Hodgkin And Reed-Sternberg Research Articles

Mutation analysis of tumor necrosis factor alpha-induced protein 3 gene in Hodgkin lymphoma

AimsSurvival and proliferation of Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (CHL), are dependent on constitutive activation of nuclear factor kB (NF-κB). A20, encoded by TNF alpha-induced protein 3 (TNFAIP3), one of the inhibitors of NF-kB, was found to be inactivated by deletions and/or point mutations in CHL. MethodsTNFAIP3 mutations were examined in 37 patients with CHL by using PCR and direct sequencing. In addition, protein expression of A20 was evaluated by immunohistochemistry. Epstein–Barr virus (EBV) status of HL samples was determined by EBV EBER chromogenic in situ hybridization (ISH). ResultsWe identified 8 mutation positive cases in a collective of 37 investigated cases (22%). Mutations were most frequent in the nodular sclerosis subtype. Our results revealed the tendency that cases harboring A20 mutations were negative for A20 staining. None of A20 mutation-positive CHL cases showed EBV infection. ConclusionsOur study confirms the involvement of the TNFAIP3 tumor suppressor gene in CHL. A20 may represent a suppressor of human lymphoma and provide a critical molecular link between chronic inflammation and cancer. None of A20 mutation-positive CHL cases showed EBV infection. This fact suggests complementing functions of TNFAIP3 inactivation and EBV infection in CHL pathogenesis and may represent an interesting point of further investigations.

Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma

Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed–Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis.

Role of mast cells in fibrosis of classical Hodgkin lymphoma

The underlying mechanism of fibrosis in classical Hodgkin lymphoma (CHL) remains uncertain. This study aimed to investigate the association of fibrosis in the lymph nodes of patients with CHL through histological examination of the expression of cytokines associated with fibrosis and mast cell proliferation. Additionally, we sought to determine the degree of mast cell infiltration in a nodular sclerosis subtype of CHL (NSCHL) compared with that in non-NSCHL. We analyzed lymph nodes from 22 patients with CHL, of which eight were of the NSCHL and 14 of the non-NSCHL subtype, using immunohistochemical staining of forkhead box P3 (FOXP3), transforming growth factor (TGF)-β, interleukin (IL)-3, IL-13, and stem cell factor (SCF). Mast cells were positive for TGF-β and IL-13, and FOXP3-positive cells were negative for TGF-β. Only the expression of IL-13 in Hodgkin and Reed-Sternberg (HRS) cells was significantly more frequently observed in NSCHL than that in non-NSCHL (P = 0.0028) and was associated with a higher rate of fibrosis (P = 0.0097). The number of mast cells was significantly higher in NSCHL than that in non-NSCHL (P = 0.0001). A significantly positive correlation was observed between the rate of fibrosis and the number of mast cells (correlation coefficient, 0.8524; 95% CI, 0.6725-0.9372) (P <0.0001). The number of mast cells was significantly higher in the group with IL-13-positive HRS cells than that in the group with IL-13-negative HRS cells (P = 0.0157). Based on these findings, we hypothesize that IL-13 production by HRS cells may lead to fibrosis, and furthermore, promote mast cell proliferation and infiltration. This in turn might further produce the fibrotic cytokines IL-13 and TGF-β, resulting in fibrosis typical of NSCHL.

NF-κB deregulation in Hodgkin lymphoma.

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (HL) show constitutive activity of both the canonical and non-canonical NF-κB signaling pathways. The central pathogenetic role of this activity is indicated from studies with HL cell lines, which undergo apoptosis upon NF-κB inhibition. Multiple factors contribute to the strong NF-κB activity of HRS cells. This includes interaction with other cells in the lymphoma microenvironment through CD30, CD40, BCMA and other receptors, but also recurrent somatic genetic lesions in various factors of the NF-κB pathway, including destructive mutations in negative regulators of NF-κB signaling (e.g. TNFAIP3, NFKBIA), and copy number gains of genes encoding positive regulators (e.g. REL, MAP3K14). In Epstein-Barr virus-positive cases of classical HL, the virus-encoded latent membrane protein 1 causes NF-κB activation by mimicking an active CD40 receptor. NF-κB activity is also seen in the tumor cells of the rare nodular lymphocyte predominant form of HL, but the causes for this activity are largely unclear.

