Hodgkin Lymphoma Cell Research Articles

Abstract 4032: The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma

Abstract The refractory and relapsed disease is currently the challenge in Hodgkin Lymphoma (HL) treatment. There is no specific therapy but rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the HL molecular biology and the screening for therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by Rel-B and NIK, to survive. Its constitutive activation seems to be involved in the refractory and relapsed disease. To determine the specific Rel-B target genes we performed ChIP-Seq in UH01 and L1236 human HL cell lines for Rel-A, Rel-B, cRel, p50 and p52. We found 29,414 Rel-B peaks genome-wide distributed. To further identify genes with higher probabilities of being directly regulated by Rel-A, Rel-B, and/or cRel we concentrated on the peaks that localized within a +/- 2 kb window, relative to the gene transcription start site. The +/- 2 kb Rel-B peaks were distributed on 4,509 genes; meanwhile the +/- 2 kb cRel peaks were on 1,994 genes and the Rel-A peaks were on 830 genes. Out of the 4,509 genes with the Rel-B peaks, only 6% overlapped with the Rel-A peaks and 11% with the cRel peaks. The Rel factors distribution showed a Rel-B DNA-binding hierarchy in HL. The ChIP-Seq data were merged with gene expression arrays results. The gene expression assays were analyzed by comparing the signal from cells infected with noninduced shRNAs for the Rel factors to that from cells infected with induced shRNAs. Differentially expressed genes were identified via ANOVA analysis. Genes that were downregulated more than 2-fold with a p<0.001 were considered significant. We found that the Rel-B downstream controlled gene set was enriched for cell cycle and cell death regulation and DNA damage and repair signatures, among others. One of the exclusively Rel-B target genes was BCL2. We showed that exogenous BCL2 was able to partially rescue HL cells from dying in response to Re-B depletion. We also found that BCL2 was useful as a prognosis marker in terms of overall survival in a cohort of 96 HL patients [Log Rank Test (p=0.002)]. In this retrospective study [follow up period 47,4 (6-136) months], BCL2 was able to identify at diagnosis patients that were refractory to conventional first line treatment and patients that relapsed. The HL cell lines U-H01, L1236, SUPDHL1, KM-H2 and L540 were sensitive to the BCL2 inhibitor venetoclax. The HDM-2 cell line, which does not express BCL2, was a good control of target specificity since it did not die in response to venetoclax. In summary, we found that the alternative NFkB pathway plays an important role in the refractory and relapsed Hodgkin disease, being BCL2 a key downstream target. BCL2 performed well as a prognosis marker identifying refractory patients and those that would relapse. We propose BCL2 expression assessment in the lymph node biopsy at diagnosis and BCL2 as a directed therapy. Citation Format: Angélica María Gamboa-Cedeño, Mariángeles castillo, Victoria Otero, Natalia Schutz, Dorotea Fantl, Federico Jauk Vitali, Hernán García Rivello, Myriam Nuñez, Stella Maris Ranuncolo. The alternative NF-kB pathway activity in refractory and relapsed Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4032.

PROSPECTIVE STUDY OF 3T WHOLE BODY DIFFUSION WEIGHTED MAGNETIC RESONANCE (WB DWI MRI) COMPARED TO PET IN STAGING AND ASSESSMENT OF TREATMENT RESPONSE IN FDG AVID LYMPHOMAS

