Microenvironment Of Classical Hodgkin Lymphoma Research Articles

Prognostic Markers within the Tumour Microenvironment in Classical Hodgkin Lymphoma.

Classical Hodgkin lymphoma (cHL) accounts for 0.4% of all new cancer cases globally. Despite high cure rates with standard treatment, approximately 15% of patients still experience relapsed or refractory (RR) disease, and many of these eventually die from lymphoma-related causes. Exciting new targeted agents such as anti-PD-1 agents and brentuximab vedotin have changed the therapeutic paradigm beyond chemotherapy and radiotherapy alone. Advances in understanding of the molecular biology are providing insights in the context of novel therapies. The signature histology of cHL requires the presence of scant malignant Hodgkin Reed-Sternberg cells (HRSCs) surrounded by a complex immune-rich tumour microenvironment (TME). The TME cellular composition strongly influences outcomes, yet knowledge of the precise characteristics of TME cells and their interactions with HRSCs is evolving. Novel high-throughput technologies and single-cell sequencing allow deeper analyses of the TME and mechanisms elicited by HRSCs to propagate growth and avoid immune response. In this review, we explore the evolution of knowledge on the prognostic role of immune cells within the TME and provide an up-to-date overview of emerging prognostic data on cHL from new technologies that are starting to unwind the complexity of the cHL TME and provide translational insights into how to improve therapy in the clinic.

The Association of Plasma Cell Density of the Microenvironment with Classical Hodgkin Lymphoma Stage

Background
 Plasma cells are important prognostic factors in various malignancies including Classical Hodgkin lymphoma (CHL). In the CHL microenvironment, plasma cells are the main prognostic factors, but research of relationship between plasma cell density in the microenvironment and CHL stage is still very limited. This study aims to determine the relationship between plasma cell density in the microenvironment and the CHL stage.
 
 Methods
 A case series procedure was conducted. The samples were paraffin blocks of CHL patients at Anatomic Pathology Department of Faculty of Medicine Universitas Sriwijaya/Mohammad Hoesin General Hospital. Among 47 cases of CHL diagnosed from 1st Januari 2018 to 30th November 2022, only 30 cases were included. Immunohistochemistry was performed by using specific antibody of plasma cells (anti-CD138 antibody). The number of plasma cells in each sample was calculated based on the expression of CD138 in the cell membrane and cytoplasm with weak-strong intensity. Then the cut-off point value was determined based on the ROC curve to classify the high and low plasma cell densities. The density of plasma cells was correlated to clinicopathological characteristics including age, sex, tumor location, CHL subtypes and stage. Statistical analysis was performed using Spearman test of SPSS with significant consideration if p<0.05.
 
 Results
 A high proportion (>10,9) of tumour-associated plasma cells of the tumor microenvironment was found in CHL advanced stage. Spearman test resulted in a significant correlation between density of plasma cell with advanced stage classical Hodgkin lymphoma (p=0.000).
 
 Conclusions
 There is significant relationship between high density of plasma cells with CHL stage.

Abstract 2038: Epstein-Barr virus shapes the tumor microenvironment in classical Hodgkin lymphoma

Abstract The histopathologic appearance of classical Hodgkin lymphoma (cHL) is characterized by rare multinucleated malignant Hodgkin Reed-Sternberg (HRS) cells within a rich inflammatory tumor microenvironment (TME). A subset of cHL cases are associated with Epstein-Barr virus (EBV) infection, which is considered to play a part in cHL pathogenesis. Since EBV+ and EBV- cHL exhibit differing mechanisms of pathogenesis, we hypothesized that the composition and spatial architecture of the TME in EBV-associated cHL differs from that of EBV- cHL, with implications for stratifying patients for specific treatment regimens based on EBV positivity. Here, we designed a multimodal approach using spatially resolved multiplexed protein profiling, flow cytometry, immunohistochemistry, and transcriptomics to profile EBV positive and EBV negative cHL tumors. We measured over 27 million single cells and 1,000 unique proteomic cell states derived using 29 proteins in addition to the transcriptome in 43 patient tumors. We uncovered significant differences between EBV+ and EBV- cHL in relative abundance of specific immune cell types, immune activation state, and spatial organization of the HRS cell neighborhoods. We present a multidimensional characterization of cHL in relation to EBV with implications for therapeutic stratification of patients with virus-associated cHL. Citation Format: Maryam Pourmaleki, Caitlin J. Jones, Sabrina D. Mellinghoff, Brian Greenstein, Priyadarshini Kumar, Mikhail Roshal, Travis J. Hollmann, Nicholas D. Socci, Ahmet Dogan, Ingo K. Mellinghoff. Epstein-Barr virus shapes the tumor microenvironment in classical Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2038.

