Hodgkin Lymphoma Tumor Cells Research Articles

Polyamine catabolism is concentrated in tumor-associated histiocytes in diffuse large B-cell lymphoma and classic Hodgkin lymphoma

Polyamines are cationic molecules necessary for cell survival, growth, and replication [1–5]. Polyamines come in a variety of structural forms and are principally regulated by two enzymes, spermine/spermidine acetyltransferase-1 (SAT1) and ornithine decarboxylase-1 (ODC1). SAT1 targets the polyamines spermidine and spermine for degradation via acetylation, while ODC1 is involved in converting the polyamine precursor molecule to more complex polyamines [6–8]. Polyamines and their regulatory enzymes have been implicated in tumor metastasis [9,10] and in crosstalk between oncogenes [11–13] in numerous types of cancer, but their role has never been evaluated in B-cell malignancies. In this study, we examine the expression of SAT1 in diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma (HL). We found that SAT1 is expressed in all examined cases of DLBCL (n = 15) and HL (n = 5), though the levels of expression across cases vary. We also note that SAT1 expression appears to be concentrated in tumor-associated histiocytes, rather than tumor cells in both DLBCL and HL. We propose that these findings indicate that the polyamine catabolic enzyme, SAT1, plays an unappreciated role in the pathogenesis of B-cell neoplasms.

Aberrant synaptophysin expression in classic Hodgkin lymphoma

BackgroundSynaptophysin is an immunohistochemical marker for neuroendocrine differentiation and is widely used in pathologic diagnosis. Its expression in malignant lymphoma has not yet been described. However, we experienced an index case of classic Hodgkin lymphoma with synaptophysin expression. This experience prompted us to investigate synaptophysin expression in classic Hodgkin lymphoma.MethodImmunohistochemical staining of synaptophysin was performed in 59 diagnosed cases of classic Hodgkin lymphoma, 10 anaplastic large cell lymphomas, 16 diffuse large B-cell lymphomas, and 5 extranodal marginal zone lymphoma of the mucosa-associated tissue. Synaptophysin-positive cases were stained for both chromogranin and CD56a.ResultOf 59 classic Hodgkin lymphoma cases, 11 (19%) were positive for synaptophysin. None of the anaplastic large cell lymphomas expressed synaptophysin. Synaptophysin showed weak but specific expression in the cytoplasm of the Hodgkin lymphoma tumor cells. Other background inflammatory cells (such as macrophages, B-, and T-lymphocytes) were all negative for synaptophysin expression. Chromogranin and CD56a were not expressed in the synaptophysin-positive classic Hodgkin lymphomas.ConclusionsSynaptophysin is an integral glycoprotein present in presynaptic vesicles of neurons and neuroendocrine cells. It is a diagnostic marker for neuroendocrine tumors. Aberrant synaptophysin expression has been reported in non-neuroendocrine tumors but not in lymphoma or leukemia. To the best of our knowledge, synaptophysin positivity has only been reported in a single case of precursor T-lymphoblastic leukemia/lymphoma to date. Our study showed that aberrant synaptophysin expression in classic Hodgkin lymphoma is an unexpectedly frequent finding. The mechanism underlying, and prognostic significance of, such aberrant expression is unclear. Thus, in a small biopsy, aberrant synaptophysin expression could be a diagnostic pitfall and should be carefully avoided.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.

RNA Sequencing Reveals Mechanisms Underlying Modulation of Hodgkin Lymphoma Cells and Tumor Microenvironment By the PI3K δ/γ Inhibitor, RP6530