Repression of TCF3/E2A contributes to Hodgkin lymphomagenesis.

Although Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) derived from germinal or post germinal B cells, they have lost the B cell phenotype in the process of lymphomagenesis. The phenomenon can be at least partially explained by repression of B-cell-specific transcription factors including TCF3, early B-cell factor 1 (EBF1), SPI1/PU.1, and FOXO1, which are down-regulated by genetic and epigenetic mechanisms. The unique phenotype has been suspected to be advantageous for survival of HRS cells. Ectopic expression of some of these transcription factors (EBF1, PU.1, FOXO1) indeed impaired survival of cHL cells. Here we show that forced expression of TCF3 causes cell death and cell cycle arrest in cHL cell lines. Mechanistically, TCF3 overexpression modulated expression of multiple pro-apoptotic genes including BIK, APAF1, FASLG, BOK, and TNFRSF10A/DR4. We conclude that TCF3 inactivation contributes not only to extinguishing of B cell phenotype but also to cHL oncogenesis.

HLA-G expression and role in advanced-stage classical Hodgkin lymphoma.

Non-classical human leucocyte antigen (HLA)-G class I molecules have an important role in tumor immune escape mechanisms. We investigated HLA-G expression in lymphonode biopsies taken from 8 controls and 20 patients with advanced-stage classical Hodgkin lymphoma (cHL), in relationship to clinical outcomes and the HLA-G 14-basepair (14-bp) deletion-insertion (del-ins) polymorphism. Lymphnode tissue sections were stained using a specific murine monoclonal HLA-G antibody. HLA-G protein expression was higher in cHL patients than controls. In the group of PET-2 positive (positron emission tomography carried out after 2 cycles of standard chemotherapy) patients with a 2-year progression-free survival rate (PFS) of 40%, we observed high HLA-G protein expression within the tumor microenvironment with low expression on Hodgkin and Reed-Sternberg (HRS) cells. Conversely, PET-2 negative patients with a PFS of 86% had higher HLA-G protein expression levels on HRS cells compared to the microenvironment. Lower expression on HRS cells was significantly associated with the HLA-G 14-bp ins/ins genotype.These preliminary data suggest that the immunohistochemical pattern of HLA-G protein expression may represent a useful tool for a tailored therapy in patients with cHL, based on the modulation of HLA-G expression in relation to achievement of negative PET-2.

Tumor-infiltrating HLA-matched CD4+ T cells retargeted against Hodgkin and Reed–Sternberg cells

ABSTRACTHodgkin lymphoma (HL) presents with a unique histologic pattern. Pathognomonic Hodgkin and Reed–Sternberg (HRS) cells usually account for less than 1% of the tumor and are embedded in a reactive infiltrate mainly comprised of CD4+ T cells. HRS cells induce an immunosuppressive microenvironment and thereby escape antitumor immunity. To investigate the impact of interactions between HRS cells and T cells, we performed long-term co-culture studies that were further translated into a xenograft model. Surprisingly, we revealed a strong antitumor potential of allogeneic CD4+ T cells against HL cell lines. HRS and CD4+ T cells interact by adhesion complexes similar to immunological synapses. Tumor-cell killing was likely based on the recognition of allogeneic major histocompatibility complex class II (MHC-II) receptor, while CD4+ T cells from MHC-II compatible donors did not develop any antitumor potential in case of HL cell line L428. However, gene expression profiling (GEP) of co-cultured HRS cells as well as tumor infiltration of matched CD4+ T cells indicated cellular interactions. Moreover, matched CD4+ T cells could be activated to kill CD30+ HRS cells when redirected with a CD30-specific chimeric antigen receptor. Our work gives novel insights into the crosstalk between HRS and CD4+ T cells, suggesting the latter as potent effector cells in the adoptive cell therapy of HL.

Recurrent somatic loss of TNFRSF14 in classical Hodgkin lymphoma.

Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed-Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole-genome sequencing-mediated copy number analysis of purified HRS cells. We performed low-coverage sequencing of small numbers of intact HRS cells and paired non-neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS-derived cell lines (KM-H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD-1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236-4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases.