P038: HLA expression patterns of Hodgkin-Reed-Sternberg cells shape a spatially arranged tumor microenvironment in classical Hodgkin lymphoma

P038: HLA expression patterns of Hodgkin-Reed-Sternberg cells shape a spatially arranged tumor microenvironment in classical Hodgkin lymphoma

FOXP3+/CD68+ ratio within the tumor microenvironment may serve as a potential prognostic factor in classical Hodgkin lymphoma

Classical Hodgkin lymphoma (CHL) is characterized by extensive inflammatory immune cells, which predict the disease prognosis. Therefore, this study aimed to explore the significance of different tumor-infiltrated immune cells and subpopulation ratios observed in the tumor microenvironment of CHL, particularly relating to the disease's prognosis-focusing on overall survival (OS) and event-free survival (EFS). Utilizing immunohistochemistry, the quantification and exploration of selected immune cells' subsets, including CD3+, CD4+, CD8+, FOXP3+, CD20+, and CD68+ were conducted on 102 histological samples with primary CHL. Eosinophils were pathologically assessed. Besides, we determined the ratios between different tumor-infiltrated immune cells for each patient. Kaplan-Meier method and Cox regression modeling were used for survival analysis. We demonstrated that among all ratios and immune cells individually, only a higher FOXP3+/CD68+ ratio (≥1.36 cutoff) displayed a tendency towards a favorable OS (p = 0.057, HR = 0.43 [0.18–1.02]) and EFS (p = 0.067, HR = 0.44 [0.18–1.06]) using Cox regression modeling. Moreover, the Kaplan-Meier method showed an association of a higher FOXP3+/CD68+ ratio with a longer 5-years OS (p = 0.037) and a tendency to a better EFS (p = 0.051); however, neither the combined FOXP3+ and CD68+ nor FOXP3+ or CD68+ separately was correlated to the CHL survival. Together, these results demonstrated that the FOXP3+/CD68+ ratio could predict the outcomes of CHL, providing more informative significance than FOXP3+ and CD68+ combined or FOXP3+ and CD68+ individually and might be a potential indicator of risk stratification, which has an important value for guiding the clinical treatment.

PDL1 shapes the classical Hodgkin lymphoma microenvironment without inducing T-cell exhaustion.

Classical Hodgkin lymphoma (CHL) is unusually sensitive to PD1 inhibition and PDL1 is highly expressed on CHL cells and in the tumor microenvironment. This could be interpreted as evidence of exhaustion, but paradoxically, PD1+ lymphocyte infiltration does not predict response to PD1 inhibitors and no increase in cytotoxic markers is seen after PD1 therapy as might be expected with reversal of exhaustion. In contrast to PD1, elevated PDL1 does predict response to PD1 inhibitors and recent data associate both retained CHL MHC-II expression and increased T helper (TH) T-cell receptor diversity with response, suggesting a connection to the TH compartment. We performed a phenotypic, spatial and functional assessment of T-cell exhaustion in CHL and found co-expression of an exhaustion marker and lower PD1 expression in CHL than in reactive nodes whereas the proliferative and cytokine production capacity were similar in CHL and the reactive nodes. We found no correlation between PDL1 expression and exhaustion signatures. Instead, we identified a strong association between PDL1 expression and CHL MHC-II expression, TH recruitment, and enrichment of TH1 regulatory cells. These data suggest that a dominant effect of PDL1 expression in CHL may be TH engagement and promotion of a regulatory microenvironment rather than maintenance of exhaustion.