INTRODUCTION: Aberrant activation of the phosphoinositide 3-kinase (PI3K) pathway plays a central role in Hodgkin Lymphoma (HL) pathogenesis. The pleiotropic activities of the δ and γ isoforms of PI3K in regulating the interactions of Hodgkin/Reed-Sternberg (HRS) cells with the tumor microenvironment suggest thatPI3K inhibitors might exert a therapeutic potential in the treatment of HL. The dual PI3K δ/γ inhibitor RP6530 was proven to affect HL tumor cells and to exert immunosuppressive activities. However, a genome-wide search for molecular mediators of RP6530 is largely unexplored in HL. The present study was therefore aimed at evaluating the anti-lymphoma functions and the underlying genes affected by RP6530 in HL cell lines.METHODS: We identified differentially expressed genes (DEGs) in two HL cell lines (L-540 and KM-H2) treated with RP6530 for 6 and 24 h by next generation RNA sequencing (RNA-Seq). The expression level of some of these genes was verified in HL cell lines using real time-PCR. Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to distinct canonical pathways and to predict the biological functions of the transcriptional changes. Gene silencing experiments were used to investigate and validate potential predictive biomarkers of response to RP6530 treatment. Additionally, serum levels of Thymus- and activation-regulated chemokine (TARC/CCL17) were analyzed by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in RP6530-treated HL patients enrolled in a phase 1 study.RESULTS: RNA-Seq analysis following treatment of L-540 cells for 6 h caused an upregulation of 1544 and downregulation of 839 genes. After a 24 h treatment period, 625 genes remained upregulated while the number of genes that were downregulated increased to 1362 (FDR ≤ 0.1). In KM-H2 cells, treatment with RP6530 for 6 or 24 h upregulated 137 and 980 genes while downregulating 139 and 833 genes, respectively (FDR ≤ 0.1). We further classified modulated genes according to their canonical pathways using the IPA suite. The most significantly upregulated pathways detected in both L-540 and KM-H2 cell lines included apoptosis, p53, death receptor signaling and G1/S cell cycle checkpoint regulation. In contrast, PI3K/AKT, ERK/MAPK, JAK/Stat, CD40, IL-4, IL-6, IL-10 and iNOS signaling were downregulated. We also identified 118 downregulated genes shared between the two cell lines that were associated with glycolysis, HIF1, chemokine and cytokine signaling. Upon RP6530 incubation, RNA-seq analysis showed a strikingly reduced expression of Pyruvate kinase muscle isozyme M2 (PKM2), which is a limiting glycolytic enzyme that catalyzes the final step in glycolysis. Here we show that lactic acid produced by tumor cells, as a by-product of glycolysis, has a critical function in inducing the M2-like polarization of tumor-associated macrophages (TAMs). Furthermore, we demonstrated that this effect was mediated by PKM2 and was inhibited after RP6530 treatment.We also found that the lactate-induced expression of M2 markers by macrophages, i.e., CCL-17 and CCL-22, were markedly inhibited both by RP6530 [-30% and -50% (mean of HL cells), respectively] and PKM2 silencing [-50% and -30% (mean of HL cells), respectively]. However, RP6530 increased the expression of M1 markers, switching the activation of macrophages from an immunosuppressive M2-like phenotype to an inflammatory M1-like state. PKM2 pathway therefore appears to be a critical determinant of RP6530-mediated inhibition of M2 macrophage polarization. Finally, RP6530 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines and angiogenic factors (including, TARC/CCL17, CCL22, CCL5, IL-7, IL-13, and VEGF) known to be secreted by HRS cells to promote a pro-tumoral microenvironment. As the chemokine TARC/CCL17 is considered a biomarker of disease activity in HL patients, we observed a severe reduction of serum TARC/CCL17 levels in RP6530-treated HL patients that achieved CR or PR, but not in PD patients. Collectively, these findings suggest that RP6530 affects a mechanism of communication between HL tumor cells and tumor microenvironment.CONCLUSIONS: Results demonstrate that RP6530 can affect both HL tumor cells and HL tumor microenvironment, suggesting that a novel therapeutic opportunity may be achievable for the treatment of HL patients. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; Rhizen Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding.

Bioinformatics analysis of whole slide images reveals significant neighborhood preferences of tumor cells in Hodgkin lymphoma.

In pathology, tissue images are evaluated using a light microscope, relying on the expertise and experience of pathologists. There is a great need for computational methods to quantify and standardize histological observations. Computational quantification methods become more and more essential to evaluate tissue images. In particular, the distribution of tumor cells and their microenvironment are of special interest. Here, we systematically investigated tumor cell properties and their spatial neighborhood relations by a new application of statistical analysis to whole slide images of Hodgkin lymphoma, a tumor arising in lymph nodes, and inflammation of lymph nodes called lymphadenitis. We considered properties of more than 400, 000 immunohistochemically stained, CD30-positive cells in 35 whole slide images of tissue sections from subtypes of the classical Hodgkin lymphoma, nodular sclerosis and mixed cellularity, as well as from lymphadenitis. We found that cells of specific morphology exhibited significantly favored and unfavored spatial neighborhood relations of cells in dependence of their morphology. This information is important to evaluate differences between Hodgkin lymph nodes infiltrated by tumor cells (Hodgkin lymphoma) and inflamed lymph nodes, concerning the neighborhood relations of cells and the sizes of cells. The quantification of neighborhood relations revealed new insights of relations of CD30-positive cells in different diagnosis cases. The approach is general and can easily be applied to whole slide image analysis of other tumor types.

Genomic Analysis of Hodgkin Lymphoma

The genetic analysis of the Hodgkin and Reed/Sternberg (HRS) tumor cells in Hodgkin lymphoma (HL) is very much hampered by the rarity of these cells, typically accounting for only about 1% of cells in the lymphoma tissue. Moreover, only very few cell lines were established from HL, partly with an uncertain origin. Thus, for the identification of somatic mutations in HRS cells, methods had to be established to isolate the rare HRS cells by microdissection from tissue sections or by cell sorting from cell suspensions. Initial studies were focussed on candidate gene approaches, often inspired by results from the prior analysis of HL cell lines. These early studies revealed numerous recurrent mutations in members of the NF-κB and JAK/STAT pathways, causing or at least contributing to the constitutive activation of these signaling pathways.1 The pathobiological relevance of the constitutive activation of NF-κB and JAK/STAT factors was validated by functional studies with HL cell lines. A first whole exome sequencing analysis of flow-sorted HRS cells was published by Reichel and colleagues.2 B2M was found to be inactivated by somatic mutations in 7/10 cases, and caused loss of major histocompatibility complex 1 expression. Although later studies with larger case series identified such mutations in only about 20-30% of cases,3,4 B2M inactivation is nevertheless an important factor for immune evasion of HRS cells and a frequently mutated gene in HL. A second exome sequencing study of HRS cells used microdissected tumor cells and revealed as a major novel finding frequent somatic mutations in the STAT6 gene (ca. 30% of cases).3 The genetic lesions in STAT6 are gain-of-function mutations. Overall nearly 90% of HL cases show genetic alterations in members of the JAK/STAT pathway, further demonstrating the major role of this pathway in HL pathogenesis. Our group is currently performing studies of microdissected HRS cells by whole genome sequencing, aiming to identify further genetic lesions in HRS cells beyond those affecting exons. An alternative attractive approach to uncover mutations in HRS cells is the genetic analysis of circulating DNA in plasma, because HRS cell-derived DNA fragments can be identified in most patients at the time of diagnosis. A targeted sequencing study of circulating tumor DNA showed the feasibility of this approach and validated, for example, the frequent occurrence of STAT6 mutations in HL. In rare instances, a HL can co-occur with another lymphoma in the same patient. Such composite lymphomas frequently show a common clonal origin of both lymphomas from a mature B cell.5 Composite lymphomas are therefore elegant models to study the multi-step transformation process in lymphomagenesis. We are currently performing a whole exome sequencing analysis of composite classical HL and B-cell Non-Hodgkin lymphomas, to reveal the pattern of early shared mutations and later distinct mutations. In each of the cases under investigations, such shared and distinct mutations were indeed identified. Küppers R, Engert A, Hansmann ML. Hodgkin lymphoma. J Clin Invest. 2012; 122: 3439. Reichel J, Chadburn A, Rubinstein PG, et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood. 2015; 125: 1061. Tiacci E, Ladewig E, Schiavoni G, et al. Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma. Blood. 2018; 131: 2454. Spina V, Bruscaggin A, Cuccaro A, et al. Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkinlymphoma. Blood. 2018; 131: 2413. Küppers R, Dührsen U, Hansmann ML. Pathogenesis, diagnosis, and treatment of composite lymphomas. Lancet Oncol. 2014; 15: e435. Disclosures No relevant conflicts of interest to declare.

