Hodgkin And Reed-Sternberg Cells Cells Research Articles

Targeting DNA Repair to Overcome Drug Resistance in Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by rare tumor-initiating Hodgkin and Reed-Sternberg cells (HRS) surrounded by a microenvironment with a reactional immune cells infiltrate. cHL accounts for 15% to 25% of all lymphomas. This neoplasm is curable in the majority of cases with chemotherapy including ABVD (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) or BEACOPP (Bleomycin, etoposide, Doxorubicin, cyclophosphamide, Vincristine, procarbazine and Prednisone) and/or radiation. However, 15-20% of high-risk patients ultimately relapse and high dose chemotherapy in combination with autologous stem-cell transplantation is successful in only half of the patients with relapsed/refractory cHL (Eichenaueret al., Eur. Soc. Med. Oncol,2018). HRS cells are characterized by genetic instability, abnormal DNA damage response and repair that may play a role in drug resistance in high-risk cHL (Reichelet al., Blood,2015, Monroyet al. Mol. Carcinog,2011). In this work, we hypothesized that inhibiting DNA repair mechanisms using small molecules might represent a promising strategy to overcome drug resistance to genotoxic agents in cHL, such as cyclophosphamide and topoisomerase II inhibitors (O'Connor,Mol. Cell, 2015; Shaheenet al., Blood 2011). We characterized the drug-response of 4 cHL cell lines to DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1 inhibitor), AZD6738 (ATR inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor) and genotoxic agents used in standard chemotherapy (cyclophosphamide, doxorubicin and etoposide). We showed that DNA repair inhibitors targeting ATR, CHK1 or Wee1 significantly induced apoptosis of cHL cell lines (AnnexinV staining, p<0.05), significant inhibition of proliferation (BrdU incorporation). ATR inhibitor induces a significant accumulation of cHL cells in G0/G1 phase (p<0.05) whereas CHK1 and Wee1 inhibitors induced a significant accumulation in G2/M phase (p<0.01). Moreover, ATR, CHK1 and Wee1 inhibitors induced double strand breaks monitored by 53BP1 foci formation, γH2A.X staining (p<0.01) and chromosomic instability characterized by a higher frequency of micronucleation (p<0.05) and nucleoplasmic bridges formation (p<0.01). Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 DNA repair targeted treatments with conventional genotoxic agents in cHL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone (Srivaset al., Mol. Cell, 2016), a total of two synthetic lethal combinations in cHL cells have been identified: CHK1 inhibitor (PF477736) combined with a Wee1 inhibitor (AZD1775) (IC50 reduced from 5.00 µM to 1.75 µM for L-428 and from 4.81 µM to 1.86 µM for KMH2) and cyclophosphamide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 3.26 µM to 1.12 µM for L-428 and from 1.27µM to 0.10µM for KMH2). In addition, we identified 3 synergistic/additive combinations (combination index < 1): doxorubicin combined with a DNAPK inhibitor (NU7441) (IC50 reduced from 44 nM to 22 nM for L-428 and from 27 nM to 8 nM for KMH2), cyclophosphamide combined with CHK1 inhibitor (PF477736) (IC50 reduced from 2.9 µM to 2.4 µM for L-428 and from 1.2µM to 0.9µM for KMH2) and etoposide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 420 nM to 195 nM for KMH2). The treatment of Hodgkin's lymphoma is successful for a majority of patients. However, patients with advanced-stage or high-risk disease are only cured in ∼70% of cases. Alternative treatment options for the subsets of patients for whom first- or second-line therapies fail are needed. These results open new perspectives to improve the treatment of relasped/refractory cHL patients and provide new strategies to overcome drug resistance. Disclosures Moreaux: Diag2Tec:Consultancy.

