Hodgkin And Reed-Sternberg Cells Research Articles

Expression Patterns of GATA3 in Classical Hodgkin Lymphoma: A Clinico-Pathological Study.

GATA3 is a transcription factor involved in T-cell maturation and has been previously shown to be aberrantly overexpressed in malignant Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). However, the immunophenotypes of the cell types expressing GATA3 have not been precisely characterized so far in cHL tissues. In this single-center retrospective cohort study we analyzed the expression patterns of GATA3 alone and in combination with B, T, NK or macrophage-associated markers in 73 cases with newly diagnosed cHL and investigated for a possible correlation with clinical and laboratory parameters. Immunohistochemistry (single and double) was performed using GATA3 alone and in combination with CD20, CD3, CD56, CD68, CD30 or CD15. Clinical and laboratory parameters were collected and correlated with the expression of GATA 3. GATA3 nuclear expression was found in HRS cells in 39/73 (54%) cases of cHL. The Nodular Sclerosis (NS) subtype showed the highest positivity rate (35/56, 63%), followed by mixed cellularity (MC; 4/14, 29%) and lymphocyte rich (LR; 0/3). Double immunostainings showed that GATA3 was expressed by CD30+ or CD15+ HRS cells and a few CD3+ T-cells, whereas GATA3 expression was not detected in CD20, CD56 or CD68+ cells. GATA3-negative cHL was significantly associated with unfavorable prognostic factors such as older age at diagnosis and increased levels of serum β2-microglobulin. The heterogenous expression patterns of GATA3 in HRS cells that were observed in a substantial proportion of cHL, mainly in the NS subtype, further support the biological heterogeneity of cHL.

Spatially Resolved Tumor Microenvironment Predicts Treatment Outcomes in Relapsed/Refractory Hodgkin Lymphoma.

About a third of patients with relapsed or refractory classic Hodgkin lymphoma (r/r CHL) succumb to their disease after high-dose chemotherapy followed by autologous stem-cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL. We performed imaging mass cytometry (IMC) on 71 paired primary diagnostic and relapse biopsies using a marker panel specific to CHL biology. For each cell type in the TME, we calculated a spatial score measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within the close interaction range. Spatial scores were used as features in prognostic model development for post-ASCT outcomes. Highly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early r/r CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single-cell RNA sequencing data identified unique architecture defined by CXCR5+ Hodgkin and Reed Sternberg (HRS) cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay (RHL4S) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, CD68+ tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk versus low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay. We identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

CAR-T Cell Therapy for Classical Hodgkin Lymphoma.

Classical Hodgkin lymphoma (cHL) is a malignancy characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells within a complex tumor microenvironment (TME). Despite advances in conventional therapies, a subset of cHL patients experience relapse or refractory disease, necessitating the exploration of novel treatment strategies. Chimeric antigen receptor T cell (CAR-T cell) therapy has emerged as a promising approach for the management of cHL, harnessing the power of genetically modified T cells to recognize and eliminate tumor cells. In this article, we provide an overview of the pathogenesis of cHL, highlighting the key molecular and cellular mechanisms involved. Additionally, we discuss the rationale for the development of CAR-T cell therapy in cHL, focusing on the identification of suitable targets on HRS cells (such as CD30, CD123, LMP1, and LMP2A), clonotypic lymphoma initiating B cells (CD19, CD20), and cells within the TME (CD123, CD19, CD20) for CAR-T cell design. Furthermore, we explore various strategies employed to enhance the efficacy and safety of CAR-T cell therapies in the treatment of cHL. Finally, we present an overview of the results obtained from clinical trials evaluating the efficacy of CAR-T cell therapies in cHL, highlighting their potential as a promising therapeutic option. Collectively, this article provides a comprehensive review of the current understanding of cHL pathogenesis and the rationale for CAR-T cell therapy development, offering insights into the future directions of this rapidly evolving field.

Genomic, Transcriptional, and Immunological Validation of Distinct Molecular Subtypes of Classic Hodgkin Lymphoma through Tissue-Based and Noninvasive Methods