832MO Immune microenvironment in classical Hodgkin lymphoma: Composition and dynamics in patients with relapsed/refractory disease

Classical Hodgkin lymphoma (cHL) is characterized by the unique tumor microenvironment (TME) rich in immune cells. One of the explanations for the depressed antitumor response in cHL is hyperexpression of inhibitory immune checkpoint receptors on T cells as well as increased proportion of M2 macrophages (M2) in TME. The presence of high number of M2 has been demonstrated to worsen the prognosis of patients treated with standard chemotherapy, while data regarding the prognostic value of TME features in patients treated with PD-1 inhibitors is limited. This retrospective study included 61 primary tumor samples from pts with r/r HL, treated with Nivo, and 15 repeated samples obtained during relapse or progression of disease after immunotherapy. The PFS and the best overall response (BOR) depending on the proportion of cells positive for CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, c-MAF in the TME in primary and sequential biopsies were analyzed. Immunohistochemical staining was performed with Bond III (Leica Biosystems). The antibody cocktail (CD163/c-MAF) was used for identification of M2. The response to Nivo was assessed by PET-CT with LYRIC. ROC analysis established 8,5% cut-off value for CD163 expression and 11,5% for CD68 expression, dividing patients with high and low number of positive cells. In the CD163low group, 4-year PFS was 24,1% (95% CI 9,3%-42,6%) with a median PFS of 11,6 months (95% CI 7,2-24,8); in the CD163high group - 42,6% (95% CI 20,2%-63,4%) with a median of 24,8 months (95% CI 20,4 - NA), p = 0,0086. Complete remission (CR) achievement as BOR to Nivo was associated with a lower level of M2 in primary biopsies (p = 0.047). In the analysis of sequential samples, an increase of PD- 1+ and LAG-3+ T-cells and depletion of CD68+ and CD163+ cells in repeated biopsies was observed (median PD-1–1.0% vs 7.0%; LAG-3–5.0% vs 8.0%; CD68–10.0% vs 7.0%; CD163–9.0% vs 3.0%, p<0,05). We found that low number of CD163+ cells in primary samples were associated with inferior PFS during Nivo therapy, while a lower level of M2 was correlated with achievement of CR. In repeated biopsies after Nivo therapy the cell profile in the TME changed: number of PD-1+ and Lag-3+ T-cells increased and number of CD68+ and CD163+ macrophages decreased.

Revisiting IL-6 expression in the tumor microenvironment of classical Hodgkin lymphoma

Interleukin-6 (IL-6) can induce therapeutic resistance for several cancer agents currently used to treat classical Hodgkin lymphoma (cHL). We aimed to investigate whether the presence of IL-6+ leukocytes and IL-6+ Hodgkin-Reed-Sternberg (HRS) cells in the tumor microenvironment (TME) was associated with adverse survival outcomes, expression of other immune markers, and serum IL-6 levels. We used a contemporarily treated cohort (n = 136), with a median follow-up of 13.8 years (range, 0.59-15.9 years). We performed immunohistochemistry with an IL-6 antibody on tissue microarrays from diagnostic biopsies of cHL patients. Patients with IL-6+ leukocytes ≥1% (n = 54 of 136) had inferior event-free survival (hazard ratio [HR] = 3.58; 95% confidence interval [CI], 1.80-7.15) and overall survival (HR = 6.71; 95% CI, 2.51-17.99). The adverse survival was maintained in multivariate Cox regression and propensity score-matched analyses, adjusting for well-known poor-prognostic covariates. The presence of IL-6+ HRS cells and high serum IL-6 levels were not associated with survival. IL-6+ leukocytes correlated with increased proportions of IL-6+ HRS cells (P < .01), CD138+ plasma cells (P < .01), CD68+ macrophages (P = .02), and tryptase-positive mast cells (P < .01). IL-6+ HRS cells correlated with increased proportions of CD68+ macrophages (P = .03), programmed death-ligand 1–positive (PD-L1+) leukocytes (P = .04), and PD-L1+ HRS cells (P < .01). Serum-IL-6 lacked correlation with IL-6 expression in the TME. This is the first study highlighting the adverse prognostic impact of IL-6+ leukocytes in the TME in a cohort of contemporarily treated adult patients with cHL.