Introduction: PET is considered the gold standard for staging and evaluation of treatment response in FDG avid Lymphoma. 3T WB-DWI-MRI may represent an option for detection and better characterisation of tumoral lesions without use of FDG or iodated/paramagnetic contrast medium. We compared 3T WB-DWI-MRI with PET in the evaluation of Hodgkin lymphoma (HL) and Diffuse large cell lymphoma patients (DLBCL). Methods: From may 2016, 25 patients (14 cases of HL and 11 cases of DLBCL) were diagnosed at our institution. PET and 3T WB-DWI-MRI was perfomed at diagnosis and post treatment for a total of 118 nodal sites and 21 extranodal valued sites. DWI imaging was obtained with multi shot sequences (thickness 4 mm) multi-b (b-value: 0, 150, 1000) and ADC cut off value 1.2 for functional evaluation. Morphological images were obtained by using sequences fast spin-echo short time inversion recovery (STIR with thickness 4mm) and Turbo spin-echo (T2-TSE with thickness 4mm) and sequnces T1-FAST-Field-echo (T1-FFE) in and out - phase (thickness 4mm). MRI were compared with PET imaging performed at the same time and assessed blind by radiologist and Nuclear Medicine physician. Results: The 3T WB-DWI-MRI and PET results were concordant at the diagnosis after evaluation of 118 nodal site and 21 extra nodal site with Cohen's Kappa coefficient showed agreement to be excellent (K = 0.82 and 0.84 respectively). When performed after treatment the two methods were not concordant due to the greater sensitivity and specificity of the RMI (high positive and negative predictive value. In 5/25 cases of nodal site and in 2/16 cases of extra nodal site after chemotherapy MRI and PET results were discordant and PET failed complete remission finding (doubts residues after therapy not confirmed at subsequent follow-up). Conclusions: These data suggest that 3T WB-DWI-MRI can be considered a sensitive and specific method for assessing treatment response in Lymphoma without the use of ionising radiation or administration of F-18 Flurodeoxyglucose. In this population, MRI appears to obtain a better definition of the response after chemotherapy especially in mediastinal, hepatic, waldeyer ring and bone sites. Further studies are needed to evaluate the optimum ADC values in assessing treatment response in a more large number of patients. Keywords: diffuse large B-cell lymphoma (DLBCL); Hodgkin lymphoma (HL); positron emission tomography (PET).

Proteomic analysis and verification of protein expression after upregulation of human CD99 in Hodgkin lymphoma cell line L428

目的探索稳定过表达CD99前后霍奇金淋巴瘤细胞株L428细胞中差异表达的蛋白，并分析验证差异蛋白的功能。方法采用荧光差异凝胶双向电泳和质谱分析技术检测L428细胞稳定过表达CD99前后蛋白表达的差异，采用GOfact开放软件对差异蛋白进行聚类分析，筛选在细胞转化过程中起重要作用的蛋白，并分析验证其功能。结果筛选鉴定得到38个差异蛋白，其中在稳定过表达CD99的霍奇金淋巴瘤细胞株L428（L428-CD99细胞）中与CD99表达呈正相关的蛋白21个，呈负相关的蛋白17个，所得的38个差异蛋白中有32个蛋白参与了细胞的生物过程，35个蛋白参与了细胞的组成和构建，28个参与了抗氧化、蛋白结合、催化活性、酶调节、信号转导、结构分子、翻译调节和离子转运等多个方面。结论筛选得到38个差异蛋白，其中关于细胞骨架、细胞分化、信号通路、调节基因表达等蛋白的改变与H/RS细胞和B淋巴瘤细胞转化相关。

Epstein–Barr virus-encoded LMP1 induces ectopic CD137 expression on Hodgkin and Reed–Sternberg cells via the PI3K-AKT-mTOR pathway

CD137 is a potent co-stimulatory molecule on activated T cells, and its ligand (CD137L) is expressed on antigen presenting cells (APC). Ectopic expression of CD137 has been identified on Hodgkin Reed–Sternberg (HRS) cells, the malignant cells in Hodgkin Lymphoma (HL), and CD137 on HRS cells was found to support growth of HRS cells and escape from immune surveillance. HRS cells are mostly derived from B cells, which poses the question of how B cells acquire ectopic CD137 expression during the transformation process. HL is associated with Epstein–Barr virus (EBV) infection. We show that the EBV latent membrane protein 1 (LMP1) induces expression of CD137 in HRS cell lines. In a HL tissue microarray, 96% of the CD137-positive HL cases stained positive for LMP1. LMP1 utilizes the PI3K-AKT-mTOR pathway for inducing CD137 expression. These findings support the role of EBV in HL pathogenesis.

Impact of rs12917 MGMT Polymorphism on [18F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL).

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34months. We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.