Novel Immunotherapeutic Approaches for Hodgkin Lymphoma

The outcome of patients with relapsed/refractory Hodgkin Lymphoma (HL) for whom salvage therapy or stem cell transplant have failed is poor. HL tumor cells (HRS) cells universally express CD30, which is an excellent target for directed therapy such as the antibody drug conjugate brentuximab vedotin (BV). Patients with relapsed HL can also respond to checkpoint inhibitors, emphasizing the susceptibility of this tumor to T-cell-mediated immune control. The adoptive transfer of chimeric antigen receptor T (CAR-T) cells, which combine antibody-mediated antigen specificity with the effector function and replication potential of T lymphocytes, offers the opportunity to infuse large numbers of T cells with defined antigen-specificity and MHC-independent tumor targeting. In two parallel and simultaneously conducted phase 1/2 dose-escalation studies, we administered CD30-specific CAR-T (CD30.CAR-T) cells after lymphodepletion to patients with HL that had relapsed after multiple lines of therapy, including BV, checkpoint inhibitors and stem cell transplant. We observed an excellent safety profile, with modest and self-limited cytokine release syndrome and no neurologic toxicity. The overall response rate was 78%, including 18 patients (67%) with complete responses, and the 1 year progression free survival was 47% (95% CI: 25% - 67%). Despite these promising results, unsolved obstacles include CAR-T cell trafficking and persistence in the tumor site. HL is characterized by a unique tumor microenvironment that creates a physical barrier and a hostile niche for CD30.CAR-T cells. Expression of PD-1 on CD30.CAR-T cells indicates that these cells likely remain susceptible to inhibition by PD-L1 in the tumor, and suggests that the combination of CD30.CAR-T cells with checkpoint blockade should be investigated. Enhancing CAR-T cell trafficking to tumor sites should also tip the delicate balance between effector and inhibitory mechanisms towards the former. Since the tumor microenvironment of HL is rich in TARC (thymus and activation regulated chemokine/CC chemokine ligand 17), we have initiated a clinical study in which CD30.CAR-T cells are further modified to co-express the cognate receptor (CCR4) for TARC to improve their tumor homing (NCT03062157). Disclosures Savoldo: Baylor College of Medicine: Patents & Royalties: CAR.CD30 patent; Bluebirdbio: Other: research agreement; Cell Medica: Other: Research Agreement; Bellicum: Other: Research Agreement. Grover:Seattle Genetics: Consultancy. Ramos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding.

Overexpression of survivin in pediatric Hodgkin lymphoma tumor cells: Characterization of protein expression and splice-variants transcription profile

Survivin is abundantly expressed during fetal development but absent in most differentiated adult tissues; an exception being components of the immune system, such as B and T lymphocytes. Beyond acting as a master regulator of the cell cycle, survivin acts as an inhibitor of apoptosis and is overexpressed in almost all carcinoma types; however, its expression in lymphomas is lesser-explored. Survivin's role in carcinogenesis was subjected to its sub-cellular localization and splice transcripts expression, namely wild-type survivin, survivin-∆Ex3 and survivin-2B. To assess survivin's expression and sub-cellular localization in Epstein Barr virus positive and negative biopsies from treatment naïve pediatric patients with Hodgkin lymphoma (HL), samples were stained for survivin protein by immunofluorescence. The proportion of survivin+ cells was calculated, survivin sub-cellular localization assessed and its fluorescence intensity quantified. Transcription profile of survivin mRNA variants was studied by RT-qPCR. Survivin was overexpressed in the nucleus of tumor cells, and also in a greater proportion of tumor cells, in comparison with the non-tumoral infiltrating cells. Although a higher expression of survivin was observed in advanced clinical stages, no correlation was found between the expression level of survivin and a proliferation marker, or event-free survival. Instead, survivin was related to apoptosis inhibition in tumor cells. Additionally, survivin's transcriptional variants displayed similar expression levels. Present results suggest that although survivin is overexpressed in Hodgkin's tumor cells, it may not play a central role in the progression of classic HL, or act as a suitable progression biomarker, as suggested for most carcinomas.