Expression of the Immune Checkpoint Regulators LAG-3 and TIM-3 in Classical Hodgkin Lymphoma

IntroductionThe role of the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis is well established in classical Hodgkin lymphoma (HL), where PD-1 blockade demonstrated spectacular efficacy in relapsed/refractory disease. However, little is known about the frequency and cellular distribution of other immune checkpoints in HL samples. Patients and MethodsUsing immunohistochemistry, we investigated, along with PD-L1 and PD-1, the expression of lymphocyte-activation gene 3 (LAG-3) and T-cell immunoglobulin and mucin-domain containing 3 (TIM-3) in 57 biopsy samples of patients with classical HL. ResultsHodgkin and Reed/Sternberg (HRS) cells were strongly positive for PD-L1 in nearly all cases. HRS cells were TIM-3 positive in 36% of samples, whereas LAG-3 was rarely expressed (5.2%). In the microenvironment, PD-1, LAG-3, and TIM-3 were expressed by ≥ 5% of cells in 65%, 98%, and 96% of cases, respectively. T-cell rosettes surrounding HRS cells consisted of CD4+ FoxP3− helper T cells expressing both PD-1 and LAG-3, with a variable expression of TIM-3. ConclusionThis study demonstrates for the first time that LAG-3 and TIM-3 are nearly always expressed in the microenvironment of classical HL. This may constitute the basis for targeting LAG-3 or TIM-3 in combination with anti–PD-1 antibodies in the treatment of relapsed/refractory HL.

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

Identification of LAG3+ T Cell Populations in the Tumor Microenvironment of Classical Hodgkin Lymphoma and B-Cell Non-Hodgkin Lymphoma

An Integrative Unbiased Global Analysis of the Metabolic Adaptive Response to Tryptophan Deprivation in Classical Hodgkin Lymphoma

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

Abstract IA04: The immune cell microenvironment of classical Hodgkin lymphoma by single-cell analysis

Abstract Background: Over the past decade knowledge about the mechanisms by which the neoplastic Hodgkin and Reed-Sternberg (HRS) cells recruit and manipulate the extensive tumor microenvironment (TME) of classical Hodgkin lymphoma (cHL) has significantly expanded. While some findings support the concept that characteristics of the host immune system contribute to the composition of the TME, there is increasing evidence that somatically acquired genetic alterations and phenotypes of HRS cells influence disease evolution and underlie specific TME interactions. Integrating these concepts, the cHL TME might be the reflection of a symbiotic ecosystem containing the malignant cells and host immune cells. Dissection of this symbiotic relationship at single-cell resolution will be critical for a better understanding of cHL pathogenesis with relevance for improved therapeutic targeting. Knowledge Gained: We present single-cell transcriptome sequencing data derived from cell suspensions of 22 Hodgkin lymphoma patients (representative of the nodular sclerosis and mixed cellularity subtype) that confirmed the previously identified predominance of Th1 polarized T regulatory cells in the TME. In comparison to 5 reactive lymph node samples, we describe a highly enriched, HL-specific T-cell subset that is characterized by high LAG3 expression and an immunosuppressive cytokine profile, consistent with type 1 regulatory (Tr1) T cells. Analyzing co-expression of inhibitory T-cell makers on the single-cell level, LAG3 expression occurred mostly in a mutually exclusive pattern to the canonical Treg marker FOXP3. Furthermore, using imaging mass cytometry and multicolor immunohistochemistry, we revealed a spatial pattern of rosetting LAG3+ cells in the direct vicinity of MHC-II negative HRS cells, suggesting differential microenvironment architecture dependent on malignant cell phenotypes. Comparative analysis of LAG3 expression across a spectrum of B-cell lymphomas confirmed the high abundance of these cells in cHL, but also suggest that LAG3+ CD4+ and LAG3+ CD8+ T-cell subsets play a role in the related entity of primary mediastinal large B-cell lymphoma (PMBCL) and diffuse large B-cell lymphoma (DLBCL). Expanding our studies to the lymphocyte-rich (LR) subtype of cHL, we also revealed subtype-specific differences in background B cell and T follicular helper (TFH) cell content, suggesting an important role of the CXCR5-CXCL13 axis in cHL. Significance: Characterization of the TME using multiparametric single-cell technologies, including multicolor immunohistochemistry, flow-based mass cytometry, imaging mass cytometry, single-cell RNAseq, and proximity ligation assays, has provided unprecedented insight into HL biology. These studies fuel hope for accelerated development of immunotherapies targeting the TME and predictive biomarker development that might guide the appropriate selection of checkpoint inhibitors. Citation Format: Christian Steidl. The immune cell microenvironment of classical Hodgkin lymphoma by single-cell analysis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA04.