Introduction: The scarcity of malignant Hodgkin and Reed-Sternberg (HRS) cells has hampered comprehensive genomic profiling of classic Hodgkin lymphoma (cHL) from tumor tissue. Multiple recent studies have demonstrated that plasma cfDNA profiling facilitates molecular characterization of cHL. Leveraging noninvasive genotypes and Latent Dirichlet Allocation, we recently defined 2 genetic cHL subtypes in a large international cohort comprising 366 patients including pediatric and adult patients of all ages (Alig et al., ASH 2022 and ICML 2023). Cluster H1 comprised ~2/3 of cases and was dominated by a high somatic mutational burden, and non-silent mutations in genes canonically involved in NF-κB, JAK/STAT and PI3K signaling as well as in B2M. Conversely, cluster H2 (~1/3 of cases) was primarily characterized by recurrent somatic copy number aberrations as well as mutations in TP53 and KMT2D. Herein, we validate these previously identified genetic subtypes in external datasets as well as through orthogonal methods including tissue-based and noninvasive transcriptional and immune profiling. Methods: To validate genetic cHL subtypes, we first leveraged public cHL genotypes from 61 patients obtained through whole genome/exome sequencing (WGS/WES) of flow-sorted HRS cells (Maura et al, Blood Cancer Disc. 2023). Using our previously locked down probabilistic classifier, we assigned the H1/H2 subtype, and then correlated cluster assignments with clinical variables. To explore transcriptional differences between genetic subtypes, we profiled baseline plasma samples (n=113) from the larger plasma genotyping cohort (n=293) using EPIC-Seq (Esfahani et al, Nat Biotechnol 2022), which allows for noninvasive gene expression profiling from cfDNA fragmentation patterns at transcription start sites. Further, we used SABER (Sworder, Cancer Cell 2023) to enumerate T-cell receptor (TCR) rearrangements in cfDNA (cfTCR, n=292). Finally, to assess subtype-specific immune infiltrate patterns, we profiled cHL tumor specimens using RNA-Seq (n=64), and applied CIBERSORTx. Results: Similar to observations in our cfDNA discovery cohort, H1 was found to be the more prevalent subtype in the external validation set comprising 56% of tumors, while 44% were classified as H2. Recurrence frequencies of genetic features were comparable to and significantly correlated with those from our plasma discovery cohort ( R S=0.59 [H1] and R S=0.63 [H2], P<0.001 each). Of note, when considering the whole genome space, the higher mutational burden of H1 tumors could be confirmed ( P<0.01), and this association was independent of the tumor EBV status. In agreement with the discovery cohort, the bimodal age distribution and increased EBV positivity of the H2 subtype could also be recapitulated (31% vs 6% EBV+, P<0.01). To explore transcriptional differences between H1/H2 subtypes, we took advantage of the plasma enrichment of HRS cell ctDNA and utilized EPIC-Seq to noninvasively infer expression of 1,676 targeted genes. Tracking signatures derived from scRNA-Sequencing in plasma samples, we found that both HRS cells and the cHL tumor microenvironment can be successfully profiled by EPIC-Seq. Strikingly, we found substantial enrichment of a cytokine response signature in H1 tumors, while T-cell activation was among the top upregulated signatures in H2 tumors ( Fig. A). Importantly, the T-cell signature found in H2 was accompanied by a higher abundance of T-cell clones as quantified by SABER in baseline plasma samples ( P<0.001, Fig. B). Notably, cfTCR fragment length profiles resembled the mutant ctDNA profiles, strongly suggesting a tumor origin of the TCR rearrangements detected in plasma. Lastly, immune cell deconvolution of bulk RNA-Seq specimens indicated a higher abundance of CD8+ T-cells in H2 tumors ( P<0.01), further confirming our prior observations. Conclusions: Collectively, these results serve to validate H1 and H2 as distinct cHL subtypes, to confirm the characteristic genotypes defining them, and recapitulate their distinctive associations with key clinical and pathological variables including age and EBV status. We further validate the subtypes using orthogonal methods revealing dominant cytokine driven signaling in H1. Conversely, H2 tumors, which largely lack B2M mutations, despite their lower mutational burden, are rather immunogenic triggering T-cell responses.

Unraveling the Immune Microenvironment in Classic Hodgkin Lymphoma: Prognostic and Therapeutic Implications.

Classic Hodgkin lymphoma (cHL) is a lymphoid neoplasm composed of rare neoplastic Hodgkin and Reed-Sternberg (HRS) cells surrounded by a reactive tumor microenvironment (TME) with suppressive properties against anti-tumor immunity. TME is mainly composed of T cells (CD4 helper, CD8 cytotoxic and regulatory) and tumor-associated macrophages (TAMs), but the impact of these cells on the natural course of the disease is not absolutely understood. TME contributes to the immune evasion of neoplastic HRS cells through the production of various cytokines and/or the aberrant expression of immune checkpoint molecules in ways that have not been fully understood yet. Herein, we present a comprehensive review of findings regarding the cellular components and the molecular features of the immune TME in cHL, its correlation with treatment response and prognosis, as well as the potential targeting of the TME with novel therapies. Among all cells, macrophages appear to be a most appealing target for immunomodulatory therapies, based on their functional plasticity and antitumor potency.

Proteomic profiling differentiates classic Hodgkin lymphoma with and without skeletal involvement at the time of diagnosis