Clinical Impact of CD8+ Cells in Hodgkin Lymphoma Lymphadenopaties. Contribution of Flow Cytometry

CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma.OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL.DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software.SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells).PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years.INTERVENTIONS :This was a retrospective observational study.MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse.RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years > 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and>12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9).CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The molecular pathology of classical Hodgkin lymphoma

In 1832, Dr. Thomas Hodgkin reported the first cases with this malignancy, which came to be named Hodgkin's disease. The cells that are a hallmark of this disease, Hodgkin and Reed-Sternberg (HRS) cells, account for only 1% of those in tumor tissues, with the majority of cells in Hodgkin lymphoma being of various inflammatory types. Advances in molecular techniques have contributed to molecular biological analysis of HRS cells. Intriguingly, HRS cells are derived from germinal center B-cells, but have lost their B-cell gene-expression and co-express non-B-cell genes. Multiple signaling pathways, including the NFκB and JAK/STAT pathways, show deregulated activity in HRS cells, suggesting an important role for these pathways in the pathogenesis of Hodgkin lymphoma. This article describes the molecular pathological characteristics of HRS cells: 1) the cellular origin of HRS cells, 2) deregulated gene expression in HRS cells, 3) genetic alterations and 4) epigenetic alterations in HRS cells, 5) the lost B-cell phenotype of HRS cells, 6) the role of EBV in Hodgkin lymphoma pathogenesis, and 7) micro-environmental interactions between HRS and reactive cells.

Expression of Interleukin-1 and Interleukin-1 Receptors Type 1 and Type 2 in Hodgkin Lymphoma.

Signaling through the IL-1-receptor type 1 (IL-1R1), IL-1 is required for initiation and maintenance of diverse activities of the immune system. A second receptor, IL-1R2, blocks IL-1 signal transduction. We studied expression of IL-1beta, IL-1R1, and IL-1R2 in 17 Hodgkin lymphomas (HL) by in situ hybridization (ISH). IL-1beta expressing cells, morphologically consistent with endothelial cells and fibroblasts, occurred in all HL tissues with elevated transcript levels in areas of active fibrosis. Hodgkin and Reed-Sternberg (HRS) cells of all cases expressed low IL-1R1 transcript levels in some tumor cells, and high levels of IL-1R2 in large proportions of HRS cells. Only few bystander cells showed low levels of IL-1R1 and IL-1R2 RNA. Supernatants of 4 out of 7 HL-derived cell lines contained soluble IL-1R2 protein at high levels. HL patient sera carried variably amounts of IL-1R2 protein with significantly increased titers in patients with active disease compared to patients in complete remission and control individuals without HL. Western blots and co-immunoprecipitations showed binding of the IL-1R2 to the intracellular IL-1R-accessory protein (IL-1IRAcP). These data suggest functions of the IL-1R2 as a „decoy-receptor” sequestrating paracrine IL-1 extracellularly and intracellularly by engaging IL-1IRAcP, thus depriving IL1-R1 molecules of their extracellular and intracellular ligands. Expression of IL1-R2 by HRS cells seems to contribute to local and systemic modulation of immune function in HL.

Abstract 4062: Role of CD137 signaling in immune evasion of Hodgkin and Reed-Sternberg cells

Abstract Hodgkin lymphoma (HL), a lymphoid malignancy, is a frequently occurring cancer among adolescents and young adults. HL is marked by the presence of malignant Hodgkin and Reed-Sternberg (HRS) cells, which constitute only a small fraction of the tumor mass. HRS cells are present in a complex stroma of inflammatory leukocytes, but are often able to evade the host immune system. These cells express various receptors to recruit leukocytes and hijack them for supporting the tumor growth. Our group has reported the overexpression of CD137, a member of the tumor necrosis factor receptor (TNFR) superfamily, on the HRS cells of HL tumor samples. CD137 is a co-stimulatory molecule expressed by T cells, only upon activation. We speculated that the expression of CD137 on the HRS cells may be associated with its malignancy, as HRS cells are clonally derived from B cells which are not expressing CD137. Our study aims to analyse the functional role of CD137 expression behind the survival, progression, and cytokine release of the malignant HRS cells. We found that the induction of CD137 signaling in HRS cell lines enhanced the secretion of cytokines such as IL-6, TNF-α and IL-13. In order to investigate the effect of these cytokines on the immune microenvironment, we cultured peripheral blood mononuclear cells (PBMCs) in the supernatants derived from CD137-induced HRS cells. PBMCs, when cultured in the above mentioned conditioned supernatants, secreted lesser levels of IFN-γ (a Th1 cytokine essential for T cell and NK cell cytotoxicity), implying a suppressed state of immune cells. Further, neutralisation of IL-13 (a Th2 cytokine) in the conditioned supernatants reversed the reduction of IFN-γ observed in PBMCs. This shows that IL-13 secreted by the HRS cells upon CD137 signaling is responsible for the inhibition of IFN-γ secretion by PBMCs. To summarize, CD137 signaling in HRS cells leads to the secretion of cytokines, which drive the polarization of helper T cells towards Th2 type, thus reducing Th1 immune responses against cancers. Taken together, our findings reveal the role of CD137 signaling as a novel mechanism of immune escape in HRS cells. Hence, targeting CD137 can be a potential therapeutic option for HL patients. Citation Format: Sakthi Rajendran, Herbert Schwarz. Role of CD137 signaling in immune evasion of Hodgkin and Reed-Sternberg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2015-4062

