Genetic Variants of Interleukin 10 (IL10) are Associated with Clinical Outcome and Tumor Microenvironment Composition of Classical Hodgkin Lymphoma (cHL)

Background.Tumor tissue in classical Hodgkin Lymphoma (cHL) contains 1-10% malignant Hodgkin/Reed-Sternberg cells and a significant number of immune cells in the tumor microenvironment that are characterized by expression of inhibitory molecules (PD-1, CTLA-4, LAG-3, TIM-3, TIGIT). Despite overall effectiveness of anti-PD-1 treatment many patients still have relapsed or refractory (r/r) disease, therefore the search for predictive/prognostic biomarkers in patients on immunotherapy is highly demanded. Materials and methods.The study included 39 primary tumor specimens from patients with r/r cHL obtained before starting the treatment with nivolumab (primary biopsies). Specimens from 11 patients were studied before and after treatment (sequential biopsies). Treatment response was evaluated by PET-CT according to LYRIC criteria. Immunohistochemical staining for CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT was performed with an automated staining system (Bond III; Leica Biosystems). The slides were scanned with Aperio ScanScope XT (AperioTechnologies Inc.) and were analyzed with ImageScope Analysis software (Aperio Technologies) и Qupath (https://qupath.github.io). We explored progression-free survival (PFS) depending on the proportion of cells positive for CD68, CD163, PD-1, LAG-3, TIM-3, CTLA-4, TIGIT in the tumor microenvironment and analyzed the changes of these parameters between primary and sequential biopsies after treatment with nivolumab. Statistical analysis was performed using SPSS software (v.23). Data on sequential biopsies were analyzed with the Wilcoxon signed-rank test. PFS was calculated with the Kaplan-Meier method. The significance level was p ≤ 0.05. Results.A significant correlation was found in primary biopsies group between the value of CD163 and CTLA-4 (correlation coefficient -0,62, p < 0.05). There was no significant association between PFS and proportion of cells positive for CD68, PD-1, TIM-3, CTLA-4, TIGIT, LAG-3 in primary biopsies group. ROC analysis allowed to establish a 9% cut-off value of CD163 expression, dividing these patients into subgroups of CD163high and CD163low. In the CD163low group, the two-year PFS was 19,1% (95% CI 6%-37,7%) with a median PFS of 8,8 months (95% CI 5,7-12) and in the CD163high group - 53,8% (95% CI 28,4%-73,7%) with a median of 24,8 months (95% CI 18,8 - 39,2). In sequential biopsies, a statistically significant increase in numbers of PD-1+ and TIGIT+ T-cells and depletion of CD68+ and CD163+ cells was observed compared to corresponding cell counts in primary biopsies (median PD-1 - 3% vs 10%; median TIGIT - 10% vs 14%; median CD68 - 10% vs 7%; median CD163- 8% vs 3,5%; р <0,05). Conclusion.A comprehensive analysis of expression of CD68, CD163, LAG-3, TIGIT, CTLA4, TIM-3, PD-1 was performed in patients with r/r cHL before and after treatment with nivolumab. Significant association was found between the expression of CD163 and CTLA4. The results of the study indicate inferior PFS among patients with low expression (<9%) of CD163 in lymph node samples before immunotherapy. Biopsies taken after treatment with nivolumab showed a statistically significant increase in the number of PD-1+ and TIGIT+ cells and a decrease in the number of CD68+ and CD163+ cells compared with data from primary biopsies. The results of the study may contribute to our knowledge regarding biology of classical Hodgkin lymphoma and the mechanisms of resistance to therapy with immune checkpoints inhibitors. This study was supported by BMS research grant CA209-8EG Disclosures Ionova: Takeda:Other: principal investigator of the observational studies sponsored by Takeda;BMS:Other: principal investigator of the observational studies sponsored by BMS.