Autocrine LTA signaling drives NF-κB and JAK-STAT activity and myeloid gene expression in Hodgkin lymphoma

AbstractPersistent NF-κB activation is a hallmark of the malignant Hodgkin/Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL). Genomic lesions, Epstein-Barr virus infection, soluble factors, and tumor–microenvironment interactions contribute to this activation. Here, in an unbiased approach to identify the cHL cell-secreted key factors for NF-κB activation, we have dissected the secretome of cultured cHL cells by chromatography and subsequent mass spectrometry. We identified lymphotoxin-α (LTA) as the causative factor for autocrine and paracrine activation of canonical and noncanonical NF-κB in cHL cell lines. In addition to inducing NF-κB, LTA promotes JAK2/STAT6 signaling. LTA and its receptor TNFRSF14 are transcriptionally activated by noncanonical NF-κB, creating a continuous feedback loop. Furthermore, LTA shapes the expression of cytokines, receptors, immune checkpoint ligands and adhesion molecules, including CSF2, CD40, PD-L1/PD-L2, and VCAM1. Comparison with single-cell gene-activity profiles of human hematopoietic cells showed that LTA induces genes restricted to the lymphoid lineage, as well as those largely restricted to the myeloid lineage. Thus, LTA sustains autocrine NF-κB activation, impacts activation of several signaling pathways, and drives expression of genes essential for microenvironmental interactions and lineage ambiguity. These data provide a robust rationale for targeting LTA as a treatment strategy for cHL patients.

Immunological Agents Used in Cancer Treatment.

Immune checkpoint inhibitors (ICI) are monoclonal antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1), or PD-1 ligand (PD-L1). ICI are approved for the treatment of malign melanoma, non-small cell lung cancer, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, and renal cell carcinoma. They can lead to long-term anti-tumor responses by deactivating the brake mechanism in the immune system. Ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab are examples of ICI. CTLA-4 is a brake mechanism in immune response. Ipilimumab and tremelimumab are antibodies against CTLA-4. PD-1 is another important immune checkpoint co-inhibitor receptor that is expressed by activated T cells in the peripheral tissue. As a result of blockage of the PD-1/PD-L1 pathway, local tumor-specific immune response augments, and long-term tumor control can be achieved. In recent years, ICI are approved for the treatment of various malignities. They may be responsible for specific toxicities called immune-related adverse events (irAEs). irAEs are a consequence infiltration of normal tissues by activated T lymphocytes that are responsible for autoimmunity. Corticosteroids and anti-tumor necrosis factor agents, such as infliximab and mycophenolate mofetil, are effective in the treatment of irAEs. Immune checkpoint inhibition with monoclonal antibodies against CTLA-4 and/or PD-1/PD-L1 by single agent or combination treatments became a new option in various solid tumors. However, ICI have unique adverse events, and these adverse events should be considered in any new onset clinical situation and should be managed properly. Future prospective randomized clinical trials will clarify recent questions.

Temporal Splicing Switches in Elements of the TNF-Pathway Identified by Computational Analysis of Transcriptome Data for Human Cell Lines.

Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages (HD-MY-Z, stage IIIb and L-1236, stage IV) compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes TRAF1, TNFRSF12A and NFKB2. Our results point to a role of temporal alternative splicing in isoform production, which may alter the outcome of the TNF pathway and impact on tumorigenesis.

Identification of human endogenous retrovirus transcripts in Hodgkin Lymphoma cells.

During the last decades, the prognosis for patients with Hodgkin Lymphoma (HL) has been steadily improved. Nevertheless, new and less toxic therapy strategies have to be developed especially for patients with advanced disease. The activation of human endogenous retroviruses (HERV) is suspected to occur in HL and therefore, HERV might represent interesting target structures. In order to identify transcribed HERV of the HERV-H and HERV-K families in HL we used a reverse transcription-polymerase chain reaction based cloning approach. In addition to unspliced HERV-H and HERV-K transcripts, we detected spliced HERV-K transcripts that matched genomic sequences with the expected splicing-donor and splicing-acceptor sites. Of particular interest was the expression of HERV-K18 related transcripts at the CD48 locus. Our data indicate transcriptional activity of several HERV loci in HL cells.

Targeting Cancer Cells and Tumor Microenvironment in Preclinical and Clinical Models of Hodgkin Lymphoma Using the Dual PI3Kδ/γ Inhibitor RP6530.