Abstract A023: Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma

Abstract Background: Up to 20% of patients with advanced Hodgkin lymphoma (HL) are not cured with current chemo-immunotherapy treatments. Adoptive immunotherapy (AIT) with mature T-cells modified with a chimeric antigen receptor (CAR) holds promise for the treatment of various types of B cell lymphoma. The CD30 antigen, expressed in virtually all HL tumor cells, is absent in most healthy tissues, thus representing an ideal target for AIT of HL. In contrast to CAR19 for B cell malignancies, efficacy of CAR30 T-cells for Hodgkin and other CD30 lymphomas remains modest. Thus, novel approaches are needed to significantly improve the clinical efficacy of this approach. To this end, we used a novel antigen binding domain of a CD30 mAb, unaffected by soluble CD30 protein, as part of a chimeric antigen receptor (CAR) coupled to the 4.1BB and ζ chain endodomains, and transduced memory stem T-cells (TSCM) in an effort to ensure efficient engraftment, prolonged persistence, and enhancement of in vivo antitumor activity. To evaluate the feasibility of ex vivo expansion and antitumor efficacy of genetically-modified TSCM with a novel CD30-specific CAR that recognizes a membrane-proximal epitope of the CD30 molecule for the treatment of HL. Methods: A second-generation CD30-41BBz-EGFRt CAR was developed using a scFv that recognizes a membrane-proximal epitope of the CD30 protein. TSCM cells were generated with IL-7, IL-15 and IL-21, and transduced on day 2 of culture with a third-generation lentiviral vector encoding the CD30-CAR. The HL derived cell line L540 was used as tumor model. Tumor-specific cytotoxicity and cytokines were analysed at 24 hours. The in vivo antitumor efficacy was tested in a NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse model of HL xenografted with 2,5x10^6 L450 cells. Results: After 10 days, TSCM represented the majority of T-cells in culture (50.38±5.47 % for CD4+ and 70.84 ± 3.36 % for CD8+ TSCM, respectively) with a very high expression of CD30-CAR (77 ± 3.64 % in CD4+ and 83.12 ± 4.25 % in CD8+ TSCM, respectively). Expression of CD30 protein in TSCM increased over the culture period, peaking at day 8 in both CD4+ (17.5 ± 4.17 %) and CD8+ TSCM (42.37 ± 5.75 %), respectively. Despite these high levels of CD30 expression, TSCM expanded ex vivo very efficiently (CD4+ CD30-CAR: 97.24 ± 26.2 fold expansion, and CD8+ CD30-CAR: 96.79 ± 22.84 fold expansion, respectively), although lower than TSCM transduced with a CD19-CAR (CD4+ CD19-CAR: 161.96±35.9 fold expansion, and CD8+ CD19-CAR: 163.25 ± 26.51, fold expansion, respectively). Bulky CD30-CAR TSCM mediated very efficient in vitro tumor cytotoxicity (tumor cell death at 5:1 ratio: 85.25% vs. 0% with untransduced TSCM), while control CD30- targeT-cells were not killed. In addition, we evaluated the antitumor efficacy of CD30-CAR TSCM in vivo. Upon adoptive transfer of 10x10^6 bulk (CD4+ and CD8+) TSCM into NSG mice engrafted with L540 (2,5x10^6 subcutaneously), complete eradication of HL was observed. In addition, in a stress L540 model where equal numbers of CAR30 T-cells were infused at a low, suboptimal dose (i.e, 50% of effective T-cell dose to induce complete remission), highly enriched CAR30 TSCM (>70% of TSCM) had significantly antitumor effect compared to the infusion of CAR30 T-cells predominantly represented by effector cells (<30% TSCM) (mean survival 54 days vs 40 days, respectively; P< 0.01). Conclusions: We demonstrate that TSCM can be efficiently transduced and ex vivo expanded with a novel CD30-CAR that surpasses fratricide killing of CD30 expressing T-cells and confers potent in vivo antitumor efficacy against HL. Importantly, highly enriched CAR30 TSCM products have enhanced antitumor properties compared to those comprising predominantly effector cell subsets. These proof-of-concept studies support the use of a refined CAR30 with highly enriched TSCM cell products for improving clinical efficacy of adoptive immunotherapy of patients with HL. Citation Format: Carmen Alvarez-Fernández, Laura Escribà-Garcia, Ana Carolina Caballero, Jorge Sierra, Javier Briones. Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A023.

CCR5 antagonism by maraviroc inhibits Hodgkin lymphoma microenvironment interactions and xenograft growth.