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Immune and Inflammatory Cells of the Tumor Microenvironment Represent Novel Therapeutic Targets in Classical Hodgkin Lymphoma

International Journal of Molecular Sciences ◽

10.3390/ijms20215503 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5503 ◽

Cited By ~ 8

Author(s):

Eleonora Calabretta ◽

Francesco d’Amore ◽

Carmelo Carlo-Stella

Keyword(s):

Tumor Microenvironment ◽

Hodgkin Lymphoma ◽

Checkpoint Inhibitors ◽

Therapeutic Targets ◽

Suppressor Cells ◽

Inflammatory Cells ◽

Brentuximab Vedotin ◽

New Drugs ◽

Classical Hodgkin Lymphoma ◽

Relevant Role

Classical Hodgkin Lymphoma (cHL) is a B-cell malignancy that, typically, responds well to standard therapies. However, patients who relapse after standard regimens or are refractory to induction therapy have a dismal outcome. The implementation of novel therapies such as the anti-CD30 monoclonal antibody Brentuximab Vedotin and immune checkpoint inhibitors has provided curative options for many of these patients. Nonetheless, responses are rarely durable, emphasizing the need for new agents. cHL is characterized by a unique microenvironment in which cellular and humoral components interact to promote tumor survival and dissemination. Knowledge of the complex composition of cHL microenvironment is constantly evolving; in particular, there is growing interest in certain cell subsets such as tumor-associated macrophages, myeloid-derived suppressor cells and neutrophils, all of which have a relevant role in the pathogenesis of the disease. The unique biology of the cHL microenvironment has provided opportunities to develop new drugs, many of which are currently being tested in preclinical and clinical settings. In this review, we will summarize novel insights in the crosstalk between tumor cells and non-malignant inflammatory cells. In addition, we will discuss the relevance of tumor-microenvironment interactions as potential therapeutic targets.

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The PD1/PD-L1 Axis in the Classical Hodgkin Lymphoma Microenvironment: PD-1 Is Rarely Expressed but Identifies Patients with High-Risk Disease

Blood ◽

10.1182/blood.v118.21.1560.1560 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1560-1560

Author(s):

Paul Greaves ◽

Andrew James Clear ◽

Andrew Owen ◽

Andrew Wilson ◽

Janet Matthews ◽

...

Keyword(s):