Introduction: Classic Hodgkin lymphoma (cHL) is a highly curable disease, even in advanced stages. It is controversial whether bone involvement has a negative effect on overall- and progression-free survival in patients treated with intensive chemotherapy. cHL is characterized by a unique tumor microenvironment (TME) consisting of few, scattered neoplastic Hodgkin and Reed-Sternberg (HRS) cells, embedded in an abundant background of reactive immune and stromal cells. When cHL disseminates, the new disease sites also contain both HRS cells and TME cells. Whether cases that present with bone lesions, harbour specific TME features is unknown. We investigated protein expression in lymph node biopsies from cHL patients with and without skeletal involvement at diagnosis, using nano liquid chromatography—tandem mass spectrometry (nLC-MS/MS), to identify potential markers of skeletal disease. Methods: Protein expression patterns in pre-therapeutic formalin-fixed, paraffin-embedded lymphoma samples from 69 cHL patients diagnosed at Aarhus University Hospital, Denmark between 2009 and 2018 were analysed by nLC-MS/MS. Patients were grouped according to diagnostic 18F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). FDG PET/CT scans were reviewed specifically for bone involvement. The study cohort included 50 patients with nodal cHL (n-cHL) and no skeletal involvement, and 19 patients with both skeletal and nodal disease (s-cHL). Results: We identified 2,298 proteins; comparison of the protein profiles between the s-cHL and n-cHL groups revealed 220 unique proteins significantly differentially expressed (p < 0.05) and with a fold change of at least 25%. Of these, 117 proteins were upregulated (fold change 1.25–3.94), and 103 were downregulated (fold change 0.44–0.80) in s-cHL. In hierarchal clustering based on an even higher significance threshold (p < 0.001), i.e., 25 significantly differentially expressed proteins, two clusters were observed: (i) a skeletal-group comprising 12 s-cHL and 4 n-cHL samples; and (ii) a nodal-group of 46 n-cHL and 7 s-cHL samples. Of particular interest among the differentially expressed proteins, we identified isocitrate dehydrogenase (IDH1) and WD repeat- and FYVE domain-containing protein 4 (WDFY4). This pattern of protein expression suggests disturbance in cytoplasmic NADPH production, antigen processing/presentation, and B-cell survival in cHL patients with skeletal involvement compared to those without. Conclusion: Our data show differential protein expression in cHL lymphoma tissues that correlate with the presence or absence of concomitant bone involvement at diagnosis. This indicates that certain biological pathways are affected among those presenting with disease disseminated to the skeletal system. The research was funded by: Department of Clinical Medicine, Aarhus University; Health Research Foundation of Central Denmark Region; Brødrene Hartmanns Foundation; Carl and Ellen Hertz Foundation; the Danish Lymphoma Group; Kong Christian den Tiendes Foundation; Torben and Alice Frimodts Foundation; Aase and Ejnar Danielsens Foundation; Foundation of 1870; Dagmar Marshalls Foundation; Merchant Einar Willumsen’s Memorial Foundation; Eva and Henry Frænkel’s Memorial Foundation; Master Carpenter Jørgen Holm and wife Elisa f. Hansen’s Memorial Grant. The Orbitrap Fusion Tribrid mass spectrometer was funded by A. P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal. Keywords: Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.

STING is Expressed by Hodgkin and Reed Sternberg (HRS) Cells in a Subset of Classical Hodgkin Lymphoma (cHL) and Correlates with Tumor Microenvironment and Immune Response

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through activation of the adaptor protein STING. The latter, in turn, activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The expression patterns of STING in Hodgkin and Reed-Sternberg (HRS) cells and the potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remain unknown to date. Methods: In this prospective study, STING expression was immunohistochemically analysed in a cohort of 52 previously untreated patients with cHL and available tissue for flow cytometry (FC) analysis and histology as well as available peripheral blood samples for FC. An arbitrary 10% cutoff was used for positivity in HRS. The in vitro system included 6 cHL cell lines (MDAV, L1236, L428, L540, HDLM2, KMH2). Gene (mRNA level) and protein expression/activation of cGAS-STING components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist or were subjected to silencing of STING gene using transient transfection with specific STING siRNA construct. The gene expression of cGAS-STING-associated IFNs and cytokines, including IFN-β, CXCL10, IFN-γ, STING, and a control gene (GAPDH), was analysed with RT-qPCR. Results: STING was positive in HRS cells of cHL in 22 of 52 (42%) patients. STING positivity in HRS significantly correlated with lower numbers of STING+ T-lymphocytes (p < 0.0001) and CD3+ T-lymphocytes (p = 0.04) but higher numbers of CD20+ B-cells assessed by FC in the tissues. In peripheral blood samples assessed by FC at presentation, STING expression by HRS correlated with higher numbers of CD3+ T-cells (p = 0.005) but lower numbers of NK-cells (p = 0.02). STING expression did not correlate with clinical features at presentation, including age, gender, Ann Arbor stage, B-symptoms, tumor burden, bulky disease, anemia, or other hematologic values. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonists stimulated gene expression of IFN-β and/or CXCL10 at a variable level indicating functional cGAS-STING pathway in HRS. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Conclusions: STING is expressed by HRS in a subset of cHL and significantly correlates with the lymphocytic populations in the tumor microenvironment and peripheral blood. The cGAS-STING pathway is functional in HRS suggesting that STING agonists may have therapeutic implications in patients with cHL. Keywords: Diagnostic and Prognostic Biomarkers, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.

Hodgkin lymphoma in the bone marrow aspirate of a young patient with human immunodeficiency virus.