Tumor-associated macrophages (TAMs) and the hyperactivation of the PI3K/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma and affect disease outcome. Because the δ and γ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME), we propose that the PI3Kδ/γ inhibitor RP6530 might affect both HRS cells and TME, ultimately leading to an enhanced antitumor response. Hodgkin lymphoma cell lines (L-540, KM-H2, and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in Hodgkin lymphoma cell line xenografts. In vitro, RP6530 besides killing and inhibiting the proliferation of Hodgkin lymphoma cells, downregulated lactic acid metabolism, switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing, we define tumor glycolysis as a specific PI3Kδ/γ-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator pyruvate kinase M2 as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore, we show in human tumor xenografts that RP6530 repolarizes TAMs into proinflammatory macrophages and inhibits tumor vasculature, leading to tumor regression. Interestingly, patients with Hodgkin lymphoma experiencing objective responses (complete response and partial response) in a phase I trial using RP6530 showed a significant inhibition of circulating myeloid-derived suppressor cells and an average mean reduction in serum thymus and activation-regulated chemokine levels of 40% (range, 4%-76%). Our results support PI3Kδ/γ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat patients with Hodgkin lymphoma.

Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

CD123 (IL-3Rα) is frequently expressed by malignant Hodgkin lymphoma (HL) cells. Naked monoclonal antibodies (mAb) against HL lack clinical benefit, partially due to absence of natural killer (NK) cells in the tumor microenvironment. Here we show that the combination of a fully humanized anti-CD123 mAb (CSL362) and high-affinity Fcγ-receptor NK-92 cells (haNK) effectively target and kill HL cells in vitro. First, we confirmed high expression of CD123 in 2 of the 3 HL cell lines (KM-H2 and L-428), and its absence in NK cells. Cytotoxicity of haNK cells against CD123-positive HL cells was significantly higher in the presence of CSL362. This was also shown with IL-15-activated primary NK cells, although haNK cells showed a 10.87-fold lower estimated half-maximal stimulatory effective concentration (EC50). CSL362 facilitated a significant increase in the expression of CD107a, intracellular IFN-γ and TNF-α and enhanced expression of c-JUN, PLD-1, and ARF6 by NK cells. Inhibition of the ARF6–PLD-1 axis (NAV2729), but not of the MAPK pathway (U0126), completely abrogated CSL362-facilitated antibody-dependent cell-mediated cytotoxicity (ADCC) in haNK and activated primary NK cells. Our results support CD123 as an immunotherapeutic target for HL and the combination of NK cells and CSL362 as a treatment strategy for HL.

Epstein-Barr virus LMP2A utilizes Syk and PI3K to activate NF-κB in B-cell lymphomas to increase MIP-1α production.

The incidence of Hodgkin's lymphoma (HL) is growing due to an increase in Epstein-Barr virus (EBV)-associated HL in AIDS patients. The HL tumor microenvironment is vital for the survival of the malignant Hodgkin-Reed Sternberg (HRS) cells of HL, which express the EBV protein latent membrane protein 2A (LMP2A). While previous work shows that LMP2A mimics B-cell receptor (BCR) signaling to promote the survival of HRS cells, the ability of LMP2A to establish and maintain the tumor microenvironment through the production of chemokines remains unknown. Since BCR signaling induces the production of the chemokine macrophage inflammatory protein-1α (MIP-1α), and since LMP2A is a BCR mimic, we hypothesized that LMP2A increases MIP-1α levels. A comparison of multiple LMP2A-negative and -positive cell lines demonstrates that LMP2A increases MIP-1α. Additionally, LMP2A-mutant cell lines and pharmacologic inhibitors indicate that LMP2A activates a Syk/PI3K/NF-κB pathway to enhance MIP-1α. Finally, based on the finding that an NF-κB inhibitor decreased MIP-1α RNA/protein in LMP2A-positive cells, we are the first to demonstrate that LMP2A increases the nuclear localization of the NF-κB p65 subunit using DNA-binding assays and confocal microscopy in human B cells. These findings not only have implications for the treatment of HL, but also other LMP2A-expressing B-cell tumors that overexpress NF-κB.