Unravel Inter-Tumor and Intra-Tumor Heterogeneity of Digitally Sorted Single Hodgkin and Reed Sternberg Cells Using Genome-Wide Copy Number Profiling

IntroductionImmune-checkpoint blockade has emerged as an effective therapeutic strategy in solid tumor and in hematologic malignancies, including classical Hodgkin Lymphoma (cHL).cHL represents about 11% of all malignant lymphoma and it is generally highly curable with standard frontline therapies, although about 20% of the patients will relapse or become refractory after initial treatment.The hallmark of cHL is the presence of malignant Hodgkin and Reed-Sternberg Cells (HRS) that represent only a small fraction (about 1%) of the surrounding heterogeneous immune infiltrate. Despite this extensive inflammatory microenvironment, HRS are able to escape immune surveillance using several mechanisms, including the overexpression of PD-1 ligands (PD-Ls) that bind PD-1 on reactive T-cells, inhibiting their activity and proliferation and causing ultimately T-cell exhaustion. The PD-Ls expression is upregulated in a dose-dependent manner by copy number alterations of chromosome 9p24.1, a locus encoding for PD-L1/PD-L2 as well as JAK2, which further enhances PD-Ls expression through JAK2/STAT pathway.Here we present a method for the isolation and the genetic characterization of single purified HRS, which overcomes the limitations posed by the low tumor cellularity of cHL biopsies and gives an estimation of inter-tumor and intra-tumor heterogeneity which may be useful to guide immune treatment selection.MethodsFFPE tissue sections from 4 cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Since CD30 is not expressed exclusively by malignant cells, beyond the positivity to CD30 and PD-L1 HRS were selected according to morphological criteria, such as cell size and the presence of nuclei with ploidy higher than the surrounding lymphocytes.DEPArray™ NxT system (Menarini Silicon Biosystems) was used to isolate single target cells. After recovery, single cells were whole genome amplified (Ampli1™ WGA, Menarini Silicon Biosystems), and genome-wide copy-number alterations (CNAs) profiles were obtained using Ampli1™ LowPass kits (Menarini Silicon Biosystems) on Illumina® and Ion Torrent™ platforms.ResultsFor each patient, at least 8 HRS cells and infiltrating lymphocytes were identified and isolated from lymphoid tissue using DEPArray™ NxT system.Copy-number analyses of recovered cells allowed us to precisely discriminate HRS, characterized by extensive gains and losses, from non-tumor cells, showing flat profiles as expected (Fig.1). Ploidy of HRS was automatically determined, based on best-fitting of profiles with underlying copy number levels.Hierarchical clustering showed that some alterations are highly conserved among patients, e.g. the region containing PD-L1/PD-L2/JAK2 has several copy gains in the majority of malignant cells. Interestingly, these alterations show high variable copy-number levels between different HRS even in the same patient, ranging from few copy-gains to amplifications, suggesting some level of heterogeneity.Different CNAs are also detected in regions containing genes belonging to pathways already known to be altered in cHL, like REL/NFKB and JAK/STAT pathways, which may be involved in the constitutive activation of proliferative and antiapoptotic phenotype of HRS.ConclusionSingle HRS sorting combined with low-pass whole genome sequencing offer a valuable tool to uncover genetic alterations hidden by the massive cHL immune infiltrate and to estimate inter-tumor and intra-tumor heterogeneity in cHL patients. Considering that PD-Ls locus amplifications are associated with advanced stages of the disease and with a shorter progression free survival, the analysis of purified HRS could be helpful for patient stratification for the adoption of immune therapy. [Display omitted] DisclosuresMangano:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Garonzi:Menarini Silicon Biosystems: Employment. Lanzellotto:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Ceccolini:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fici:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Gallerani:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

Strong Expression of the Immune Checkpoint Regulators LAG3 and Tim3 in Hodgkin Lymphoma