eJHaemEarly View HAEMATOLOGY IMAGESOpen Access Hodgkin lymphoma in the bone marrow aspirate of a young patient with human immunodeficiency virus Evashin Pillay, Corresponding Author Evashin Pillay evashinp@gmail.com orcid.org/0000-0003-0733-2860 University of KwaZulu-Natal & National Health Laboratory Service, Inkosi Albert Luthuli Central & King Edward VIII Hospitals, Durban, South Africa Correspondence Evashin Pillay, Department of Haematology, Inkosi Albert Luthuli Central Hospital, 4th Floor Laboratory Building, 800 Bellair Road, Durban 4091, KwaZulu-Natal, South Africa. Email: evashinp@gmail.comSearch for more papers by this authorPoobalan Naidoo, Poobalan Naidoo University of KwaZulu-Natal & KwaZulu Natal Provincial Department of Health, Inkosi Albert Luthuli Central & King Edward VIII Hospitals, Durban, South AfricaSearch for more papers by this author Evashin Pillay, Corresponding Author Evashin Pillay evashinp@gmail.com orcid.org/0000-0003-0733-2860 University of KwaZulu-Natal & National Health Laboratory Service, Inkosi Albert Luthuli Central & King Edward VIII Hospitals, Durban, South Africa Correspondence Evashin Pillay, Department of Haematology, Inkosi Albert Luthuli Central Hospital, 4th Floor Laboratory Building, 800 Bellair Road, Durban 4091, KwaZulu-Natal, South Africa. Email: evashinp@gmail.comSearch for more papers by this authorPoobalan Naidoo, Poobalan Naidoo University of KwaZulu-Natal & KwaZulu Natal Provincial Department of Health, Inkosi Albert Luthuli Central & King Edward VIII Hospitals, Durban, South AfricaSearch for more papers by this author First published: 13 March 2023 https://doi.org/10.1002/jha2.670AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat A 31-year-old South African lady, HIV-positive since 2019 (medication: fixed dose combination antiretroviral therapy – tenofovir, lamivudine and dolutegravir; CD4 count: 328 cells/µL; HIV viral load: undetectable) and who completed a full course of empiric therapy for tuberculosis (TB) 2 months prior, presented to the emergency room complaining of fatigue, loss of appetite and malaise; no bleeding. During admission, her general examination was remarkable for muscle wasting, tachycardia, pallor, pyrexia (>38°C) and generalised significant lymphadenopathy (LAD), particularly evident as a left-sided axillary mass. Systemic examination revealed a distended abdomen compatible with ascites, and reduced bi-basal breath sounds; no hepatosplenomegaly. The overall clinical impression was that of a young female with stage 4 HIV and features of lymphoma, as supported by constitutional symptoms, LAD and serositis. Blood work comprising an FBC demonstrated an isolated severe anaemia (leucocytes 5.20 × 109/L – lymphocytes 7%, haemoglobin 5.1 g/dL and platelets 314 × 109/L); corrected reticulocyte count 0.45%. A staging CT (computed tomography) scan demonstrated generalised significant LAD (>1 cm; neck, supraclavicular, axillary (largest; >4 cm in short axis), mediastinal, hilar and para-aortic), bilateral pleural effusions with bi-basal atelectasis, gross ascites and hepatomegaly (±14 cm in span); no splenomegaly but multiple non-enhancing round hypodensities were noted in the spleen. Tissue biopsies were then planned, and the excision node histology confirmed a diagnosis of nodular sclerosis classic Hodgkin lymphoma (cHL), syncytial variant. A subsequent staging bone marrow (BM) biopsy was performed and remarkably, the BM aspiration smears revealed ∼5% large- and giant-sized, mono- and multi-nuclear tumour cells that morphologically resembled Hodgkin and Reed–Sternberg (HRS) cells (Figure 1). Unfortunately, several attempts to process the BM aspirate sample for flow cytometry proved unsuccessful due to repeated clogging of the sample injection tube. Nonetheless, the corresponding BM trephine biopsy sections showed focal infiltration by neoplastic cells, in addition to many pleomorphic counterparts (Figure 2). Immunohistochemical and special stains (Figure 2) revealed the HRS cells to express the following phenotype: CD45−, CD15+, CD30+, PAX5+, MUM1+, EBER+ (Epstein–Barr virus-encoded RNA), CD3−, CD20−, CD138−, ALK− and AE1/AE3−. Moreover, identifiable in close proximity to the HRS cells were a pathognomonic mixed population of inflammatory cells (Figures 1 and 2). Trilinear haematopoiesis was relatively adequately preserved. FIGURE 1Open in figure viewer Bone marrow aspirate smear (panels A–I; May–Grünwald–Giemsa; 100× objective). Morphologically heterogeneous tumour cells including Hodgkin cells (panels A–C; white arrows), forms undergoing cell division (panel D; yellow arrow), Reed–Sternberg cells (panels E–H; black arrows) and multi-nuclear counterparts (panel I; purple arrow) comprised of abundant, deeply basophilic, agranular cytoplasm with vacuolation and clasmatosis (pseudopod formation), and containing nuclei with irregular borders, reticulated chromatin, conspicuous nucleoli (panel A) and vacuolation (panels C, F and I). Inflammatory cells comprising eosinophils (panel B; pink arrow), granulocytes (panel C; green arrow), plasma cells (panel E; red arrow) and lymphocytes (panel I; cyan arrow) in close proximity to tumour cells. FIGURE 2Open in figure viewer Bone marrow trephine sections (panels A–I; 100× objective). Hodgkin cells (panels A and B; haematoxylin and eosin [H&E]; white arrows), Reed–Sternberg cells (panels B–E; H&E black arrows) and multi-nuclear tumour cell variants (panels C, E and F; H&E purple arrows). A lacunar cell (panel A; white arrow), hallmark-like cell (panel B; blue arrow) and mitotic Reed–Sternberg cell with ‘cherry-red’ nucleoli (panel D; yellow arrow). Immunostaining of tumour cells for CD15 (panel G; black arrow), CD30 (panel H; black arrow) and Epstein–Barr virus-encoded RNA (EBER) (panel I; in situ hybridisation [ISH]; black arrow). Inflammatory cells comprising eosinophils (panel A; pink arrow), plasma cells (panel A; red arrow), granulocytes (panel B; green arrow) and lymphocytes (panel B; cyan arrow) in close proximity to tumour cells. Finally, an international prognostic score of 4 was concluded (serum albumin 1.3 g/dL, haemoglobin 5.1 g/dL, stage IV disease and lymphocytes 7%). Notably, microbiological investigations for SARS-CoV-2 and TB were negative. Unfortunately, the patient succumbed to a hospital-acquired pneumonia despite exhaustive supportive therapy and empiric antibiotics, and prior to initiating chemotherapy. It is exceedingly rare to observe well preserved HRS cells in a BM aspirate film. The accompanying fibrotic milieu of the infiltrated BM probably causes tumour cell fragmentation and/or impairs their effective aspiration [1-4]. Another consideration is that of scattered focal bony lesions which may not be biopsied during the process of attempting aspiration [5, 6]. This case highlights an exceptional clinicopathologic entity by demonstrating HRS cells in the BM aspirate film of a young HIV-positive patient presenting with the syncytial variant of nodular sclerosis cHL. It has enabled careful examination of numerous HRS cells thereby contributing to appreciation of their morphological heterogeneity. Haemato-pathologists should be cognizant of such cases to prevent misdiagnosis as metastatic non-haematological neoplasms (particularly carcinoma) or other haematological neoplasms (particularly non-Hodgkin lymphoma) [7, 8]. Accordingly, timeous diagnosis may prove to be lifesaving, especially in resource-constrained settings. ACKNOWLEDGEMENTS The authors thank the hospital administrative and laboratory staff for their assistance in preparing this case. Verbal consent was duly obtained from the patient for purposes of publication. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. ETHICS STATEMENT Ethical approval was obtained from the Biomedical Research Ethics Committee (BREC) affiliated with the University. Open Research DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. REFERENCES 1Swart L, Shuttleworth MH, Opie JJ, van Schalkwyk W. Paediatric Hodgkin lymphoma with Reed-Sternberg and mononuclear Hodgkin cells in the bone marrow aspirate. Br J Haematol. 2012; 157: 2. 2Xu Z, Burns BF, Faught C. Reed-Sternberg cells detected in the bone marrow aspirate in a young patient with nodular sclerosis Hodgkin lymphoma. Eur J Haematol. 2013; 92: 365– 6. 3Parsons K, Leach M, Bain BJ. Hodgkin lymphoma in a bone marrow aspirate. Am J Hematol. 2020; 95: 328– 9. 4Domínguez-Muñoz MÁ, Morales-Camacho RM, Prats-Martín C, Ávila R, Vargas MT, Burillo S, et al. Unusual co-occurrence of Hodgkin lymphoma and hemophagocytic lymphohistiocytosis in a bone marrow aspirate. Ann Hematol. 2016; 95: 1019– 21. 5Lee S-G, Paik S-Y, Sohn H-J, Kim SY, Kong S-Y. Numerous Reed-Sternberg cells in bone marrow aspirate from a patient with syncytial variant of nodular sclerosis classical Hodgkin lymphoma. Int J Hematol. 2015; 101: 107– 8. 6Kini JR, Suresh PK, Sinha R, Sahu K, Kumar S, Prasad K. Value of bone marrow examination in Hodgkin lymphoma: report of three cases with review of literature. J Cancer Res Ther. 2012; 8(3): 457– 9. 7Strickler JG, Michie SA, Warnke RA, Dorfman RF. The “syncytial variant” of nodular sclerosing Hodgkin's disease. Am J Surg Pathol. 1986; 10(7): 470– 7. 8Conlin PA, Blann MM, Morgan DL. Pathologic quiz case: supraclavicular lymphadenopathy in a 30-year-old man. Arch Pathol Lab Med. 2000; 124: 1837– 8. Early ViewOnline Version of Record before inclusion in an issue FiguresReferencesRelatedInformation