CD30 expression in neoplastic T cells of follicular T cell lymphoma is a helpful diagnostic tool in the differential diagnosis of Hodgkin lymphoma

Follicular T cell lymphoma is derived from follicular T-helper cells. In many cases, neoplastic T cells form rosettes around Hodgkin–Reed–Sternberg-like cells, which can lead to the misdiagnosis of classical Hodgkin lymphoma. The aim of the present study was to obtain a better understanding of this rosetting phenomenon and to recognize features that are helpful in the differential diagnosis of classical Hodgkin lymphoma. Sixteen mostly elderly follicular T cell lymphoma patients (mean 66 years) were analyzed. Fifteen of the 16 follicular T cell lymphoma cases presented with Hodgkin–Reed–Sternberg-like cells, which were CD20-positive in 27% of the cases and Epstein–Barr virus-infected in nearly all cases. Frequently, the immunophenotype of rosetting neoplastic T cells differed from the bulk neoplastic cells with less numerous T-follicular helper cell markers expressed, suggesting a modulation of T-follicular helper cell marker expression in the neoplastic T cells. In 75% of the cases, variable CD30 expression was encountered in the neoplastic T cells, likely reflecting an activation state in these cells. Hodgkin–Reed–Sternberg-like cells were positive for CCL17, and follicular T cell lymphoma tumor cells expressed its receptor CCR4 at variable intensity, thus potentially explaining the phenomenon of the tumor cells' rosetting around Hodgkin–Reed–Sternberg-like cells. In summary, this study confirms the presence of Hodgkin–Reed–Sternberg-like cells in a high number of cases of follicular T cell lymphoma, suggesting that Hodgkin–Reed–Sternberg-like cells may contribute to the development of this lymphoma. Hodgkin–Reed–Sternberg-like cells in follicular T cell lymphoma cannot reliably be differentiated from the Hodgkin–Reed–Sternberg cells of classical Hodgkin lymphoma based on their immunophenotype. In contrast, demonstration of a T-follicular helper cell phenotype with CD10 and frequent CD30 expression in the neoplastic T cell population can help to establish the diagnosis of follicular T cell lymphoma, and may even indicate CD30 as a therapeutic target for these patients.

Flow Cytometry for Non-Hodgkin and Hodgkin Lymphomas.

Multiparametric flow cytometry is a powerful diagnostic tool that permits rapid assessment of cellular antigen expression to quickly provide immunophenotypic information suitable for disease classification. This chapter describes a general approach for the identification of abnormal lymphoid populations by flow cytometry, including B, T, NK, and Hodgkin lymphoma cells suitable for the clinical and research environment. Knowledge of the common patterns of antigen expression of normal lymphoid cells is critical to permit identification of abnormal populations at disease presentation and for minimal residual disease assessment. We highlight an overview of procedures for processing and immunophenotyping non-Hodgkin B- and T-cell lymphomas and also describe our strategy for the sensitive and specific diagnosis of classical Hodgkin lymphoma and nodular lymphocyte predominant Hodgkin lymphoma.

Synthesis and biological evaluation of N-biphenyl-nicotinic based moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin Lymphoma

We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas, they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis. PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted an antiangiogenic effect by suppressing microvascular formation.

Nouvelles perspectives dans l’immunothérapie des cancers pédiatriques

New therapeutic paradigms are needed to improve the survival of children and adolescents with high-risk malignancies, and to reduce the sequelae associated with treatment. Immunotherapies, targeting tumor cells and/or the immune system to enhance existing anti-tumor immunity or induce novel anti-tumor immune responses, are becoming increasingly successful in adult oncology. Based on the results obtained with anti-ganglioside2 antibodies in neuroblastoma, rituximab in mature B malignancies, immune checkpoint inhibitors in lymphoma and especially in Hodgkin lymphoma, blinatumomab and CAR-T CD19 cells for B-cell acute lymphoblastic leukemia, immunotherapy has demonstrated irrefutable benefits in pediatric patients. However, these results are currently limited to a minority of patients and histologies. Current and ongoing trials tend to focus on a single type of immunotherapy, but it is likely that combinations of immunotherapies with different mechanisms of action or combination with other classes of anti-cancer treatments will be additives or even synergistic. The development of this new class of drugs in the treatment of pediatric cancers has multiple challenges: to better evaluate the response to treatment, to define the optimal doses and schedules, to manage immuno-mediated toxicities, to identify its specific sequelae, and, finally, to better understand the strategies of immune evasion of pediatric cancers in order to develop efficient immunotherapies.

