigen presentation by HRS cells in cHL.