Classic Hodgkin lymphoma tumor cells express a functional CCR5 receptor, and tumor tissues express high CCL5 levels, suggesting that CCL5-CCR5 signaling is involved in tumor-microenvironment formation and tumor growth. Using the CCR5 antagonist, maraviroc, and a neutralizing anti-CCL5 antibody, we found that CCL5 secreted by classic Hodgkin lymphoma cells recruited mesenchymal stromal cells and monocytes. The “education” of mesenchymal stromal cells by tumor cell-conditioned medium enhanced mesenchymal stromal cells’ proliferation and CCL5 secretion. In turn, educated mesenchymal stromal cell-conditioned medium increased the clonogenic growth of tumor cells and monocyte migration, but these effects were reduced by maraviroc. Monocyte education by tumor cell-conditioned medium induced their growth and reprogrammed them towards immunosuppressive tumor-associated macrophages that expressed IDO and PD-L1 and secreted IL-10, CCL17 and TGF-β. Educated monocyte-conditioned medium slowed the growth of phytohemagglutinin-activated lymphocytes. Maraviroc decreased tumor cell growth and synergized with doxorubicin and brentuximab vedotin. A three-dimensional heterospheroid assay showed that maraviroc counteracted both the formation and viability of heterospheroids generated by co-cultivation of tumor cells with mesenchymal stromal cells and monocytes. In mice bearing tumor cell xenografts, maraviroc reduced tumor growth by more than 50% and inhibited monocyte accumulation, without weight loss. Finally, in classic Hodgkin lymphoma human tumor tissues, CCL5 and CD68 expression correlated positively, and patients with high CCL5 levels had poor prognosis. In conclusion, since the present challenges are to find molecules counteracting the formation of the immunosuppressive tumor microenvironment or new, less toxic drug combinations, the repurposed drug maraviroc may represent a new opportunity for classic Hodgkin lym phoma treatment.

Ectopic expression of transcription factor BATF3 induces B-cell lymphomas in a murine B-cell transplantation model.

The mechanisms involved in malignant transformation of mature B and T lymphocytes are still poorly understood. In a previous study, we compared gene expression profiles of the tumor cells of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) to their normal cellular counterparts and found the basic leucine zipper protein ATF-like 3 (BATF3) to be significantly upregulated in the tumor cells of both entities. To assess the oncogenic potential of BATF3 in lymphomagenesis and to dissect the molecular interactions of BATF3 in lymphoma cells, we retrovirally transduced murine mature T and B cells with a BATF3-encoding viral vector and transplanted each population into Rag1-deficient recipients. Intriguingly, BATF3-expressing B lymphocytes readily induced B-cell lymphomas after characteristic latencies, whereas T-cell transplanted animals remained healthy throughout the observation time. Further analyses revealed a germinal center B-cell-like phenotype of most BATF3-initiated lymphomas. In a multiple myeloma cell line, BATF3 inhibited BLIMP1 expression, potentially illuminating an oncogenic action of BATF3 in B-cell lymphomagenesis. In conclusion, BATF3 overexpression induces malignant transformation of mature B cells and might serve as a potential target in B-cell lymphoma treatment.

CD83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma.

Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83−T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma.

Clinical significance of tryptophan catabolism in Hodgkin lymphoma.

Indoleamine 2,3‐dioxygenase 1 (IDO) is an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway. The purpose of the present study was to determine the clinical significance of Trp catabolism in newly diagnosed Hodgkin lymphoma (HL) patients. We quantified serum Trp and Kyn in 52 HL patients, and analyzed their associations with different clinical parameters including serum soluble CD30 concentration. The IDO expression was evaluated in the patients’ affected lymph nodes. The cohort comprised 22 male and 30 female patients (age range, 15‐81 years; median, 45 years), with a 5‐year overall survival (OS) of 88.6%. The OS was significantly shorter for patients with a high Kyn/Trp ratio (OS at 5 years, 60.0% vs 92.2%), for those with stage IV disease, and for those with lymphocytopenia (<600/mm3 and/or <8% white blood cell count). The latter two parameters are components of the international prognostic score for advanced HL. In contrast, there were no significant differences in OS according to age, serum albumin, hemoglobin, sex, white blood cell count, or serum soluble CD30 (≥ or <285.6 ng/mL). Multivariate analysis using the three variables stage, lymphocytopenia, and serum Kyn/Trp ratio showed that only the latter significantly affected OS. Indoleamine 2,3‐dioxygenase 1 was produced by macrophages/dendritic cells, but not by HL tumor cells, and IDO levels determined by immunohistochemistry had a significant positive correlation with the serum Kyn/Trp ratio. In conclusion, quantification of serum Kyn and Trp is useful for predicting prognosis of individual HL patients.

The Role of B Regulatory Cells in the Immunological Escape of Tumor Cells in Hodgkin Lymphoma

Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin Lymphoma (HL) are surrounded by a rich inflammatory infiltrate which aids in their survival and escape from cytotoxic CD8+ T cells (CTLs) and Natural Killer cells (NKs). Within HL environment, T regulatory cells (Tregs) directly suppress the activity of CTLs and NKs, enhancing the tolerance against HRS cells. B regulatory cells (Bregs) have been shown to support the differentiation of Tregs through IL-10 production; thus, we hypothesized that they could have a role in the pathophysiology of HL.We evaluated 30 classic HL patients (M/F: 18/12; median age, 31 years, range 15-62) and 5 healthy controls (HC) for circulating peripheral blood (PB) Bregs, Tregs, CTLs, NKs, and NKTs. Twenty-four of them were new-diagnosed patients (NwHL) and 6 received a previous diagnosis of HL but were in complete remission (CR) for more than 12 months (PvHL). NwHL patients were divided according to the International Prognostic Score (IPS) and the Ann-Arbor Staging System. All subjects were treated following the ABVD protocol (doxorubicin 25 mg/m2, bleomycin 10 U/m2, vinblastine 6 mg/m2, and dacarbazine 375 mg/m2). Flow cytometry was performed on heparinized PB samples with a 5-color Beckman Coulter Cytomics FC500 flow cytometer. Breg (CD19+CD24+), Treg (CD3+CD4+CD25+), CTL (CD3+CD8+), NK (CD3-CD56+), and NKT (CD3+CD56+) levels were measured simultaneously with the PET/CT evaluations, ie at diagnosis, at the end of the second ABVD administration, at the end of treatment, and at 6 and/or 12 months off-therapy. Moreover, Breg levels were compared to IPS and Ann-Arbor staging groups, and also were correlated to the erythrocyte sedimentation rate (ESR) and to the absolute lymphocyte count (ALC).We found decreased circulating Bregs in NwHL and PvHL patients compared to controls (0.39% vs 0.875% vs 1.813%, respectively, p<0.0001). In addition, we found decreased CTLs in NwHL compared to PvHL and HC (2.815% vs 4.057% vs 5.780%, respectively, p=0.0228). Thus, all HL patients showed lower numbers of Bregs, but there were no differences between patients of different IPS or stage. Besides, lower Breg levels were correlated with higher ESR values (r2=0.3193, p=0.0117), while no correlation was found for ALC (r2=0.0414, p=0.3898). The levels of Tregs, NKs, and NKTs were not significantly different between patients and controls at diagnosis. As a result, the greater reduction of Bregs compared to Tregs in PB caused a larger Bregs/Tregs ratio in NwHL and PvHL patients compared to controls (1.014 and 1.003 vs. 5.975, p=0.0094). Further reduction of circulating Bregs was observed in 58% of NwHL patients at the end of the second ABVD administration for patients experiencing both CR and partial remission (PR) (based on PET/CT scans) and in 80% at the end of treatment (71% CR, 29% PR). Interestingly, post-treatment Breg levels of NwHL were initially lower than that of PvHL patients and controls (NwHL post-treatment 0.4062% vs PvHL 0.8750% vs HC1.813%, p=0.0030) but normalized after 6 months off-therapy (1.444%, p=0.6189). Similar trends were observed for CTLs (NwHL post-treatment 4.615% vs NwHL 6 months off therapy 5.603% vs PvHL4.057% vs HC 5.780%, p=0.7558) and Bregs/Tregs ratio (0.9613 vs 5.975, p=0.0678). These data suggest that the B cell depletion phase during chemotherapy may interrupt the positive feedback between B and T cell compartments and the normalization of Bregs and CTLs after treatment may be linked to the restoration of a normal immune response, supporting by the achievement of CR in all patients.Our preliminary data suggest involvement of Bregs in the escape and survival of HRS cells during active disease. Peripheral blood may mirror disease activity in lymphoid tissues. Thus, the decrease of circulating Bregs may be related to the recruitment of these cells to the tumor site; amplification of the Bregs/Tregs ratio may result in a greater Breg-dependent Treg activation with subsequent inhibition of CTL and NK function. Additionally, the normalization of Bregs and the Bregs/Tregs ratio after chemotherapy could be used to predict disease remission. While larger prospective studies are required to validate these results, we present intriguing evidence of the involvement of Bregs in the pathophysiology of HL. DisclosuresNo relevant conflicts of interest to declare.

CD58 mutations are common in Hodgkin lymphoma cell lines and loss of CD58 expression in tumor cells occurs in Hodgkin lymphoma patients who relapse.

CD58 is involved in immune recognition of tumor cells via binding of the CD2 receptor expressed on cytotoxic T cells. In diffuse large B-cell lymphoma, mutations of the CD58 gene are reported to contribute to immune evasion of the tumor cells. We previously showed CD58 mutations in three Hodgkin lymphoma (HL) cell lines by whole-exome sequencing. In this study, we confirmed the mutations by Sanger sequencing at the DNA and RNA level and showed low levels or total loss of CD58 mRNA expression in two of the three cell lines. CD58 protein expression as determined by flow cytometry, western blotting and immunohistochemistry was absent in all three mutated HL cell lines. In primary tissue samples, loss of CD58 expression was observed in 11% of the patients who relapse. These data suggest that loss of CD58 is a potential immune escape mechanism of HL tumor cells, especially in clinically aggressive disease.

Expression of CD137 in tumor cells of Hodgkin lymphoma from Northern China and its application in pathological differential diagnosis

