T-cell States, Repertoire, and Function in Classical Hodgkin Lymphoma Revealed through Single-Cell Analyses.

The classical Hodgkin lymphoma (cHL) environment is comprised of a dense and complex immune cell infiltrate interspersed with rare malignant Hodgkin-Reed-Sternberg (HRS) cells. HRS cells are actively surveilled by endogenous T cells, but data linking phenotypic and functional T-cell states with clonality at the single-cell level in cHL is lacking. To address this knowledge gap, we performed paired single-cell RNA and T-cell receptor sequencing on 14 cHL and 5 reactive lymphoid tissue specimens. Conventional CD4+ T cells dominated the cHL landscape. However, recurrent clonal expansion within effector and exhausted CD8+ T-cell and regulatory T-cell clusters was uniquely observed in cHL specimens. Multiplex flow cytometric analysis revealed that most lymphoma-resident T cells produced effector cytokines upon ex vivo restimulation, arguing against a profound dysfunctional T-cell state in cHL. Our results raise new questions about the nature of T cells that mediate the antilymphoma response following programmed cell death protein 1 (PD-1) blockade therapy in cHL.

Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Hodgkin Lymphoma: A disease shaped by the tumor micro- and macroenvironment.

The tumor microenvironment (TMicroE) and tumor macroenvironment (TMacroE) are defining features of classical Hodgkin lymphoma (cHL). They are of critical importance to clinicians since they explain the common signs and symptoms, allow us to classify these neoplasms, develop prognostic and predictive biomarkers, bioimaging and novel treatments. The TMicroE is defined by effects of cancer cells to their immediate surrounding and within the tumor. Effects of cancer cells at a distance or outside of the tumor define the TMacroE. Paraneoplastic syndromes are signs and symptoms due to effects of cancer at a distance or the TMacroE, which are not due to direct cancer cell infiltration. The most common paraneoplastic symptoms are B-symptoms, which manifest as fevers, chills, drenching night sweats, and/or weight loss. Less common paraneoplastic syndromes include those that affect the central nervous system, skin, kidney, and hematological autoimmune phenomena including hemophagocytic lymphohistiocytosis (HLH). Paraneoplastic signs such as leukocytosis, lymphopenia, anemia, and hypoalbuminemia are prognostic biomarkers. The neoplastic cells in cHL are the Hodgkin and Reed Sternberg (HRS) cells, which are preapoptotic germinal center B cells with a high mutational burden and almost universal genetic alterations at the 9p24.1 locus primarily through copy gain and amplification with strong activation of signaling via PD-L1, JAK-STAT, NFkB, and c-MYC. In the majority of cases of cHL over 95% of the tumor cells are non-neoplastic. In the TMicroE, HRS cells recruit and mold non-neoplastic cells vigorously via extracellular vesicles, chemokines, cytokines and growth factors such as CCL5, CCL17, IL6, and TGF-ß to promote a feed-forward inflammatory loop, which drives cancer aggressiveness and anti-cancer immune evasion. Novel single cell profiling techniques provide critical information on the role in cHL of monocytes-macrophages, neutrophils, T helper, Tregs, cytotoxic CD8+ T cells, eosinophils, mast cells and fibroblasts. Here, we summarize the effects of EBV on the TMicroE and TMacroE. In addition, how the metabolism of the TMicroE of cHL affects bioimaging and contributes to cancer aggressiveness is reviewed. Finally, we discuss how the TMicroE is being leveraged for risk adapted treatment strategies based on bioimaging results and novel immune therapies. In sum, it is clear that we cannot effectively manage patients with cHL without understanding the TMicroE and TMacroE and its clinical importance is expected to continue to grow rapidly.

Ultra-Deep Sequencing Reveals the Mutational Landscape of Classical Hodgkin Lymphoma.