Background: Recent results of immune checkpoint blockade trials have provided a proof of concept for immunotherapy in classical Hodgkin lymphoma (cHL) with more than two third of relapsed/refractory patients responding to blockade of the PD1/PDL1 axis. Unfortunately, there is still a proportion of patients who will present primary or secondary resistance to immunotherapy. Besides the PD1/PDL1 axis, several other molecules are critical regulators of the immune response and may be the target of therapeutic intervention. Combined immune checkpoint targeting has shown interesting results in preclinical and clinical trials in several types of tumors.Methods: Patients with initially diagnosed or relapsed cHL for whom formalin fixed paraffin embedded (FFPE) tissue was available at our institution were identified. Fifty-seven cases were selected depending solely on the availability and the quality of the FFPE blocks. Expression of the following immune checkpoints PD1, PDL1, LAG3, TIM3 was assessed using immunohistochemical methods with a threshold of 5% set for positivity.Results: Complete results for 25 cases were available at the time the abstract was written. Hodgkin and Reed Sternberg cells (HRS) were identified morphologically upon microscopic examination. Consistently with data published in the literature, HRS stained positively and intensely for PDL1 in 100% of the cases (25/25). HRS were positive for Tim3 in 36% (9/25) of cases but with more varying intensities. No PD1 or LAG3 expression was found on HRS cells except for a single case where 5% of HRS stained weakly for LAG3. In the tumor microenvironment, PD1 expression was detected in 65% of cases (15/23) and PDL1 in 60% of cases (15/25). Impressively, LAG3 and TIM3 stained positively in 96% (23/24) and 92% (24/25) of cases respectively. Lymphocyte-rosetting was present in 9/25 cases. These CD4+ FoxP3- T cells surrounding HRS were positive for PD1 in 5 cases, for LAG3 in 2 cases and for both PD1 and LAG3 in 2 cases, suggesting they represented exhausted T-cells. Concomitant expression of PD1 and PDL1 in the tumor microenvironment was present in 43% of cases (10/23).Conclusion: LAG3 and TIM3 are nearly universally expressed in the tumor microenvironment of cHL. These findings provide a strong rationale for their blockade alone or in combination in relapsed/refractory patients with cHL. The role of TIM3 expression by HRS remains unclear. Correlation of these findings with clinical data and survival outcome of the patients will be done for the whole sample. DisclosuresRibrag:NanoString: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees; ArgenX: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Analysis of the Genomic Heterogeneity in Hodgkin Lymphoma Using Next Generation Sequencing

Classical Hodgkin´s lymphoma (cHL) is a clonal proliferation of the characteristic Hodgkin and Reed Sternberg cells (HRS), diluted in a reactive inflammatory background, and characterized by a defective B cell immunophenotype. Few studies have been aimed to the identification of the driver mutations in this disease, and only occasional gene mutations in members of the NF-kappaB and JAK/STAT pathways, and, more recently B2M, have been previously described.We analyzed 57 cHL tumor samples (FFPE) using massive parallel sequencing (Ion Torrent™ semiconductor technology). To increase coverage a previous tumor cell enrichment process by punch tissue cores from selected tumor-rich areas was implemented. In addition, we sequenced 7 cHL cell lines using the same technical approach. Bioinformatics analysis, including alignment with germline sequences and SNPs filtering, were done using the Torrent Suite Software and Variant Caller. Pathogenic prediction of single nucleotide variants (SNVs) and mutation interpretation were performed using the Alamut and Provean softwares. All experiments were done in duplicate to minimize false positive rates.Overall, the results show very high genomic heterogeneity with a range of 10-400 SNVs per sample, most of them (~60%) missense type. We found a relatively large number of genes recurrently mutated at low frequency and only a few genes mutated in up to 15-20% of the patients, reflecting a high level of genomic instability in the neoplasm. Specific mutations in genes previously described in cHL (NFKBIA, TNFRSF14) and in diffuse large B-cell lymphomas (CARD11, STAT6, CREBBP, CMYB) were consistently found, as well as new SNVs in genes not previously described (BTK, NFKB2). Mutations affecting selected genes (B2M, CARD11, NFKBIA, CSF2RB, and STAT3) in cell lines were additionally validated by Sanger sequencing.Of note, we found high prevalence of mutations affecting BTK and the BCR pathway, suggesting some dependencies of active BCR signaling albeit the absence of BCR expression by HRS cells. Consistent with this interpretation, incubationof a panel of cHL cellular models with either Ibrutininb or AVL-292, selective-BTK inhibitors, in vitro constrains cell proliferation and causes cell death. Interestingly, the concentration to induce cell apoptosis in cHL was comparable to other cell models of diffuse large B-cell and mantle cell lymphoma sustaining the requirement of an active BCR signaling for cHL cell viability.In conclusion, cHL is characterized by high genomic instability, including numerous mutations in genes related with B-cell function and specific signaling pathways. Pathogenic predictions of the different SNVs identify potential driver mutations that can be associated with the pathogenesis of the disease and might represent new therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Contribution of the Epstein-Barr Virus to the Pathogenesis of Hodgkin Lymphoma.