Checkpoint inhibitors in Hodgkin lymphoma

Checkpoint blockade contributes to the immunosuppressive microenvironment in classical Hodgkin lymphoma (cHL) and in particular the interaction of Hodgkin cells and macrophages with T‑cells and natural killer cells via programmed cell death1 (PD-1) and programmed cell death1 ligand1 (PD-L1). The aim of this article is the evaluation the role and potential of checkpoint blockade in cHL as compared with the results of standard chemo- and radiotherapy. We analyzed preclinical and clinical data from phaseI and phaseII studies with checkpoint blockade in cHL. In 60-70% of patients with chemotherapy-refractory cHL, PD‑1 blockade results in responses. Overall survival is excellent and asmall number of patients achieve persistent response. Thus, the use of anti-PD‑1 monoclonal antibodies has become an important treatment approach in relapsed cHL in line with the label. The results of first-line therapy are still preliminary; initial phaseII studies using nivolumab in combination with doxorubicin (=adriamycin), vinblastin and dacarbazin (AVD) in early unfavorable or advanced stages showed response rates of up to 90%. Thus, implementing immunomodulatory approaches using PD 1‑blockade have resulted in asignificant reduction of chemotherapy. This might represent aparadigm shift in the therapy of cHL.

PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma

Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents &amp; Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents &amp; Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Beyond Exhaustion: The PDL1-PD1 Axis Shapes the Classical Hodgkin Lymphoma Microenvironment