Abstract 4750: Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma

Abstract Introduction: Most genomic studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines due to the difficulty of isolating sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. One approach to evaluate primary cases has been to microdissect HRS cells from fresh-frozen tissue biopsies, which has been used for gene expression profiling and array comparative genomic hybridization to assess copy number alterations. However, laser capture microdissection is technically challenging, does not provide a pure tumor cell population, and yields very small amounts of degraded nucleic acids that may not be adequate for whole-transcriptome level RNA sequencing. Methods: We used a flow cytometric cell isolation method, which has enabled rapid isolation of thousands of viable HRS cells from primary cHL tumors (Fromm, et al., Am J Clin Pathol 126, 2006). Here we combined flow cytometry using CD64, CD95, CD30, CD5, CD20, CD15, CD40, and CD45, with the Arcturus PicoPure RNA Isolation kit to isolate high quality RNA from viable HRS cells and intratumor B cells from nine primary cases of CHL respectively. We applied the Clontech SMARTer Ultra Low Input RNA Kit followed by Kapa Library Preparation kit to generate what is to our knowledge the first full transcriptome RNA sequence data from primary cases of cHL. Four cell lines were sequenced using the same methodology. We performed differential expression and gene fusion analyses, and conducted a search for activated pathways. Results: We report the expression signature of purified primary HRS cells. PCA analysis showed that these segregate from HRS cell lines and primary B cells. We confirmed at the RNA level overexpression of relevant amplified genes for which inhibitors are available (AURKA and EZH2), and underexpression of transcripts encoded by genes previously found to be deleted (for example ATM). Pathway analysis revealed extensive downregulation of antigen presentation, consistent with our previous exome sequence analysis showing mutations in beta-2 microglobulin as the most common genetic alteration. Results of the mutational and transcriptional landscape of the HRS cells will be presented. Conclusions: Transcriptome-level analysis of flow-sorted HRS cells allows for expanded study of CHL pathogenesis, new target identification based on altered signaling pathways and potentially individualized approaches to CHL therapy. Citation Format: Jonathan Reichel, Joshua Brody, Olivier Elemento, Raul Rabadan, Ethel Cesarman. Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2015-4750

Abstract 1263: Cellular senescence in Hodgkin's lymphoma

Abstract Hodgkin's lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most frequent lymphomas in the Western world. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. The origin of the pro-inflammatory signals remains elusive, and represents a critical gap in our knowledge of the pathogenesis of HL. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease. Citation Format: Jacob Gopas, Udi Zurgil, Estee Zeev, Daniel Benharoch, Jodi Ezratty, Giora Shubinsky, Etta Livneh. Cellular senescence in Hodgkin's lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2015-1263

Alterations of the CD58 gene in classical Hodgkin lymphoma.