Lymphocyte Co-Expression of CD38/HLA-DR Is a Marker for Anti-Tumor Activity in Hodgkin Lymphoma in Vitro: Evidence for a PD1-Independent Mechanism

IntroductionThe immunosuppressive tumor microenvironment has become of increasing interest in Hodgkin lymphoma (HL), in particular due to the recent success of checkpoint inhibitors (CI) as part of therapeutic strategies. The mechanism of these agents action in HL, however, remains elusive. Some studies have shown that cytotoxic T-lymphocytes may not be responsible for clinical efficacy, and that tumor-associated macrophages may also be targeted by these agents. We recently described a positive association between increasing proportion of CD38/HLA-DR co-positive lymphocyte in affected nodal tissue and clinical outcome in children with HL. We developed an in vitro model to further evaluate our findings, in this study.MethodsPeripheral blood mononuclear cells collected from healthy volunteers were used to generate effector cytoxic T lymphocytes (CTL), natural killer (NK) and CD4 positive T (CD4+T) cells by incubating with IL-15 and IL-2. Both a short-term (4-day) incubation and a longer incubation was used to generate lymphocytes with low-level and higher CD38/HLA-DR co-positive cells, respectively. CD3/CD8 co-positive CTL, NK (CD56-positive/CD3-negative) and CD4-positive T cells were isolated using MACS system. In addition to CD38/HLA-DR expression, isolated cells were evaluated for expression of CD279 (PD-1) and CD274 (PDL-1) by flow cytometry. Two HL cell lines, HDLM-2 (nodular sclerosis HL) and KMH-2 (mixed cellular HL) were used as targets in effector cell-mediated cytotoxicity experiments. Cells were incubated at various effector:target ratios, and HL cell death was measured with a flow cytometric cell-mediated cytotoxicity assay. The resuts were corrected for alloreactive cell elimination. Blocking antibodies against PD-1 and PDL-1 were used for cytoxicity experiments, using CTL and CD4-positive T cells as effector cells, as well.ResultsHigher CD38/HLA-DR co-expression was seen in CTL after longer incubation (day 11) with IL-2 and IL-15, while peak expression was reached earlier in NK cells (day 4). Both CTL and NK cells demonstrate cytotoxicity against HDLM-2 and KMH-2 cell lines. Cytotoxicity was increased, as evidenced by lower (lethal unit 20%) LU20 effector:target ratio levels, at day 11 of incubation (compared to day 5) for CTL for both cell lines: 0.5 (day 5) vs 0.29 (day 11) for KMH-2 (p=0.02) and 1.1 (day 5) vs. 0.4 (day 11) for HDLM-2 (p=0.15) cells. There was no difference between cytotoxicity with CTL compared to NK cells for either cell line at day 11.CTL, NK cells, and CD4-positive T cells all expressed both PD-1 and PDL-1, with no difference between cell types in percent positivity or mean channel intensity after cytokine incubation. PD-1 expression increased with incubation time in CTL, peaking at 50.5% on day 10, as opposed to NK cells, where it peaked at day 5-7. The co-expression of CD38/HLA-DR was higher in CTL compared to CD4-positive T cells (79% vs. 37% at day 7). PD-1 blockade did not inhibit CTL or CD4-positive T cell-mediated cytotoxicity in either cell line. This was also the case after PDL-1 blockade on tumor cells, indicating PD-1/PDL-1 pathway-independent HL cell elimination by CD38/HLA-DR co-positive CTL and CD4-positive T cells.DiscussionOur results indicate that higher CD38/HLA-DR co-expression in CTL was associated with superior elimination of HL cells in vitro supporting our recent in vivo findings. Induced co-expression of CD38/HLA-DR was higher in CTL compared with NK cells and reached a peak level earlier in NK cells. Increasing expression of PD-1 and PDL-1 was observed for all three effectors cells with longer incubation time. Interestingly, there was no change in CTL or CD4-positive T cell-mediated cytoxicity of HL cells following PD-1 and PDL-1 blockade in vitro. In conclusion, both CTL and NK cells are effective against HL cells. The anti-tumor activity of CTL correlated with increasing levels of CD38/HLA-DR expression in our experimental model. Cytotoxicity was enhanced despite increased expression of PD-1, and, therefore, appears to be independent of the PD-1/PDL-1 pathway, suggesting involvement of other operational mechanisms. This model could be useful in further elucidating the interactions between immune effectors and HL cells. DisclosuresNo relevant conflicts of interest to declare.