IL-6 as an Inflammatory Marker in Hodgkin Lymphoma

Hodgkin lymphoma (HL) is a lymphoid tumor that develops from embryological or post-germinal center B-cells. The disease was originally recognized in 1832 by Thomas Hodgkin. Globally, 0.4% of clinically diagnosed cancers in 2018 were caused by HL. Regardless of age, ethnicity, or location, the prevalence of HL varies. It's a particular kind of malignant tumor in which the cancerous Hodgkin and Reed-Sternberg (HRS) cells are surrounded by a significant cellular infiltration of immune cells (lymphocytes, macrophages, eosinophils, mast cells, plasma cells, and collagen cells), which also contribute to the microenvironment of the tumor. Numerous chemotherapy drugs and low-dose field radiation have been found to significantly reduce illness mortality [1].&#x0D; High levels of cytokines that stimulate the immune system, such as interleukin (IL)-6, tumor necrosis factor (TNF), and eotaxin are produced by developing tumor cells and/or by reactive immune cells associated with tumors. These markers are anticipated to promote B-cell activation and related high-risk DNA-modifying activities, which will lead to the growth of B-cell lymphoma. IL-6 is a critical cytokine that stimulates the growth of hematological and solid tumors and is synthesized by a wide range of cell types, including fibroblasts, ECs, epithelial cells, and monocytes as hematological tumor lines. Through the activation of JAK, it stimulates the transcription factors STAT. In NHL, lower progression-free survival and overall survival rates have been linked to higher blood IL-6 levels, and IL-6 production in HL cell lines has been demonstrated. Numerous investigations revealed that IL-6 expression correlated with early onset vascular development events, indicating that it may be involved in angiogenesis [2].&#x0D; Interleukin-6 (IL-6) is thought to play a crucial pathobiological function in classical HL among the biologic variables like cytokines. According to reports, adult patients with HL who have elevated serum interleukin-6 (IL-6) have better response rates, longer survival times, and B symptoms. TH-17 response may be elicited by HRS cells that express IL-6, but TH-2 response may be elicited by HRS cells that do not express IL-6. T-lymphocytes are known to be attracted by IL-6, and depending on how it interacts with other cytokines like TGF-beta, the lymphocytes may induce a TH-2 or TH-17 response. A significant cancer hallmark known as immune evasion can be brought on in cancer patients by the TH-2 response [3].