Immune evasion plays a central role in the pathophysiology of classical Hodgkin lymphoma (cHL). As mutations of the CD58 gene contribute to immune evasion of diffuse large B cell lymphoma tumor cells, we studied whether alterations of the CD58 gene also occur in Hodgkin and Reed/Sternberg (HRS) cells of cHL. Single nucleotide polymorphism chip analysis revealed homozygous deletions within the CD58 gene in two cHL cell lines (SUP-HD1 and U-HO1). Sequencing of the CD58 gene in seven cHL cell lines disclosed in addition a homozygous splice site mutation in cell line KM-H2. None of the three mutated lines expressed CD58 protein on their surface. Thus, three of seven cHL cell lines analyzed harbor destructive CD58 mutations. Molecular analysis of isolated HRS cells from 10 primary cases of cHL; however, did not reveal any case with a CD58 mutation. A FICTION study indicated heterozygous deletions of CD58 in 3 of 13 cHL analyzed. Overall, we report frequent inactivating mutations of CD58 in cHL cell lines, but their rare occurrence in primary HRS cells. As the three cHL cell lines with CD58 mutations were all established from HRS cells located in pleural effusions, i.e., outside the normal lymph node microenvironment, in end-stages of the disease, CD58 inactivation in cHL might be predominantly prevalent to such situations.

Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells

AbstractClassical Hodgkin lymphoma (cHL) is characterized by sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells amid reactive host background, complicating the acquisition of neoplastic DNA without extensive background contamination. We overcame this limitation by using flow-sorted HRS and intratumor T cells and optimized low-input exome sequencing of 10 patient samples to reveal alterations in genes involved in antigen presentation, chromosome integrity, transcriptional regulation, and ubiquitination. β-2-microglobulin (B2M) is the most commonly altered gene in HRS cells, with 7 of 10 cases having inactivating mutations that lead to loss of major histocompatibility complex class I (MHC-I) expression. Enforced wild-type B2M expression in a cHL cell line restored MHC-I expression. In an extended cohort of 145 patients, the absence of B2M protein in the HRS cells was associated with lower stage of disease, younger age at diagnosis, and better overall and progression-free survival. B2M-deficient cases encompassed most of the nodular sclerosis subtype cases and only a minority of mixed cellularity cases, suggesting that B2M deficiency determines the tumor microenvironment and may define a major subset of cHL that has more uniform clinical and morphologic features. In addition, we report previously unknown genetic alterations that may render selected patients sensitive to specific targeted therapies.

Targeting Functionality of Cancer Cell-Derived Extra cellular vesicles to Immune Cells of the Tumor Microenvironment

In most cases, cancer cells cannot proliferate alone. They receive support form stromal cells and recruit and reprogram non-malignant immune cells not to damage the cancer cells but to support the tumor growth. Classical Hodgkin lymphoma (cHL) is a good example, as its growth critically depends on tumor-supporting immune cells. The affected lymphoid tissue contains very few disseminated malignant Hodgkin- and Reed-Sternberg (H-RS) cells, which are outnumbered by a massive infiltrate of lymphocytes, fibroblasts and innate immune cells. [1]. The H-RS cells need this environment to survive and proliferate because they are usually not detectable in the peripheral blood and they have difficulties to grow in immune-deficient mice [2]. H-RS cells manipulate the bystander cells for better development of their malignant phenotype and evasion of the host defense [3]. Among the infiltrated immune cells, the eosinophils and mast cells play an important role and their cell count indicates a negative prognostic feature [4,5]. In vitro, the direct interaction with cancer cells communicates survival in cancer cells and the production of cancersupporting factors in immune cells. In the last years, extracellular vesicles (EVs) emerged as important vehicles to shuttle between different the cell types and communicate as a surrogate cellular crosstalk in trans [6,7].

Insulin-like growth factor I is expressed in classical and nodular lymphocyte-predominant Hodgkin's lymphoma tumour and microenvironmental cells.

Hodgkin's lymphoma (HL) is among the most frequent nodal lymphomas in the Western world and is classified into two disease entities: nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) and classical Hodgkin's lymphoma (cHL, 95% of all HL). HL lesions are characterised by a minority of clonal neoplastic cells, namely Hodgkin and Reed-Sternberg (HRS) cells and their variants in cHL and lymphocyte-predominant (LP) cells in NLPHL, both occurring within a microenvironment of, for example, reactive T and B cells, macrophages and granulocytes that are assumed to support the proliferation and maintenance of neoplastic cells through cytokines, chemokines and growth factors. Insulin-like growth factor I (IGF-I) is an important growth factor involved in proliferation, differentiation, apoptosis and cell survival of numerous (including immune) tissues and probably has a role in tumour pathogenesis and maintenance. Although HL is characterised by disturbed cell differentiation and apoptosis mechanisms, with the involvement of the IGF-I receptor (IGF-1R), the distinct location of IGF-I in HL has not yet been defined. We localise IGF-I by double-immunofluorescence in frequent neoplastic cells of all cHL and NLPHL cases investigated. Additionally, IGF-I immunoreactivity is detected in high endothelial venules and various immune cells within the surrounding tissue of cHL including neutrophils and macrophages. IGF-1R immunoreactivity of variable intensity is found in HRS cells and high endothelial venules within the microenvironment in cHL. We assume that autocrine and paracrine IGF-I plays an anti-apoptotic role in tumour pathogenesis and in shaping the tumour microenvironment.