The morphology of the pathognomonic Hodgkin and Reed-Sternberg cells (HRS) of Hodgkin lymphoma was described over a century ago, yet it was only relatively recently that the B-cell origin of these cells was identified. In a proportion of cases, HRS cells harbour monoclonal forms of the B lymphotropic Epstein-Barr virus (EBV). This review summarises current knowledge of the pathogenesis of Hodgkin lymphoma with a particular emphasis on the contribution of EBV.

C-JUN N-terminal kinase (JNK) is activated and contributes to tumor cell proliferation in classical Hodgkin lymphoma.

c-JUN N-terminal Kinase (JNK) is activated/phosphorylated by upstream MAPK kinases (MKK), and, in turn, phosphorylates and activates its major substrate c-JUN, a member of the activator protein-1 (AP-1) transcription factors. c-JUN is overexpressed and activated in Hodgkin and Reed Sternberg cells (HRS) of classical Hodgkin lymphoma (cHL), however, the mechanism of its activation remains unknown. JNK activation was immunohistochemically assessed in 60 cases of HL and in a control group of 151 B-cell non-Hodgkin lymphomas. The biologic effects of JNK activation in cultured HRS cells were investigated using colony formation, cell growth and viability assays and cell cycle analysis by flow cytometry. Western blotting was used to assess protein levels. p-JNK was expressed in 90% of HL, 83% of Burkitt lymphomas, 28% of mantle cell lymphomas, 23% of diffuse large B-cell lymphomas, 19% of follicular lymphomas, and 18% of extranodal marginal zone lymphomas of MALT type. None of the 48 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma and 18 cases of plasma cell myeloma showed JNK phosphorylation (P < 001, Kruskall-Wallis test). Pharmacological inhibition of JNK activity in cultured HRS cells resulted in a significant decrease of cell growth, which was associated with cell cycle arrest at the G2/M phase. The cell cycle effects were linked to deactivation of c-JUN and upregulation of its known target, the cyclin-dependent kinase inhibitor p21. JNK is highly activated in HRS cells, and may contribute to uncontrolled cell cycle progression and proliferation of tumor cells in cHL.

Multiple primary enoral soft tissue manifestations of a Hodgkin lymphoma—case report and literature review

Hodgkin lymphoma (HL) are lymphoproliferative neoplasms, histologically comprising of mononuclear and multinucleated Hodgkin and Reed Sternberg cells (HRS). About 4 % of all lymphatic malignancies of the head and neck are HL. The typical disease presents itself as a nodal lesion. Extranodal, enoral soft tissue involvement by HL is very rare. A 73-year-old man with a suspect, ulcerating lesion in the left retromolar region of the mandible was assigned to our hospital. Prior anti-inflammatory therapy has been without success. Subsequently, three biopsies were taken which could only show inflammation. Finally, two biopsies from the left retromolar region and the left inner cheek showed HRS cells with positive expressions of CD15 and CD30 corresponding to a Hodgkin lymphoma. No lymphatic node or bone involvement could be detected. The patient was designated to receive radio-chemotherapy, but died 3 weeks after diagnosis of multiple organ failure. In a literature review, together with this report, nine cases were found concerning primary HL of the oral mucosa. Accordingly, this is the first case of primary multiple extranodal HL in the oral mucosa in absence of lymphatic node involvement. Neither clinical features nor radiological appearances of HL presenting as primary enoral lesions are pathognomonic. Especially when only small biopsy specimens are available, histological diagnosis remains challenging, may lead to a delay in therapy and may result in a significant worse prognosis.