目的明确CD137在北方地区经典型霍奇金淋巴瘤（cHL）中的表达，探讨其作为cHL辅助病理鉴别诊断新指标的可能应用价值。方法收集54例cHL患者资料，以55例伴有“HRS样细胞”的非cHL患者为对照。在病理组织标本中选取“HRS细胞”或“HRS样细胞”丰富的区域制作组织芯片；以“HRS细胞”或“HRS样细胞”为观察对象，cHL组应用CD30、CD15、CD20、PAX5、CD3免疫组织化学染色；同时对两组患者标本进行CD137（BBK-2）抗体免疫组织化学染色及采用EBV编码的小RNA（EBER）原位杂交法检测EBV感染状态。结果54例cHL患者均为淋巴结内原发，中位年龄45.5（22.0~68.0）岁；男女比例1.7∶1；对照组患者结内54例，结外（皮肤）1例，中位年龄50.0（12.0~81.0）岁；男女比例1.9∶1。54例cHL患者均表达CD30，HRS细胞主要诊断相关免疫标志物CD30、CD15、CD20、CD3阳性表达率依次为100.0%、70.4%、18.5％和0，可见PAX5弱至中等强度表达，阳性率70.4%；EBV感染阳性率25.9%（对照组阳性率21.8%）。cHL组CD137阳性率57.4%，对照组阳性率14.5%，差异有统计学意义（P<0.001）。将cHL组及对照组按照患者年龄（≥60/<60岁）、性别、有无EBV感染、组织学亚型以及主要诊断相关标志物的表达与否进行分组，CD137阳性率差异均无统计学意义（P值均> 0.05）。以2013年为界进行分组，2013年前后两组cHL患者的CD137阳性率差异有统计学意义（39.4%对85.7%，P=0.001），对照组差异无统计学意义（12.5％对16.1%，P=0.705）；2013以后存档的标本中cHL组与对照组患者CD137阳性率差异有统计学意义（85.7％对16.1%，P<0.001）。结论通过研究初步证实北方地区大多数cHL患者的HRS细胞表达CD137，而对照组患者“HRS样细胞”CD137阳性率较低。保存期3年以内较保存期3年以上的cHL患者标本CD137阳性率高，更适于进行CD137免疫组织化学染色检测。CD137有望作为辅助cHL病理鉴别诊断的新指标。

Continuous Exposure to Bendamustine (BDM) Results in Stable Upregulation of CD30 and Increased Sensitivity to Brentuximab Vedotin (BV) in Tumor Cells of Hodgkin Lymphoma (HL)

Background: Given the consistent antitumor activity and favorable toxicity profile in heavily pretreated patients, BDM represents a suitable platform for combination with target-based agents in the setting of recurrent HL. The anti-CD30 antibody-monomethyl auristatin E (MMAE) conjugate brentuximab vedotin (BV) appears a most valuable candidate by coupling an impressive clinical activity with the lack of serious overlapping toxicities with BDM. While the combination of BDM and BV is being evaluated in Phase II studies, no information is available as to possible mechanisms through which BDM may regulate the antitumor efficacy of BV towards HL cells.Methods: We evaluated the effects of acute and extended exposure to BDM on CD30 expression and sensitivity to the cytotoxic activity BV in the HL cell line L1236. Cells were cultured in the presence of BDM at its IC50 for different time points of acute exposure. Through continuous exposure to increasing concentrations (25.0, 50.0, 75.0, 100 micromol/L) of BDM, we then performed a serial in vitro selection for BDM-resistant (R) L1236 cell clones and determined their CD30 expression by flow cytometry, qRT-PCR, and Western analysis.Results: Acute exposure to BDM (48 to 72 hrs) of L1236 cells led to a sizeable upregulation of CD30 as shown by flow cytometry. This effect was unsustained since CD30 intensity returned to baseline levels upon culturing in BDM-free medium for one week. Expression of other surface antigens, i.e. HLA-DR, was unaffected by BDM while acute exposure to doxorubicin and other cytotoxic drugs did not modify the CD30 expression. We established four L1236-derived cell clones (R25, R50, R75 and R100) able to proliferate across the different concentrations of BDM with growth/viability curves superimposable to native cells. Clonal identity among clones and with parental cells was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. All R-clones displayed an up to 900% increase in CD30 median fluorescence intensity, relative to native L1236 cells (Figure 1A). The sustained CD30 upregulation was confirmed by qRT-PCR and Western blotting since R-clones expressed up to 10-fold higher levels of CD30-specific mRNA and CD30-specific 120 kDa components as compared to parental cells. To rule out a general upregulation of surface molecules as a result of the chronic exposure to BDM we evaluated the expression of other surface antigens. As opposed to CD30, HLA-DR and PDL-1 relative transcript levels and cell surface expression were significantly reduced in R-cells. Intriguingly, BDM-induced CD30 upregulation was specific to L1236 cells since extended exposure to BDM did not modify expression levels of CD30 in other lymphoma cell types (SUDHL1, JEKO). BDM-induced CD30 overexpression significantly enhanced the sensitivity of HL cells to the cytotoxic effects of BV. MTS assay showed the IC50 of R100 cells to BV shifted to 0.21 ± 0.06 mcg/mL (48 hrs) and 0.19 ± 0.05 mcg/mL (72 hrs) vs. 3.16 ± 0.75 mcg/mL (48 hrs) and 3.87 ± 0.68 mcg/mL (72 hrs) of parental cells, a 15- and 20-fold increase, respectively (p<0.001; Figure 1B). Finally, qRT-PCR studies disclosed that R-clones did not show any upregulation of the drug exporter MDR1 gene, involved in MMAE extrusion and BV resistance in HL cells, as compared to parental L1236 cells.Conclusions: Whileacute exposure to BDM induced a transient upregulation of CD30, extended exposure to the agent resulted in the selection of L1236 subclones with a stable overexpression of surface CD30 and reduced levels of HLA-DR and PDL-1. Upregulation of CD30 was associated with a significantly enhanced sensitivity to cytotoxic effects of BV. Translated into a clinical context these findings suggest that HL patients progressing upon BDM treatment may turn exquisitely sensitive to BV and prompt the exploration of a sequential treatment with BDM and BV in late and earlier disease phases. Results also provide a mechanistic insight as to the efficacy of the BDM-BV combination in HL, highlighting non-overlapping resistance pathways between BDM and MMAE. BDM-induced selection of CD30hi, HLA-DRlow/PDL-1low tumor cell clones might be relevant to the design of novel treatment strategies for HL based on the combination of BDM with BV and anti-PD1/PDL-1 antibodies. [Display omitted] [Display omitted] DisclosuresPinto:Takeda: Research Funding; Takeda, Celgene, Roche, TEVA: Honoraria.