The malignant Hodgkin and Reed Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) are scarce in affected lymph nodes, creating a challenge to detect driver somatic mutations. As an alternative to cell purification techniques, we hypothesized that ultra-deep exome sequencing would allow genomic study of HRS cells, thereby streamlining analysis and avoiding technical pitfalls. To test this, 31 cHL tumor/normal pairs were exome sequenced to approximately 1,000× median depth of coverage. An orthogonal error-corrected sequencing approach verified >95% of the discovered mutations. We identified mutations in genes novel to cHL including: CDH5 and PCDH7, novel stop gain mutations in IL4R, and a novel pattern of recurrent mutations in pathways regulating Hippo signaling. As a further application of our exome sequencing, we attempted to identify expressed somatic single-nucleotide variants (SNV) in single-nuclei RNA sequencing (snRNA-seq) data generated from a patient in our cohort. Our snRNA analysis identified a clear cluster of cells containing a somatic SNV identified in our deep exome data. This cluster has differentially expressed genes that are consistent with genes known to be dysregulated in HRS cells (e.g., PIM1 and PIM3). The cluster also contains cells with an expanded B-cell clonotype further supporting a malignant phenotype. This study provides proof-of-principle that ultra-deep exome sequencing can be utilized to identify recurrent mutations in HRS cells and demonstrates the feasibility of snRNA-seq in the context of cHL. These studies provide the foundation for the further analysis of genomic variants in large cohorts of patients with cHL. SIGNIFICANCE: Our data demonstrate the utility of ultra-deep exome sequencing in uncovering somatic variants in Hodgkin lymphoma, creating new opportunities to define the genes that are recurrently mutated in this disease. We also show for the first time the successful application of snRNA-seq in Hodgkin lymphoma and describe the expression profile of a putative cluster of HRS cells in a single patient.

The Polyvalent Role of CD30 for Cancer Diagnosis and Treatment.

CD30, also known as TNFRSF8 (tumor necrosis factor receptor superfamily member 8), is a protein receptor that is heavily glycosylated inside the Golgi apparatus, as well as a tumor marker that is found on the surface of specific cells in the body, including certain immune cells and cancer ones. This review aims to shed light on the critical importance of CD30, from its emergence in the cell to its position in diagnosing various diseases, including Hodgkin lymphoma, where it is expressed on Hodgkin and Reed-Sternberg cells, as well as embryonal carcinoma, anaplastic large cell lymphoma (ALCL), and cutaneous T-cell lymphoma (CTCL). In addition to its role in positive diagnosis, targeting CD30 has been a promising approach treating CD30-positive lymphomas, and there is ongoing research into the potential use of CD30-targeted therapies for autoimmune disorders. We aim to elaborate on CD30's roles as a tumor marker, supporting thus the hypothesis that this receptor might be the aim of cytostatic treatment.

Classic Hodgkin lymphoma in young people.

Classic Hodgkin lymphoma (CHL) is a unique form of lymphoid cancer featuring a heterogeneous tumor microenvironment and a relative paucity of malignant Hodgkin and Reed-Sternberg (HRS) cells with characteristic phenotype. Younger individuals (children, adolescents and young adults) are affected as often as the elderly, producing a peculiar bimodal age-incidence profile that has generated immense interest in this disease and its origins. Decades of epidemiological investigations have documented the populations most susceptible and identified multiple risk factors that can be broadly categorized as either biological or environmental in nature. Most risk factors result in overt immunodeficiency or confer more subtle alterations to baseline health, physiology or immune function. Epstein Barr virus, however, is both a risk factor and well-established driver of lymphomagenesis in a significant subset of cases. Epigenetic changes, along with the accumulation of somatic driver mutations and cytogenetic abnormalities are required for the malignant transformation of germinal center-experienced HRS cell precursors. Chromosomal instability and the influence of endogenous mutational processes are critical in this regard, by impacting genes involved in key signaling pathways that promote the survival and proliferation of HRS cells and their escape from immune destruction. Here we review the principal features, known risk factors and lymphomagenic mechanisms relevant to newly diagnosed CHL, with an emphasis on those most applicable to young people.

BET inhibitors induce NF-κB and E2F downregulation in Hodgkin and Reed-Sternberg cells.

The prognosis of patients with relapsed and/or refractory classic Hodgkin lymphoma (cHL) continues to be poor. Therefore, there is a continuing need to develop novel therapies and to rationalize the use of target combinations. In recent years there has been growing interest in epigenetic targets for hematological malignancies under the rationale of the presence of common alterations in epigenetic transcriptional regulation. Since Hodgkin and Reed-Sternberg (HRS) cells have frequent inactivating mutations of the CREBBP and EP300 acetyltransferases, bromodomain and extra-terminal (BET) inhibitors can be a rational therapy for cHL. Here we aimed to confirm the efficacy of BET inhibitors (iBETs) using representative cell models and functional experiments, and to further explore biological mechanisms under iBET treatment using whole-transcriptome analyses. Our results reveal cytostatic rather than cytotoxic activity through the induction of G1/S and G2/M cell-cycle arrest, in addition to variable MYC downregulation. Additionally, massive changes in the transcriptome induced by the treatment include downregulation of relevant pathways in cHL disease: NF-kB and E2F, among others. Our findings support the therapeutic use of iBETs in selected cHL patients and reveal previously unknown biological mechanisms and consequences of pan-BET inhibition.