Mirna EXPRESSION SIGNATURES as PROGNOSTIC Markers IN ADVANCED Classical HODGKIN LYMPHOMA

AbstractAbstract 4157In spite of the progress in the therapy of advanced stage classic Hodgkin lymphoma (cHL), still a significant proportion of patients fail to respond or relapse after complete remission. MicroRNAs (miRNA) are essential regulators of cell differentiation, emerging as robust predictor and prognostic markers. Specific miRNA signatures from the Hodgkin and Reed Sternberg cells (HRS) cells and their microenvironment have been proposed, but the potential prognostic role of them remains unclear. Here, we investigated whether miRNA signatures can allow to a better understanding of cHL pathogenesis and being applied in patient outcome prognostication.We have used mirRNA gene expression data from 32 samples of advanced cHL patients and 5 cHL derived cell lines (L540, L1236, L428, HDLM2, and KHMZ) to identify specific miRNA profiles from the tumoral cells and their non-tumoral microenvironment. A miRNA signature was identified in the cHL cell lines, probably reflecting functional properties of the HRS, whereas the others informed from properties of the reactive infiltrate. Selected miRNAs were further validated by performing laser capture microdissection using frozen samples in order to look for specific miRNAS preferentially expressed for either HRS cells and their surrounding microenvironment.In addition, logistic regression and Cox analysis allowed us to identify a set of 102 miRNAs differentially expressed (p<0.05) among patients with favourable and unfavourable outcome that included members of the miR-17-92 cluster such us mir-17, mir-92a and mir-92b* and other miRNAs known to be involved in cancerogenesis. A restricted group of 32 miRNAS with better prognostic capacity was further selected to set up a quantitative RT-PCR assay and explore the potential diagnostic application of their expression in an independent series of 96 formalin-fixed paraffin-embedded (FFPE) cHL samples.Bioinformatics target prediction (miRBase and TargetScan) and the web based computational tool DIANA-mirPath, were used in the identification of molecular pathways and target genes potentially regulated by the expression of these mirRNAs. Combined analysis of miRNAs, paired with bioinformatic target prediction, revealed a series of genes and pathways targeted by a small number of miRNAs, including essential pathways for lymphoma survival like MAPK signaling pathway, cell cycle, Jak-STAT signaling pathway and others.In conclusion, specific miRNA signatures for the HRS cells and their microenvironment can be identified in cHL samples, and improve our understanding in cHL pathogenesis. Selected miRNAs can be used in paraffin-embedded tissue for prognosis of advanced cHL cases, and can be incorporated in an RT-PCR assay with potential clinical application in advanced cHL. Disclosures:No relevant conflicts of interest to declare.

Vorinostat inhibits STAT6-mediated TH2 cytokine and TARC production and induces cell death in Hodgkin lymphoma cell lines