Diagnostic Utility of the Germinal Center-associated Markers GCET1, HGAL, and LMO2 in Hematolymphoid Neoplasms.

Implementation of new phenotypic markers in routine diagnostics of hematolymphoid neoplasms is a challenging task with a plethora of potentially relevant proteins. We investigated 3 recently discovered proteins expressed in the germinal centers of lymph nodes (LMO2, GCET1, and HGAL) in a compilation of leukemia, lymphoma, and thymic tumor entities. Altogether, 1590 cases (1519 on tissue microarrays, 71 on conventional slides) were included. Expressions of LMO2, GCET1, and HGAL were investigated by immunohistochemistry, evaluated for their differential diagnostic relevance, and correlated with the clinical outcome of patients. In Hodgkin lymphoma (HL), the expression of LMO2, GCET1, and HGAL could be largely seen in tumor cells of nodular lymphocyte predominant HL (NLPHL) but only occasionally in classic HL. The majority of B-cell lymphoma cases was positive for LMO2 [except for Burkitt lymphoma (BL)] and HGAL with weaker to moderate staining intensity, compared with the intensely staining follicular lymphomas (FL). Except for FL (60% of cases) and diffuse large B-cell lymphomas (DLBCL, 36% of cases), all other B-cell lymphomas expressed little or no GCET1. In thymomas, the non-neoplastic immature T-cells were LMO2-negative, whereas the neoplastic lymphoblasts were LMO2-positive in more than half of the lymphoblastic lymphomas (LBL). Our findings provide new potential assistance in the differential diagnosis of FL to marginal zone lymphoma, classic HL to NLPHL and primary mediastinal B-cell lymphoma, DLBCL to BL, and thymoma to LBL. Finally, HGAL proved to be a prognostic marker for classic HL regarding the background population and in DLBCL regarding the tumor cells.

K1 and K15 of Kaposi's Sarcoma-Associated Herpesvirus Are Partial Functional Homologues of Latent Membrane Protein 2A of Epstein-Barr Virus.

The human herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are associated with Hodgkin's lymphoma (HL) and Primary effusion lymphomas (PEL), respectively, which are B cell malignancies that originate from germinal center B cells. PEL cells but also a quarter of EBV-positive HL tumor cells do not express the genuine B cell receptor (BCR), a situation incompatible with survival of normal B cells. EBV encodes LMP2A, one of EBV's viral latent membrane proteins, which likely replaces the BCR's survival signaling in HL. Whether KSHV encodes a viral BCR mimic that contributes to oncogenesis is not known because an experimental model of KSHV-mediated B cell transformation is lacking. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary BCR-negative (BCR(-)) human B cells with them. We confirmed that the survival of BCR(-) B cells and their proliferation depended on an active LMP2A signal. Like LMP2A, the expression of K1 and K15 led to the survival of BCR(-) B cells prone to apoptosis, supported their proliferation, and regulated a similar set of cellular target genes. K1 and K15 encoded proteins appear to have noncomplementing, redundant functions in this model, but our findings suggest that both KSHV proteins can replace LMP2A's key activities contributing to the survival, activation and proliferation of BCR(-) PEL cells in vivo. Several herpesviruses encode oncogenes that are receptor-like proteins. Often, they are constitutively active providing important functions to the latently infected cells. LMP2A of Epstein-Barr virus (EBV) is such a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi's sarcoma-associated herpesvirus (KSHV) expressed in virus-associated tumors, have less obvious functions. We found in infection experiments that both viral receptors of KSHV can replace LMP2A and deliver functions similar to the endogenous BCR. K1, K15, and LMP2A also control the expression of a related set of cellular genes in primary human B cells, the target cells of EBV and KSHV. The observed phenotypes, as well as the known characteristics of these genes, argue for their contributions to cellular survival, B cell activation, and proliferation. Our findings provide one possible explanation for the tumorigenicity of KSHV, which poses a severe problem in immunocompromised patients.

Mechanisms and consequences of constitutive NF-κB activation in B-cell lymphoid malignancies

The discovery of constitutive nuclear factor-κB (NF-κB) activation in Hodgkin's lymphoma tumor cells almost two decades ago was one of the first reports that directly connected deregulated NF-κB signaling to human cancer. Subsequent studies demonstrated that enhanced NF-κB signaling is a common hallmark of many lymphoid malignancies, including Hodgkin lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large B-cell lymphoma and multiple myeloma. By inducing an anti-apoptotic and pro-proliferative gene program, NF-κB is involved in lymphoma survival and growth. Identification of somatic mutations that led to activation of oncogenes and inactivation of tumor suppressor genes in the pathway revealed that specific pathogenic mechanisms are responsible for constitutive NF-κB activation in different lymphoma entities. Thus, the identification of distinct oncogenic events is reflecting the diverse cellular origins of the different lymphomas. Further, elucidation of the mechanisms that drive NF-κB in lymphoma is of high clinical relevance as it will allow the design of target-directed precision therapy. Indeed, a number of drugs that impair constitutive NF-κB activation in lymphoid malignancies are currently in preclinical or clinical development.