Spatial Tumor Microenvironment Characterization and Outcome of Relapsed/Refractory Classic Hodgkin Lymphoma

INTRODUCTION: Despite recent treatment advances, about half of relapsed or refractory classic Hodgkin lymphoma (r/r CHL) patients succumb to their disease after high dose chemotherapy followed by autologous stem cell transplantation (HDT/ASCT). Our group has previously developed a gene expression based prognostic assay (RHL30) that identifies high-risk r/r CHL patients treated with HDT/ASCT (J Clin Oncol 2017). However, RHL30 does not incorporate important information about cellular interactions, specific expression features of Hodgkin and Reed-Sternberg (HRS) cells and the spatial architecture of the tumor-microenvironment (TME). Here, we aimed to determine spatially resolved TME ecosystems and establish novel biomarkers associated with treatment failure in r/r CHL. METHODS: Using a customized marker panel specific for CHL biology, we performed imaging mass cytometry (IMC) using a tissue microarray containing formalin-fixed paraffin-embedded tissues from 169 CHL tumors, including 73 patients with paired primary and relapse samples, as well as 23 pretreatment diagnostic samples from patients in long-term remission. Segmentation was performed by the Ilastik machine learning package following the Bodenmiller IMC segmentation pipeline to obtain single cell level data. We used the Phenograph clustering algorithm to identify major cell types and functional subsets. For each cell, we calculated a spatial cell enrichment score, called 'spatial score', of a given cell type as the distance to the five nearest neighbor cells, capped at the spatial interaction range (50um). Patients were classified as having early relapse disease if their CHL progressed within 12 months after initial diagnosis. For prognostic model development, we used an empirical Bayesian approach based on LPS (linear prediction score). RESULTS: We obtained highly multiplexed images for a total of 7,146,042 cells that could be broadly clustered into T cell, macrophage, B cell as well as HRS cells using the phenograph tool. We also identified subsets within the HRS cell cluster, such as GATA3-high HRS cells and CXCR5-high HRS cells. We, then, investigated the difference of TME characteristics according to the disease status. Overall, CHL biopsies from early relapse demonstrated shared TME patterns between diagnostic and relapse samples, and were characterized by a lesser extent of CD8+ cells (P < 0.01) as well as enrichment of a myeloid cell population (P < 0.05). In contrast, late relapse samples often demonstrated significant TME changes between diagnostic and relapse biopsies (P < 0.01). Considering the previous finding of the superiority of relapse biopsies for outcome prediction in comparison to pretreatment diagnostic samples in r/r CHL, we again focused our prognostic model development on relapse biopsies. Using LASSO based analyses, four variables, CXCR5+ HRS cells (Hazard Ratio [HR]: 2.86), PD1+ CD4+ T cells (HR: 2.80), macrophage (HR: 1.99), and CXCR5+ B cells (HR: 0.19) were identified as factors that were significantly associated with post-ASCT failure free survival (FFS). Using these 4 variables, we developed a spatial score-based prognostic model (RHL-4S) and compared performance with a classical protein%-based model. The RHL-4S produced a high-risk group of patients with significantly inferior post-ASCT FFS compared with the low-risk group (5-year post-ASCT FFS: high risk, 38% vs low risk, 96%; P < 0.0001, Figure A) as well as inferior post-ASCT overall survival (OS) (5-year post-ASCT OS: high risk, 45% vs low risk, 95%; P < 0.0025). IMC analyses furthermore identified unique spatial architecture defined by CXCR5+ HRS cells. In particular, CXCR5+ HRS cells were spatially arranged together with other HRS cells forming cell clusters as an architectural feature (P < 0.05, Figure B). CXCR5+ HRS cells in these clusters were also characterized by a lower abundance of neighboring non-malignant immune cells, including CD4+ T cells and CD20+ B cells when compared to CXCR5- HRS cells. CONCLUSION We developed a novel prognostic model, RHL-4S, applicable to relapse biopsies of r/r CHL patients who were treated with ASCT. In particular, we have shown the interaction between CXCR5 on HRS cells with ligand-expressing CXCL13+ follicular T helper cells (Aoki et al, PNAS 2021) and macrophages as a prominent crosstalk axis in relapsed disease. This insight opens new avenues to predictive biomarker development. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P043: Molecular pathogenesis of Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL) is one of the most frequent lymphomas in the Western world. Its malignant Hodgkin and Reed/Sternberg (HRS) cells are derived from pre-apoptotic germinal center B cells and only account for ca. 1% of the tumor cell mass. The surrounding inflammatory infiltrate is unable to establish an effective immune response against the HRS cells. To better understand HRS cell formation and their molecular pathogenesis, we aim to determine the mutational landscape of HRS cells. HRS cells of a total of 30 cases were isolated by microdissection or flow cytometry and subjected to exome or whole-genome sequencing. We confirmed recurrently mutated genes (e.g. SOCS1, TNFAIP3) but also found novel promising genes such as NLRC5, which is involved in MHCI expression and negative NFkB regulation. Intriguingly, mutational signatures associated with APOBEC and somatic hypermutation were identified. Moreover we are currently analyzing the WGS samples for mutations in gene regulatory regions, miRNA binding sites and gains and losses. Both WGS and WES show a wide variation in their mutational loads.