Background: PD1 inhibitor (PD1i) efficacy is taken as evidence of T cell exhaustion in Classical Hodgkin Lymphoma (CHL), however, direct evidence for exhaustion is limited. PD1 expression does not predict PD1i response in CHL. Additionally, PDL1 and PD1 expression are not correlated and are independently prognostic. This suggests a more complex picture than PD1 expression simply reflecting exhaustion induced by PDL1. Aim: To investigate PD1/PDL1 interaction in CHL Methods: 149 CHL cases were compared to 27 reactive lymph node controls (RLN) by immunohistochemistry (IHC). PD1 expression was validated with two clones. Multiplex IHC (mIHC) was performed in 47 CHL and 27 RLN by stripping, reprobing and image alignment. Imaging Mass Cytometry (IMC) was analysed by t-SNE clustering and spatial point pattern modelling (SPPM). SPPM is a powerful technique adopted from other disciplines and its use in multiplex image analysis is novel. Flow-based CFSE proliferation and cytokine assays were performed in 15 CHL and 5 RLN single cell suspensions (SCS) with CD3/28 and PMA/Ionomycin stimulation. Co-cultures were with KMH2 and L428 cell lines and healthy donor naïve T helper cells (TH). Results: We evaluated PDL1 expression by IHC and found that PDL1hi CHL cells were surrounded by PDL1+ macrophages (Mφ), consistent with the literature. Mφ PDL1 intensity correlated inversely with distance from CHL cells consistent with induction by a CHL-secreted factor (p &amp;lt;0.001), a finding we validated in vitro (p &amp;lt;0.001). Deep phenotyping using IMC identified CD14+CD16-CD163-PDL1+-, CD14-CD68+CD163-PDL1+ and CD14+CD16+CD163+PDL1- myeloid subsets by t-SNE. SPPM revealed that tumor and vessel location predicted their distribution with subsets one and two localising to tumor whilst subset three localised to vessel. Our data supports the hypothesis that CHL recruits and induces a PDL1+ myeloid environment. We next sought evidence of T cell exhaustion. PD1 expression by IHC was lower in CHL than RLN (p&amp;lt;0.001). Cells co-expressing PD1 with TIM3 or LAG3 were significantly reduced in CHL compared to RLN by mIHC. Cells co-expressing PD1 with TBET or EOMES were reduced in or showed no significant difference between CHL and RLN. No correlation was seen between PD1 and PDL1 expression. We performed functional validation, evaluating proliferative and IL2/IFNγ production capacity, and identified no significant difference between CHL and RLN SCS with or without the addition of Pembrolizumab or isotype control. This constitutes a more detailed assessment of exhaustion with higher case numbers than publications to date. Our data does not support the presence of an exhausted T population above that seen in reactive controls and finds no relationship between PD1 and PDL1 expression. The absence of exhaustion does not exclude a role for the PD1-PDL1 axis, which regulates multiple facets of T cell homeostasis including motility, differentiation (skewing towards T regulatory cells (TReg)), and TReg effector functions. Many of these are characterised by transient PD1 expression unlike the sustained expression seen in exhaustion. In contrast to PD1, CHL PDL1 and MHC class 2 expression do predict PD1i response. CHL MHC2 expression suggests antigen presentation and a role for TH cells. This led us to examine connections between PDL1 and the TH compartment. Co-culture of CHL cell lines with naïve TH led to enrichment of CD25hiCD127loFOXP3+ TReg which upregulated PD1 compared to TReg from paired CD3/28 controls (p&amp;lt;0.001). FOXP3+ cells were enriched by IHC compared to RLN (p&amp;lt;0.001). Unlike PD1, FOXP3 associated with CHL MHC2 expression (p=0.012), which in turn associated with PDL1 expression (p=0.02). SPPM of TC and TH subsets revealed that PDL1 expression predicted the location of FOXP3+ TReg and negatively predicted the location of TC and TH17. PDL1 was a stronger predictor than CHL cell density. These data support a role for PDL1 in shaping T differentiation and trafficking irrespective of observed PD1 expression. Conclusion: We find that CHL cells are PDL1hi and induce a PDL1+ myeloid environment, but detect little evidence of T exhaustion. Instead we find that PDL1 expression shapes T (particularly TReg) differentiation and trafficking. Our data challenges the narrative that PD1i in CHL act by reversing exhaustion and highlights novel mechanisms with the potential to provide insight into CHL biology and inform the hunt for biomarkers of response. Figure Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

M1-like macrophage polarization prevails in young children with classic Hodgkin Lymphoma from Argentina

The microenvironment in classical Hodgkin lymphoma (cHL) comprises a mixture of different types of cells, which are responsible for lymphoma pathogenesis and progression. Even though microenvironment composition in adult cHL has been largely studied, only few groups studied pediatric cHL, in which both Epstein Barr virus (EBV) infection and age may display a role in their pathogenesis. Furthermore, our group described that EBV is significantly associated with cHL in Argentina in patients under the age of 10 years old. For that reason, our aim was to describe the microenvironment composition in 46 pediatric cHL patients. M1-like polarization status prevailed in the whole series independently of EBV association. On the other hand, in children older than 10 years, a tolerogenic environment illustrated by higher FOXP3 expression was proved, accompanied by a macrophage polarization status towards M2. In contrast, in children younger than 10 years, M1-like was prevalent, along with an increase in cytotoxic GrB+ cells. This study supports the notion that pediatric cHL exhibits a particular tumor microenvironment composition.