Novel mRNA Detection Assay for Quantifying CD30 Expression in Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma

Introduction:Innovations in detecting and measuring antigen expression is paramount in the current era of targeted therapy and check-point inhibitors. For instance, Brentuximab Vedotin (BV), one of two FDA-approved antibody-drug conjugates, employs an anti-CD30-monomethyl auristatin E (MMAE) immunoconjugate for the treatment of refractory Hodgkin lymphoma and anaplastic large cell lymphoma with positive CD30 expression. However, a lack of correlation between objective response rate and range of CD30 expression has been previously demonstrated, and the limitations of immunohistochemistry in detecting low levels of CD30 has been implicated. Moreover, there is interest in the study of the topology of checkpoint proteins such as programmed cell death-1 ligands (PD-L1) malignant cells and surrounding PD-1 expressing immune cells, which requires assay with high sensitivity, specificity, and accurate spatial localization.In this study, we implemented a novel CD30 mRNA detection method using the BaseScopeTM assay from Advanced Cell Diagnostics to enable detection of specific CD30mRNA isoforms that correspond the different CD30 protein segments within the neoplastic cells and tumor microenvironment immune cells. Using this method, the CD30 mRNA expression patterns in classical Hodgkin lymphoma (CHL) and anaplastic large cell lymphoma (ALCL) were evaluated.Methods:Nineteen CHL and 11 ALCL cases were selected from the Roswell Park Comprehensive Cancer Institute department of pathology archives. CD30 expression by immunohistochemistry (IHC) and CD30 mRNA isoform expression by BaseScope™ assay were measured on formalin fixed paraffin embedded ( FFPE ) tissue specimens. Four probe sets spanning the exon junctions for increased specificity were used to detect domains on the protein corresponding to signal peptide (SP), transmembrane portion (TMP), cytoplasmic portion (CP), and transmembrane and cytoplasmic portion (TMP &CP) of full length CD30 protein. The hybridization, amplification, and staining of probes are based on the manufacturer's protocol. The BaseScope™ assay probe signal is detected in the form of red dots, which are localized to the neoplastic cells and tumor microenvironment immune cells. Semi-quantitative analysis of both positive and negative signals using 100X magnification was performed on 100 neoplastic cells for each probe for each case, by two hematopathologists (VN, JW). For tumor microenvironment immune cells, an average number of positive and negative cells were obtained in five 100X magnification fields.Results:In both CHL and ALCL, 100% of the neoplastic cells were positive for CD30 protein by immunohistochemistry. The table below illustrates relative expression of CD30 mRNA isoforms in CHL and ALCL neoplastic cells. Expression of CD30 mRNA in CHL the tumor microenvironment cells is also tabulated.Conclusions:Detection of CD30 mRNA isoforms using BaseScope™ assay offers a reliable and reproducible signal detection platform, as an alternative to CD30 protein expression by immunohistochemistry. The BaseScope™ assay provides a high-contrast low noise signal for easy visualization, accurate quantification, and precise positive signal localization. Different CD30 isoform patterns and signal frequency were observed within the neoplastic cells and immune cells in the tumor microenvironment. The CD30 mRNA isoform corresponding to transmembrane domain was a dominant expresser in both CHL and ALCL neoplastic cells and bystander cells in CHL. This novel mRNA-based method can potentially provide a more sensitive method for antigen expression detection and be used in new research studies. DisclosuresNo relevant conflicts of interest to declare.

SLAMF7 in Primary Effusion Lymphoma, Target for Individualized Therapy?