T028: Single-cell RNA sequencing reveals the interplay between circulating CD4 T cells, B cells and cancer-associated monocytes in classic Hodgkin lymphoma treated with PD-1 blockade

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin and Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA sequencing to characterize the peripheral immune response to PD-1 blockade and more broadly define non-CD8+ dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL treated with PD-1 blockade (nivolumab) on the CheckMate 205 clinical trial (cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed, previously untreated cHL and 13 healthy donors. Unlike healthy donors, all evaluated patients with cHL had circulating IFN stimulated adaptive and innate populations, including several distinct CD4+ T-cell effectors and an NK cell subset with reduced cytotoxic potential, and decreased numbers of B cells at all stages of differentiation. Patients with the most favorable responses to PD-1 blockade had: 1) significantly increased CD4+ T-cell receptor diversity and more abundant naïve/ central memory subsets; and 2) significantly higher B-cell receptor diversity and increased numbers of circulating B cells. The most abundant circulating CD3- population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumorigenic cytokines and chemokines, PD-L1 and SIRPa. This newly identified monocytic population was virtually absent from the blood of healthy donors. RNAscope analysis of the intact tumor microenvironment localized these tumor-infiltrating monocytes/macrophages to the immediate proximity of HRS cells. Monocytes from patients whose disease progressed following PD-1 blockade expressed significantly higher levels of immunosuppressive cytokine/chemokine signature which led to the development of a predictive transcriptional assay. We identified a comparable circulating monocyte population and transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor underscoring the broad-based significance of these findings.

Programmed death-ligand 1 (PD-L1) expression in Hodgkin’s lymphoma among Peruvian patients

Objective: Various studies on Hodgkin’s lymphoma (HL) have determined programmed death-ligand 1 (PD-L1) expression in Hodgkin and Reed-Sternberg (HRS) cells, with variable results. This protein has gained relevance for its role in theimmune response in malignant neoplasms. This work aimed to determine PD-L1 immunohistochemical expression in HRS cells and its relationship with age, sex, clinical stage and overall survival (OS) in a cohort of Peruvian patients. Materials and methods: Twenty-five (25) biopsies from patients diagnosed with HL were assessed, which allowed determining PD-L1 immunohistochemical expression in HRS cells in relation to OS and clinical data of the patients. Results: All cases showed PD-L1 expression in more than 1 % of tumor cells. There was no statistically significant difference in OS when two groups were compared in terms of PD-L1 expression with a cut-off point of 50 %, clinical stage (CS), age and sex. Conclusions: High PD-L1 expression was found in pre-treatment HL tumors. No association was found between PD-L1 expression, OS, age, sex or CS. Further studies with a larger number of patients are necessary to reassess the prognosticimpact of the expression of this protein in HL.

Autophagy and cellular senescence in classical Hodgkin lymphoma

Autophagy and cellular senescence are interrelated cellular stress responses important for cellular homeostasis and they have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL). However, the presence of autophagy and cellular senescence and their relation with clinical and laboratory parameters needs further elucidation. Thus, autophagy (LC3B and p62 immunohistochemical expression) and cellular senescence (p16 immunohistochemical expression and SenTraGor™ staining) were studied in tissue sections from 59 patients with cHL. Autophagy and cellular senescence-associated markers were detected in variable proportions of Hodgkin and Reed-Sternberg (HRS) cells in cHL cases. High nuclear p62 immunohistochemical expression in HRS cells showed significant positive correlation with relapse of the disease (p = 0.037). Heterogeneous autophagy and cellular senescence patterns were revealed in HRS cells suggesting biological heterogeneity of cHL. The detection of high nuclear p62 expression in HRS cells may identify a subset of cHL with more aggressive clinical behavior.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

AbstractClonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Current and Emerging Approaches to Study Microenvironmental Interactions and Drug Activity in Classical Hodgkin Lymphoma.

Simple SummaryIn classical Hodgkin Lymphoma (cHL), the tumor microenvironment (TME) plays an important role in tumor progression and treatment response, making its evaluation critical for determining prognosis, treatment strategies and predicting an increase in drug toxicity. Therefore, there is a need to utilize more complex systems to study the cHL-TME and its interplay with tumor cells. To evaluate new anticancer drugs and to find the mechanisms of drug resistance, this review summarizes emerging approaches for the analysis of the TME composition and to identify the state of the disease; the in vitro techniques used to determine the mechanisms involved in the building of an immunosuppressive and protective TME; new 3-dimensional (3D) models, the heterospheroids (HS), developed to mimic TME interactions. Here, we describe the present and likely future clinical applications indicated by the results of these studies and propose a classification for the in vitro culture methods used to study TME interactions in cHL.Classic Hodgkin lymphoma is characterized by a few tumor cells surrounded by a protective and immunosuppressive tumor microenvironment (TME) composed by a wide variety of noncancerous cells that are an active part of the disease. Therefore, new techniques to study the cHL-TME and new therapeutic strategies targeting specifically tumor cells, reactivating the antitumor immunity, counteracting the protective effects of the TME, were developed. Here, we describe new methods used to study the cell composition, the phenotype, and the spatial distribution of Hodgkin and Reed–Sternberg (HRS) cells and of noncancerous cells in tumor tissues. Moreover, we propose a classification, with increasing complexity, of the in vitro functional studies used to clarify the interactions leading not only to HRS cell survival, growth and drug resistance, but also to the immunosuppressive tumor education of monocytes, T lymphocytes and fibroblasts. This classification also includes new 3-dimensional (3D) models, obtained by cultivating HRS cells in extracellular matrix scaffolds or in sponge scaffolds, under non-adherent conditions with noncancerous cells to form heterospheroids (HS), implanted in developing chick eggs (ovo model). We report results obtained with these approaches and their applications in clinical setting.