Programmed cell death protein-1 (PD1) expression in the microenvironment of classical Hodgkin lymphoma is similar between favorable and adverse outcome and does not enrich over serial relapses with conventional chemotherapy

Programmed cell death protein-1 (PD1) expression in the microenvironment of classical Hodgkin lymphoma is similar between favorable and adverse outcome and does not enrich over serial relapses with conventional chemotherapy

Composition of Classical Hodgkin Lymphoma Microenvironment Is Associated with Clinical Outcome

Background.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 microenvironmental composition in clinical outcome of cHL.Methods.79 patients (pts) with cHL were included in this study (median age: 41, range: 18-65 years; males: 26, females: 53).Patients were treated with ABVD or BEACOPP (14/esc) and radiation therapy. CR/PR was achieved in 86.1% of patients. 13.9% of pts had relapse or disease progression during the therapy.Using IHC and qPCR techniques, we analyzed lymph-node biopsy obtained from patients during diagnostic to determine cellular and cytokine signature (CD3, CD19, CD11b, PD-L1, PD-L2, IDO, TGF-β, IL-13) that correlates with cHL treatment outcome.Results. Expression level of tested markers was heterogeneous across the samples and did not correlate with histological variant or stage of cHL. For most of cytokines as well as immunomodulatory molecules we did not notice significant independent effect on cHL clinical outcome, except IL-13, which appeared to be a strong and independent marker of cHL prognosis. High expression of IL-13 was associated with lower EFS and OS rates comparing to IL-13 low/negative group (p<0.05).We found that the presence of myeloid CD11b+ cells in cHL microenvironment correlates significantly with shortened EFS rate (p<0.05). Multivariate analysis has shown that the negative impact of presence of myeloid cells increases gradually when combined with elevated expression of one or more following markers - PD-L1, IDO or TGF-β (p<0.05). At the same time, we did not find any significant associations between cHL outcome and B- or T-cells populationsConclusion.In summary, our study showed the presence of CD11b+ myeloid cells together with increased expression of checkpoints molecules and cytokines (PDL1, IDO, TGF-β and IL-13)in diagnostic lymph-node samples is associated with cHL clinical outcome. DisclosuresNo relevant conflicts of interest to declare.

The Treg/Th17 Axis Is Skewed in Classical Hodgkin Lymphoma By PDL1+Ve but Not PDL1-Ve Lymphoma Cells and By Lymphoma MHC Class 2 Expression.