AbstractEpigenetic changes have been implicated in silencing several B-cell genes in Hodgkin and Reed-Sternberg cells (HRS) of Hodgkin lymphoma (HL), and this mechanism has been proposed to promote HRS survival and escape from immunosurveillance. However, the molecular and functional consequences of histone deacetylase (HDAC) inhibition in HL have not been previously described. In this study, we report that the HDAC inhibitor vorinostat induced p21 expression and decreased Bcl-xL levels causing cell-cycle arrest and apoptosis. Furthermore, vorinostat inhibited STAT6 phosphorylation and decreased its mRNA levels in a dose- and time-dependent manner, which was associated with a decrease in the expression and secretion of Thymus and Activation-Regulated Chemokine (TARC/CCL17) and interleukin (IL)–5 and an increase in IP-10 levels. Moreover, vorino-stat inhibited TARC secretion by dendritic cells that were activated by the thymic stromal lymphopoietin (TSLP). Collectively, these data suggest that pharmacologic HDAC inhibition in HL may induce favorable antitumor activity by a direct antiproliferative effect on HRS cells, and possibly by an immune mediated effect by altering cytokine and chemokines secretion in the microenvironment.

WHO classification of Hodgkin's lymphoma and its molecular pathological relevance

The current WHO classification of Hodgkin's lymphoma (HL) generally distinguishes the relatively rare variant (approximately 5% of all cases of HL) of nodular lymphocyte predominant type from a second group, which comprises classical HL and is separated into four subtypes: lymphocyte rich type, nodular sclerosis type, mixed cellularity type and lymphocyte depleted type. The classical lymphocyte rich subtype is a new entity and based on the typical morphology, can be recognized by definition only by the immunohistochemical characteristics of the Hodgkin and Reed/Sternberg cells (HRS) (CD30+, CD15+, CD20-). Molecular single cell studies are consistent with the dichotomy of HL in nodular lymphocyte predominant and classical types and stress the exceptional position of the former, which shows similarities with non-Hodgkin's lymphomas in several aspects. On the molecular biology level the tumor cells of all kinds of HL turn out to be clonal B cells derived from germinal center cells. However, tumor cells of nodular lymphocyte predominant HL differ from those of classical HL by the pattern of somatic mutations. Considering the inability of HRS cells of classical HL to express a B cell receptor, they should perish under normal conditions. However, they escape from apoptosis by mechanisms so far only partially understood, such as genomic mutations of the l-kappa B gene and the fas receptor gene or probably by down-regulation of B cell markers. In rare cases, HRS cells of HL can also be derived from T cells, as could be demonstrated by single cell analysis. Also, it could be shown by single cell PCR that HL and non-Hodgkin's lymphoma of both B and T cell types can arise from a common precursor. These results suggest that future classifications of HL will not only take into account the morphological and phenotypical profile, but also mechanisms of transformation yet to be discovered.

Epstein-Barr virus in Hodgkin disease. Relation to age and subtype.

Epstein-Barr virus (EBV) has been associated with Hodgkin disease (HD), but its relationship to the age of the patients and the histologic subtype is not well defined. The possibility that other lymphotropic herpesviruses, such as cytomegalovirus (CMV) and human herpesvirus type 6 (HHV-6) could be involved in the pathogenesis of HD is also unclear. Paraffin-embedded material from 77 cases of HD was studied. This consisted of 24 specimens from children (mean age, 11.4 years), 36 from young adults (mean age, 28.8 years), and 17 from older adults (mean age, 53.8 years). EBV was detected in Hodgkin and Reed-Sternberg cells (HR-S) by a sensitive in situ hybridization technique based on the detection of Epstein-Barr-encoded RNA. Viral activity was subsequently demonstrated in HR-S cells by using monoclonal antibodies to EBV latent membrane protein. EBV was found in a total of 25 of 77 cases (32%). There was no significant difference in EBV positivity between the different age groups. However, the prevalence of EBV varied between different subtypes: 68% of mixed cellularity cases were found to be positive by contrast with 24% nodular sclerosis, 0% lymphocyte predominant, and 14% lymphocyte depleted. Analysis of the histologic reports of a further 783 cases of HD to determine the age distribution of the different subtypes revealed an absence of an older adult second peak in the age distribution curve. In situ hybridization for CMV and HHV-6 was negative in all cases. It was concluded that EBV is predominantly associated with mixed cellularity HD, and there is no correlation with the age of the patient.