The Need for Standardization in Next-Generation Sequencing Studies for Classic Hodgkin Lymphoma: A Systematic Review.

Classic Hodgkin lymphoma (cHL) constitutes a B cell-derived neoplasm defined by a scarce tumoral population, termed Hodgkin and Reed–Sternberg (HRS) cells, submerged into a histologically heterogeneous microenvironment. The paucity of HRS cells has historically hampered genetic studies, rendering the identification of the recurrent genetic lesions and molecular pathways deregulated in this lymphoma difficult. The advent of high-throughput sequencing methods such as next-generation sequencing (NGS) could sensibly optimize the identification of the mutational landscape of cHL. However, there is no current consensus either in the design of panels for targeted NGS or in its most relevant clinical applications. In this work, we systematically review the current state of NGS studies of cHL, stressing the need for standardization both in the candidate genes to be analyzed and the bioinformatic pipelines. As different institutions have developed and implemented their own customized NGS-based protocols, to compare and systematically review the major findings of this ongoing research area could be of added value for centers that routinely perform diagnostic, monitoring and genotyping strategies in cHL samples. The results of this systematic review should contribute to the interdepartmental harmonization and achievement of a consensus in the current clinical applications of NGS studies of cHL.

Checkpoint CD47 expression in classical Hodgkin lymphoma.

SummaryThe glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed‐death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event‐free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event‐free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD‐1, PD‐L1 and PD‐L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.

The Hodgkin Lymphoma Immune Microenvironment: Turning Bad News into Good.

Simple SummaryClassic Hodgkin lymphoma (cHL) is one of the most enigmatic study models of the tumor microenvironment (TME), in which the Hodgkin–Reed Sternberg (HRS) cells are distributed throughout an abundant but ineffective immune ecosystem. The hyperactivation of HRS cells due to somatic mutations leads to complex interactions with the different subsets of immune cell populations, modeling the pathophysiology of the disease. There are remaining loose ends regarding the identification of the immune functional states in the cHL microenvironment and their influence on tumor cell survival. Here, we review the most relevant immune populations identified in the cHL context, focusing on integrative functional signatures.The classic Hodgkin lymphoma (cHL) tumor microenvironment (TME) is by far the most abundant component of tumors and is responsible for most of their biological and clinical characteristics. Recent advances in our knowledge of these networks in cellular interactions allow us to understand that the neoplastic Hodgkin and Reed Sternberg (HRS) cells, although they are in the minority, are the main architects of this dysregulated immune milieu. Here, we review the major changes that have happened in recent years: from TME as a helpless bystander, reflecting an ineffective immune response, to a dynamic tumor-promoting and immunosuppressive element. The HRS cells promote survival through interconnected intrinsic and extrinsic alterations, boosting pro-tumoral signaling pathways through genetic aberrations and autocrine growth signals, in parallel with abnormal cytokine secretion for the recruitment and selection of the best cell partners for this immunosuppressive TME. In turn, cHL is already proving to be the perfect model with which to address an immune checkpoint blockade. Preliminary data demonstrate the utility of druggable key signaling pathways in this ensemble, such as JAK-STAT, NF-κB, and others. In addition, myriad biomarkers predicting a response await validation by new in situ multiplex analytical methods, single-cell gene expression, and other techniques. Together, these components will define the functional phenotypes with which we will elucidate the molecular pathogenesis of the disease and improve the survival of patients who are refractory to conventional therapies.

The Grey Zones of Classic Hodgkin Lymphoma

Simple SummaryClassic Hodgkin lymphoma (CHL) is a well-defined lymphoid neoplasm with a minority of characteristic neoplastic cells of B cell origin, namely Hodgkin and Reed–Sternberg cells immersed in a rich reactive inflammatory infiltrate in the background. Although CHL has always been set apart from non-Hodgkin lymphomas, cases with morphological and phenotypic features intermediate between CHL and other lymphomas have been described. Whereas some of these lymphomas only represent morphological mimics, others exhibit mutational and gene expression profiles which overlap with CHL, indicating that these cases, frequently termed grey zone lymphomas, reside on the biological boundary between CHL and large B-cell lymphomas. In the present review, we aim to describe the current knowledge of these rare lymphomas, address diagnostic issues and summarize today’s concepts on the classification of grey zone lymphomas and related tumors.Classic Hodgkin lymphoma (CHL) is a well-defined neoplasm characterized by the presence of a minority of pathognomonic Hodgkin and Reed–Sternberg (HRS) cells in a reactive inflammatory background. Although genotypically of B cell origin, HRS cells exhibit a downregulated B cell program and therefore are set apart from other B cell lymphomas in the current WHO classification. However, cases with morphological and phenotypic features overlapping with CHL have been recognized, and the category of B cell lymphoma—unclassifiable—with features intermediate between diffuse large B cell lymphoma (DLBCL) and CHL, also termed grey zone lymphoma, was first introduced into the WHO classification in 2008 as provisional entity. These cases, as well as others raising a differential diagnosis of CHL can present diagnostic problems, as well as therapeutic challenges. Whereas some of these lymphomas only represent biologically unrelated morphological mimics, others, especially mediastinal grey zone lymphoma, exhibit genetic and gene expression profiles which overlap with CHL, indicating a true biological relationship. In this review, we address areas of diagnostic difficulties between CHL and other lymphoma subtypes, discuss the biological basis of true grey zone lymphoma based on recent molecular studies and delineate current concepts for the classification of these rare tumors.