Introduction: Classical Hodgkin Lymphoma (CHL) is responsive to Programmed Death 1 inhibition (PD1i). Recognition that 9p24 amplifications promote PDL1 upregulation in lymphoma cells provides insight into CHL's particular sensitivity to PD1 inhibitors, but their mechanism of action remains unclear. Although it is widely believed that PD1i act by reversing CD8 T cell exhaustion, neither CD8 infiltration, PD1 expression nor lymphoma MHC class 1 (MHC1) expression predict PD1i response in CHL. Additionally, while most CHL cells adopt CD8 evasion mechanisms such as MHC1 loss, MHC2 expression is frequently retained suggesting a vulnerability to CD4-mediated cytotoxicity. CD4 cells dominate the CHL microenvironment and recent data demonstrates that MHC2 alongside PDL1 expression on CHL cells predict PD1i response suggesting an important role for CD4 cells in PD1i action.Aim: To evaluate the impact of the PDL1 axis and MHC2 expression on the CD4 compartment in CHL and their implications for PD1i activity.Methods: We evaluated 146 CHL patients by immunohistochemistry (IHC), with comparison to 24 reactive lymph node (RLN) controls. We designed a novel spatial analysis method utilising intra-tumor cell heterogeneity to test the influence of lymphoma PDL1 expression upon the T cell infiltrate. By combining IHC dual staining with stripping and reprobing we stained sections for CD30/PDL1/CD3 and serial sections on either side for GATA3/CD8, FoxP3/CD8, RORgT/CD8 and Tbet/CD8. Images were virtually overlaid and spatial analysis was performed using Visiopharm software. Distances from T cell subtypes were measured to either CD30+PDL1- CHL cells, CD30+PDL1+ CHL cells or CD30-PDL1+ microenvironmental cells. Skewing was tested statistically by comparison to parent cell distribution (e.g. CD8+ to CD3+). Spatial analysis was performed in 10 blindly selected cases.Results/Discussion: Despite high PDL1 expression our data showed limited evidence for an exhausted PD1+CD8 population; CD8 tumor-infiltrating lymphocytes (TIL) were frequent, but PD1+TIL were rare (median 33% vs 0.08% nucleated cells respectively). No correlation was seen between PD1+TIL and CD8+TIL or PDL1. Stripping and reprobing of PD1+ IHC sections demonstrated that PD1+ cells were CD8 negative. This led us to consider alternative mechanisms of action for PD1i.Since PD1i has a profound impact on naïve CD4 differentiation and influences the type of effector generated, we next quantified CD4 effector subtypes. We found that Tbet+TIL (Th1, antitumor) and FoxP3+TIL (Treg, protumor) were enriched in CHL, whereas RORgT+TIL (Th17, antitumor) were suppressed compared to RLN (p=<0.0001, 0.0004 and <0.0001 respectively). No significant difference was seen for GATA3+TIL (Th2, protumor). We further evaluated Treg and Th17 due to their shared developmental pathway which is known to be influenced by PDL1. FoxP3:Th17 ratios were heavily skewed towards Treg in CHL compared to RLN (p=<0.0001). Spatial analysis demonstrated enrichment of FoxP3+TIL and a paucity of RORgT+TIL in proximity to CD30+ lymphoma cells and with greater enrichment around PDL1+ lymphoma cells than PDL1- lymphoma cells. This supports a hypothesis of skewing of the Treg/Th17 axis by the lymphoma cell by the PD1-PDL1 axis. Finally, we evaluated the relationship between effector subtypes and CHL MHC2 expression across our cohort and found a positive association between FoxP3+TIL numbers and lymphoma cell MHC2 expression (p=0.0014).Conclusion: The skewing of Th17/Treg via the PD1-PDL1 axis and positive association between Treg numbers and lymphoma MHC2 expression are consistent with CHL cells promoting Treg differentiation. This is supported in the literature by reports of MHC2-dependent induction of Treg by CHL cell lines. Paradoxically, Treg are associated with improved response to therapy despite evidence that they are induced by CHL cells. It is therefore hypothesised that Treg directly suppress lymphoma cell expansion. However, these data support our alternative hypothesis that PDL1 and MHC2 expression contribute to CHL cells establishing a Treg-rich and Th17-poor immunosuppressive microenvironment. Intervention with PD1i therapy or chemotherapy causing immunogenic cell death then shifts the infiltrate to a less suppressive environment leaving MHC2+ cells vulnerable to CD4-mediated cytotoxicity. We are currently modelling this in vitro. [Display omitted] DisclosuresGribben:Abbvie: Honoraria; Medical Research Council: Research Funding; Novartis: Honoraria; Cancer Research UK: Research Funding; NIH: Research Funding; Acerta Pharma: Honoraria, Research Funding; Pharmacyclics: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Honoraria; TG Therapeutics: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Wellcome Trust: Research Funding; Kite: Honoraria.

CXCR5 and ICOS expression identifies a CD8 T-cell subset with TFH features in Hodgkin lymphomas

AbstractA better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+). These cells shared phenotypic features with follicular helper T (TFH) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40. They had deficient cytotoxicity, low interferon-γ secretion, and common functional properties with intratumoral CD4+ TFH cells, such as production of interleukin-4 (IL-4), IL-21, CXCL13, and capacity to sustain B cells. Gene profiling analysis showed a significant similarity between the signatures of CD8CXCR5+ICOS+ T cells and CD4+ TFH cells. Benign lymphadenitis tissues (n = 8) were devoid of CD8CXCR5+ICOS+ cells. Among the 35 B-cell lymphoma tissues analyzed, including follicular lymphomas (n = 13), diffuse large cell lymphomas (n = 12), marginal zone lymphomas (MZLs; n = 3), mantle cell lymphomas (n = 3), and chronic lymphocytic leukemias (n = 4), only 1 MZL sample contained CD8CXCR5+ICOS+ cells. Lymphoma tumors with CD8CXCR5+ICOS+ cells shared common histopathological features including residual germinal centers, and contained high amounts of activated CD8CXCR5−ICOS+ cells. These data demonstrate a CD8 T-cell differentiation pathway leading to the acquisition of some TFH similarities. They suggest a particular immunoediting process with global CD8 activation acting mainly, but not exclusively, in HL tumors.