Primary effusion lymphoma (PEL) is a rare, aggressive form of B-cell lymphoma. With a median survival time of around six months the prognosis for PEL patients is poor. Therefore, there is a medical need for novel therapeutic strategies.We performed expression array analysis to find potential targets for antibody-based therapy. Unsupervised clustering analysis revealed that PEL cell lines grouped separate from cell lines derived from other B-non Hodgkin lymphoma (B-NHL) entities. Notably, PEL and Hodgkin Lymphoma (HL) cell lines clustered on one arm, separate from all cell lines representing less-differentiated B-NHL variants.PEL and HL cell lines were characterized by a set of common up- and downregulated genes. Typical for PEL and HL was the expression of CCND2 and the absence of Brutons tyrosine kinase and of B-cell markers including CD19, CD20, CD79A and CD79B.Highly expressed in PEL - but not in HL - were CD138, IL-10, SLAMF7 and PRDM1. PRDM1/BLIMP1 is a master regulator of terminal B-cell differentiation. Originally described as repressor, BLIMP1 can also enhance transcription of SLAMF7 in multiple myeloma (MM) and of IL-10 in type 1 regulatory T-cells. Thus, coexpression of the three genes suggests a causal relationship between transcriptionally active PRDM1/BLIMP1 and its targets SLAMF7 and IL-10 also in PEL.Expression of SLAMF7 in PEL is especially noteworthy because a monoclonal antibody targeting SLAMF7 (elotuzumab) has been approved for treatment of patients with MM. We observed that SLAMF7 is comparably expressed in PEL and in MM cell lines. If the results on cell lines can be translated to primary PEL, i.e. if PEL tumor cells express SLAMF7, the patients might benefit from an antibody-based targeted therapy against this antigen. DisclosuresNo relevant conflicts of interest to declare.

Refractory and Relapse Predictive Biomarkers in Hodgkin Lymphoma and Potential Directed-Therapy Targets

Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Nevertheless and independently of the lymphoma stage, the real challenge when treating these patients, is the refractory and relapsed disease. There is no molecular biomarker to identify patients that would be non-responsive to conventional treatment or that would relapse. Furthermore, rescue chemotherapy schemes for refractory and relapsed patients, associate with acute and late toxicity high risk. This highlights the need to deeper understand the HL molecular biology and the screening for predictive biomarkers as well as potential therapeutic directed-targets.We have previously reported that HL relies on the alternative NFkB pathway, mediated by RelB and NIK, to survive. Depletion of either RelB or NIK by shRNAs or pharmacological NIK inhibitors induce HL cell death. ChIP-Seq analysis uncovered RelB target genes showing RelB bound to BCL2 promoter. A significant downregulation of BCL2 mRNA and protein levels, following RelB or NIK knockdown was observed, indicating that RelB regulates BCL2 expression in human HL cell lines.Our molecular studies suggested that NFkB alternative pathway constitutive signaling could at least partially explain the non-responding HL cases. We aimed to analyze whether mediators of this pathway could be useful as predictive biomarkers and would represent potential targetable factors in both refractory and relapsed patients.We analyzed NIK and BCL2 citoplasm expression in Hodgkin Reed-Sternberg cells (HRS) in lymphatic node biopsies of 96 patients by inmunohistochemistry [50 female Md age and (range) 59 (6-82), 46 male 42 (9-78)]. The univariate analysis showed no correlation between NIK or BCL2 expression and the prognosis clinical and pathological parameters, neither the molecular markers routinely assayed. A positive correlation was found between NIK and BCL2 expression (p=0.01). NIK and BCL2 correlated with lack of response to conventional therapy and both early and late disease progression.The analysis of survival, applying the Kaplan-Meier Curves, showed > 60% NIK positive HRS cells associated with shorter Disease Free Survival (DFS) [Log Rank Test (p=0.000)] and predicted overall survival (OS) as well [Log Rank Test (p=0.01)]. Furthermore, > 60% BCL2 positive HRS cells correlated with poor prognosis in terms of OS [Log Rank Test (p=0.002)]. The statistical significance was maintained in the multivariate analysis [Cox Regression and Logistic Regression (p=0.001)].NIK and BCL2 performed successfully as useful predictive markers to identify refractory or risk of relapse HL patients at diagnosis. They represent attractive molecules to further analyse their potential as directed-therapy targets, since we have already reported that HL is sensitive to NIK inhibitors and BCL2 blockers have already been approved for clinical use in other hematological pathologies. DisclosuresZerga:Bristol Myers Squibb: Other: Conference fees; Janssen: Other: Conference fees; Roche: Other: Conference fees; Takeda: Other: Conference fees.