New molecular targets in Hodgkin and Reed-Sternberg cells.

Recent discoveries shed light on molecular mechanisms responsible for classical Hodgkin lymphoma (HL) development and progression, along with features of Hodgkin - Reed and Sternberg cells (HRS). Here, we summarize current knowledge on characteristic molecular alterations in HL, as well as existing targeted therapies and potential novel treatments for this disease. We discuss the importance of cluster of differentiation molecule 30 (CD30) and the programmed cell death-1 protein (PD-1) and ligands (PD-L1/2), and other molecules involved in immune modulation in HL. We highlight emerging evidence indicating that the altered function of SWI/SNF-type chromatin remodeling complexes, PRC2, and other epigenetic modifiers, contribute to variations in chromatin status, which are typical for HL. We postulate that despite of the existence of plentiful molecular data, the understanding of HL development remains incomplete. We therefore propose research directions involving analysis of reverse signaling in the PD-1/PD-L1 mechanism, chromatin remodeling, and epigenetics-related alterations, in order to identify HL features at the molecular level. Such attempts may lead to the identification of new molecular targets, and thus will likely substantially contribute to the future development of more effective targeted therapies.

Digital spatial profiling of CD4+ T cells in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (CHL) harbors a small number of Hodgkin-Reed-Sternberg (HRS) cells scattered among numerous lymphocytes. HRS cells are surrounded by distinct CD4+ T cells in a rosette-like manner. These CD4+ T cell rosettes play an important role in the tumor microenvironment (TME) of CHL. To elucidate the interaction between HRS cells and CD4+ T cell rosettes, we completed digital spatial profiling to compare the gene expression profiles of CD4+ T cell rosettes and other CD4+ T cells separated from the HRS cells. Immune checkpoint molecules including OX40, programed cell death-1 (PD-1), and cytotoxic T lymphocyte associated protein 4 (CTLA-4) expression was higher in CD4+ T cell rosettes compared to other CD4+ T cells. Immunohistochemistry confirmed variable PD-1, CTLA-4, and OX40 expression in the CD4+ T cell rosettes. This study introduced a new pathological approach to study the CHL TME, and provided deeper insight into CD4+ T cells in CHL.

CD30 induces Reed-Sternberg cell-like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines.

Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.

Increased STAT expression in Reed-Sternberg cells as a potential positive prognostication biomarker in Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) prognostication primarily relies on clinical and radiological factors. Despite this, a subset of patients still progress. Immunohistochemistry (IHC) based biomarkers on diagnostic tissue have not been routinely used for prognostication. A multicentre retrospective analysis identified 62 patients with cHL. IHC on diagnostic tissues was used to stain Reed-Sternberg cells (RS) cells for STAT1, pSTAT3, p53 and tumour microenvironment for CD68 and PD-1. IHC stains were scored by anatomical pathologists blinded to patients and their outcomes and correlated with survival. Strong intensity of STAT1 and pSTAT3 staining correlated with improved overall survival (OS), with hazard ratios (HR) of 0.21 [95% confidence interval (CI) 0.06-0.76] and 0.22 (95% CI 0.06-0.84), respectively. Similarly, the median OS for weak versus strong STAT1 or pSTAT3 staining was 8.8 years versus not reached. Other IHC stains did not correlate with prognosis. In this cohort of cHL patients, downregulation of immunohistochemical STAT1 or pSTAT3 in RS cells is associated with inferior OS, suggesting STAT transcription within the pathognomonic RS cells may have tumour suppressor function and may be a potential biomarker for cHL prognosis.

Spatial and molecular profiling of the mononuclear phagocyte network in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) has a rich immune infiltrate, which is an intrinsic component of the neoplastic process. Malignant Hodgkin Reed-Sternberg cells (HRSCs) create an immunosuppressive microenvironment by the expression of regulatory molecules, preventing T-cell activation. It has also been demonstrated that mononuclear phagocytes (MNPs) in the vicinity of HRSCs express similar regulatory mechanisms in parallel, and their presence in tissue is associated with inferior patient outcomes. MNPs in cHL have hitherto been identified by a small number of canonical markers and are usually described as tumor-associated macrophages. The organization of MNP networks and interactions with HRSCs remains unexplored at high resolution. Here, we defined the global immune-cell composition of cHL and nonlymphoma lymph nodes, integrating data across single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunofluorescence. We observed that MNPs comprise multiple subsets of monocytes, macrophages, and dendritic cells (DCs). Classical monocytes, macrophages and conventional DC2s were enriched in the vicinity of HRSCs, but plasmacytoid DCs and activated DCs were excluded. Unexpectedly, cDCs and monocytes expressed immunoregulatory checkpoints PD-L1, TIM-3, and the tryptophan-catabolizing protein IDO, at the same level as macrophages. Expression of these molecules increased with age. We also found that classical monocytes are important signaling hubs, potentially controlling the retention of cDC2 and ThExh via CCR1-, CCR4-, CCR5-, and CXCR3-dependent signaling. Enrichment of the cDC2-monocyte-macrophage network in diagnostic biopsies is associated with early treatment failure. These results reveal unanticipated complexity and spatial polarization within the MNP compartment, further demonstrating their potential roles in immune evasion by cHL.