Reed-Sternberg cell–derived lymphotoxin-α activates endothelial cells to enhance T-cell recruitment in classical Hodgkin lymphoma

AbstractIt is known that cells within the inflammatory background in classical Hodgkin lymphoma (cHL) provide signals essential for the continual survival of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells. However, the mechanisms underlying the recruitment of this inflammatory infiltrate into the involved lymph nodes are less well understood. In this study, we show in vitro that HRS cells secrete lymphotoxin-α (LTα) which acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T cell recruitment. LTα also enhances the expression of hyaluronan which preferentially contributes to the recruitment of CD4+ CD45RA+ naïve T cells under in vitro defined flow conditions. Enhanced expression of LTα in HRS cells and tissue stroma; and hyaluronan on endothelial cells are readily detected in involved lymph nodes from cHL patients. Our study also shows that although NF-κB and AP-1 are involved, the cyclooxygenase (COX) pathway is the dominant regulator of LTα production in HRS cells. Using pharmacological inhibitors, our data suggest that activity of COX1, but not of COX2, directly regulates the expression of nuclear c-Fos in HRS cells. Our findings suggest that HRS cell-derived LTα is an important mediator that contributes to T cell recruitment into lesional lymph nodes in cHL.

The Role of Microenvironment Expression of Pd-1 Ligands and Chemokines in Clinical Outcome of Hodgkin'S Lymphoma

ABSTRACT Aim: Classical Hodgkin's lymphoma (cHL) is characterized by only few malignant cells and an abundance of inflammatory cells. cHL infiltrating cells produce cytokines and growth factors that provide essential stimulatory signals for survival and proliferation of Hodgkin and Reed–Sternberg (HRS) cells. Moreover, clinical behaviour of cHL may be directly regulated by the cross-talk between HRS cells and other cells in their microenvironment. The aim of our study was to estimate the role of microenvironment expression of PD-1 ligands and chemokines in clinical outcome of cHL. Methods: The case group comprised 49 patients with cHL (stage IIA: 23, IIB: 3, III-IV: 23). The patients received chemotherapy regimens (ABVD or BEACOPP 14/esc) and radiotherapy by indications. cHL specimens were obtained from lymph node biopsies of patients at diagnosis. PD-L1, PD-L2, MIP-1a, RANTES mRNA expression levels were analyzed in fresh tissue specimens using real-time RT-PCR. Results: We observed that 26.5 % (13/49) of cHL cases were PD-L1 negative, 16.3 % (8/49) - RANTES negative and 14.3 % (7/49) – both PD-L1 and RANTES negative. MIP-1a and RANTES expression levels were slightly higher in mixed cellularity cHL (p = 0.15), whereas PD-L1 – in nodular sclerosis cHL and advanced stages of disease (p = 0.19). PD-L2 expression level was independent of histological cHL variant or disease stage. All cases with the absence of both PD-L1 and RANTES markers had a complete response to the therapy and long-term remission. For the patients with increasing of RANTES/MIP-1a rate, a trend for a higher risk of relapse was observed (P = 0.09). ROC analysis revealed that PD-L1 expression level in tumor is an important marker which is associated with clinical outcome of cHL patients (Se = 87.5 %; Sp = 64.5 %; AUC = 0.75; p = 0.002). High PD-L1 expression was associated with reduced progression-free survival (PFS) in cHL patients. The 2-year PFS rate for cHL patients with high PD-L1 expression was 47 % compared to 95 % for low or absent of PD-L1 expression (p = 0.04). Conclusions: Our results suggest that tumor microenvironment play an important role in clinical behavior of cHL. PD-L1 expression level can be used as a marker of prognosis in patients with cHL and represent an attractive target for a cHL immunotherapy in patients with poor outcome. Disclosure: All authors have declared no conflicts of interest.