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.

Epstein-Barr virus infection patterns in nodular lymphocyte-predominant Hodgkin lymphoma.

AIMS: To investigate Epstein-Barr virus (EBV) latency types in 19 cases of EBV-positive nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. METHODS AND RESULTS: Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in-situ hybridisation (ISH) for EBV-encoded RNA (EBER) was employed combined with IHC for EBV-encoded latent membrane protein (LMP)-1, EBV-encoded nuclear antigen (EBNA)-2, and EBV-encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed-Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B-cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER-positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30-negative. Five cases contained some BZLF1-positive and/or EBNA-2-positive bystander lymphocytes. CONCLUSIONS: As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV-positive NLPHL, preservation of the B-cell transcription programme, MEF2B expression combined with NLPHL-typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA-2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV-positive NLPHL.

Checkpoint CD47 expression in classical Hodgkin lymphoma.

The 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.

Prognostic Role of the Expression of Latent-Membrane Protein 1 of Epstein-Barr Virus in Classical Hodgkin Lymphoma.

The prognostic impact of the presence of Epstein-Barr virus (EBV) in classical Hodgkin lymphoma (cHL) is controversial. Previous studies reported heterogeneous results, rendering difficult the clinical validation of EBV as a prognostic biomarker in this lymphoma. The objective of this study was to evaluate the survival impact of the expression of EBV Latent-Membrane Protein 1 (EBV-LMP1) in tumoral Hodgkin-Reed-Sternberg (HRS) cells of primary diagnostic samples of cHL. Formalin-Fixed Paraffin-Embedded (FFPE) lymph node samples from 88 patients with cHL were analyzed. Patients were treated with the standard first-line chemotherapy (CT) with Adriamycin, Bleomycin, Vinblastine and Dacarbazine (ABVD) followed by radiotherapy. The Kaplan-Meier method and the Cox proportional hazards model were used for carrying out the survival analysis. In order to investigate whether the influence of EBV was age-dependent, analyses were performed both for patients of all ages and for age-stratified subgroups. In bivariate analysis, the expression of EBV was associated with older age (p = 0.011), mixed cellularity subtype cHL (p < 0.001) and high risk International Prognostic Score (IPS) (p = 0.023). Overall survival (OS) and progression-free survival (PFS) were associated with the presence of bulky disease (p = 0.009) and advanced disease at diagnosis (p = 0.016). EBV-positive cases did not present a significantly lower OS and PFS in comparison with EBV-negative cases, for all ages and when stratifying for age. When adjusted for covariates, absence of bulky disease at diagnosis (HR: 0.102, 95% CI: 0.02-0.48, p = 0.004) and limited disease stages (I-II) (HR: 0.074, 95% CI: 0.01-0.47, p = 0.006) were associated with a significant better OS. For PFS, limited-disease stages also retained prognostic impact in the multivariate Cox regression (HR: 0.145, 95% CI: 0.04-0.57, p = 0.006). These results are of importance as the early identification of prognostic biomarkers in cHL is critical for guiding and personalizing therapeutic decisions. The prognostic role of EBV in cHL could be modulated by the type of CT protocol employed and interact with the rest of presenting features.

3D connectomes of reactive and neoplastic CD30 positive lymphoid cells and surrounding cell types.

Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. It is diagnosed on the basis of histological sections by pathologists using a light microscope. The tumor cells, the Hodgkin- and Reed Sternberg cells (HRS), are visualized by morphology and positive response for the CD30-antigen. The same antigen can also be detected by immunohistochemistry on a reactive counterpart, showing CD30+ cells in special immunoreactions, such as inflammations of lymph nodes (lymphadenitis). CD30+ cells in reactive and neoplastic conditions are surrounded by lymphocytes and histiocytes, forming a micromilieu that enables the survival of the tumor cells, as well as their reactive counterparts. This study deals with an investigation of CD30+-surrounding cells using a confocal laser technology, visualizing the contacts of reactive and neoplastic CD30+ cells with CD68+ macrophages and CD163+ macrophages as well as to PD1+ lymphocytes and B cells (CD20+). CD4 immunostains were not included, because CD4+ cells were too numerous for clear dissection of single cells. 3D images visualized the, so-called, connectomes. Clear differences in the number of contacts between CD30-reactive and neoplastic cells (HRS) with macrophages and B lymphocytes were visible. Lymphadenitis and Mixed Cellularity type of classical Hodgkin Lymphoma (cHL) differed in that Mixed Cellularity (MC) cHL had more connections to macrophages (CD163+) and lower number of connections to B cells (CD20+). The connectomes of both Hodgkin variants MCcHL and Nodular Sclerosis cHL (NScHL) mainly differed in the number of contacts to CD163+ macrophages, which was higher in MCcHL. Investigating the volumes of CD30+ -reactive and neoplastic cells, we found out that reactive cells showed lesser volumes, which correlated with the number of contacts. The comparison between 2D and 3D images, including 3D prints, demonstrated clear advantages of the 3D method. 3D images visualized significantly more and clearly defined intercellular contacts. Complicated cellular networks and their contacts became especially evident in volume and surface evaluations, as well as in 3D prints.

Immunohistochemical localization of prolactin receptor (PRLR) to Hodgkin's and Reed-Sternberg cells of Hodgkin's lymphoma.

Prolactin receptor (PRLR), a type-1 cytokine receptor, is overexpressed in a number of cancer types. It has attracted much attention for putative pro-oncogenic roles, which however, remains controversial in some malignancies. In this study, we reported the localization of PRLR to the Hodgkin's and Reed-Sternberg (HRS) cells of Hodgkin's lymphoma (HL), a neoplasm of predominantly B cell origin. Immunohistochemistry performed on 5-µm thick FFPE sections revealed expression of PRLR in HRS cells. Cellular immunofluorescence experiments showed that the HL-derived cell lines, Hs445, KMH2 and L428 overexpressed PRLR. The PRLR immunofluorescent signal was depleted after treating the cell lines with 10 µM of siRNA for 48 h. We also tested whether PRLR is involved in the growth of HL, in vitro. One-way analysis of variance (ANOVA) on cell growth data obtain from WST-1 cell proliferation assay and trypan blue exclusion assay and hemocytometry showed that siRNA-depletion of PRLR expression resulted in decreased growth in all three cell lines. These results offered only a short insight into the involvement of PRLR in HL. As a result, further investigation is required to decipher the precise role(s) of PRLR in the pathogenesis of HL.

CD20-Negative Nodular Lymphocyte-Predominant Hodgkin Lymphoma: A 20-Year Consecutive Case Series From a Tertiary Cancer Center.

CONTEXT.­: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare, indolent Hodgkin lymphoma subtype with distinct clinicopathologic features and treatment paradigms. The neoplastic lymphocyte-predominant cells typically express bright CD20 and other B-cell antigens, which distinguishes them from Hodgkin/Reed-Sternberg cells of lymphocyte-rich classic Hodgkin lymphoma. OBJECTIVE.­: To characterize the clinicopathologic features of CD20-negative NLPHL at a single institution. DESIGN.­: A retrospective search for CD20-negative NLPHL in our pathology archives and medical records was conducted. RESULTS.­: Of 486 NLPHL patients identified with CD20 available for review, 14 (2.8%) had LP cells with absent CD20 expression. Patients with prior rituximab administration (n = 7) and insufficient clinical history (n = 1) were excluded, leaving 6 patients with rituximab-naïve, CD20-negative NLPHL. A broad immunohistochemical panel showed the LP cells in all cases expressed B-cell antigens, particularly Oct-2, although PAX5 and CD79a were frequently also dim. CD30, CD15, and Epstein-Barr virus-encoded small RNAs were negative in all evaluated cases. Two patients had high-risk variant immunoarchitectural pattern D. One patient had extranodal disease, involving the spleen and bone, and was suspected to have large cell transformation. Standard NLPHL therapy was given, including local radiation and/or chemotherapy. Of 5 patients with available follow-up, 4 are alive in complete remission after therapy, and 1 is alive with relapsed disease. CONCLUSIONS.­: NLPHL can lack CD20 de novo without prior rituximab therapy. In such cases, extensive immunophenotyping helps distinguish NLPHL from lymphocyte-rich classic Hodgkin lymphoma, which differ in clinical behavior and therapy. In our series, CD20-negative NLPHL showed both classic and variant histologic patterns and the expected range of clinical behavior seen in NLPHL, including 1 case with suspected large cell transformation.