Immune Response ◽

Clinical Outcome ◽

Hodgkin Lymphoma ◽

Adverse Outcome ◽

Germinal Centre ◽

Automated Image Analysis ◽

Image Analysis System ◽

Malignant Cells ◽

Classical Hodgkin Lymphoma ◽

Median Expression

Abstract Abstract 1560 Introduction: Classical Hodgkin lymphoma (CHL) is histologically unique: the malignant Hodgkin Reed-Sternberg cell (HRS) represents a fraction of tumour bulk, the majority being inflammatory cells: a combination of inadequate immune response and tumor-supportive microenvironment. PD-1 is a T-cell expressed CD28-analogue that inhibits activity of the expressing cell by interactions with ligands including the B7 analogue PD-L1. PD-L1 is upregulated in a range of immune and non-immune cells including malignant cells. Immunosuppression through this axis may contribute to failed immune response and adverse outcome in some tumors (Keir et al. Annu Rev Immunol, 2008; 26: 677–704) and there are reports regarding expression levels and pathway activity (Chemnitz et al. Blood, 2007; 110: 3226–33) as well as biological importance in CHL (Yamamoto et al. Blood, 2008; 111: 3220–3224). PD-L1 is overexpressed by HRS cells and encoded by a chromosomal region showing recurrent copy number gains in CHL (Steidl et al. Blood, 2010; 116: 418- 427). PD1/PD-L1 interactions are also essential to the survival of germinal centre B cells (Good-Jacobson et al. Nature Imm; 2010; 11: 535–42) the normal counterparts of the HRS, implying a potential direct supportive role for this pathway independent of the immune response. While the mechanistic importance of this axis in CHL is widely stated, little published evidence exists to substantiate it. This immunohistochemical (IHC) study set out to determine levels and variability of expression of PD-1 and PD-L1 at diagnosis in CHL and determine prognostic importance. Methods: IHC analysis was performed on tissue microarrays (TMAs) from 122 previously untreated CHL patients with known clinical outcome: median age 30 (range 18–80), 35% female, 71% advanced stage, median follow up 16.5 (range 2–40) years. Triplicate cores were made from areas of high cellularity avoiding fibrotic or acellular portions, arrayed and stained for PD-1 or PD-L1. Positive cells were counted across all cores using an automated image analysis system (Ariol), output confirmed by expert histopathologists, and means calculated corrected to % infiltration per 1mm2. 105 cases (86%) had tissue of sufficient quality for full IHC assessment. Groups were determined based on population distribution of infiltration levels and confirmed using the test/validation set method. Outcomes of freedom from first-line treatment failure (FFTF), disease-specific survival (DSS) and overall survival (OS) were assessed using the Kaplan Meier method with statistically significant differences between groups calculated using the log-rank test. Results: PD-1 expression was strikingly low or absent in the microenvironment of the majority of cases: <1% of cells in 40% of cases and <5% of cells in 80%. Median expression was 0.14% of all nucleated cells (range 0–8.9%). When present, this was predominantly as a low-level background infiltrate with frequent clusters/nodules of PD-1 positive lymphoid cells aggregating around HRS cells. Notably, the rare patients with high expression of PD-1 had adverse outcome. The 20% of patients with infiltration >0.5% cells had DSS 61% vs 89% at both 5 and 10 years (p=0.03). In contrast, PD-L1 was expressed at a high level in both HRS and the microenvironment in the majority of cases: >10% cells in 38% of cases, and <1% in only 5%. Median expression was 6.1% (range 0.2%–40%). Level of PD-L1 expression in the microenvironment was not associated with any measure of clinical outcome. Conclusions: PD-1 may have biological significance, as measured by impact on prognosis in a minority of patients with CHL, where overexpression is associated with an increased chance of CHL-related death. This may relate to immunosuppression, but the very low overall levels of expression and tendency to cluster around the malignant cells (a pattern seen more often in nodular lymphocyte predominant HL) imply a more localised HRS interactive role perhaps equivalent to the pathway's activity in a normal germinal centre. Its ligand PD-L1 is highly expressed in the microenvironment as well as in the HRS, which may relate to an activated rather than suppressive microenvironment, but without association with prognosis. We conclude that for the majority of patients with CHL, the definitive role for this pathway remains to be demonstrated conclusively. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Epstein-Barr Virus Status Correlates with Composition and Prognostic Impact of the Tumor Microenvironment in Classical Hodgkin Lymphoma

Blood ◽

10.1182/blood.v120.21.52.52 ◽

2012 ◽

Vol 120 (21) ◽

pp. 52-52

Author(s):

Peter Kamper ◽

Knud Bendix ◽

Stephen Jacques Hamilton-Dutoit ◽

Bent Honore ◽

Francesco d'Amore

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Hodgkin Lymphoma ◽

Epstein Barr Virus ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Cell Marker ◽

Classical Hodgkin Lymphoma ◽

Barr Virus ◽

Epstein Barr

Abstract Abstract 52 Background: Classical Hodgkin lymphoma (cHL) is often characterized by a minority of neoplastic cells surrounded by a heterogeneous background of reactive non-neoplastic cells. An increased amount of certain cell subsets, such as T-regulatory lymphocytes and macrophages, in the microenvironment of cHL tumor lesions has been found to correlate with an adverse prognosis, probably as a result of enhanced immune tolerance towards tumor cells. Furthermore, it has also been suggested that the presence of Epstein-Barr virus (EBV) infection in the Hodgkin Reed-Sternberg (HRS) cells, may modulate the composition of the tumor microenvironment. Aim: In the present study, we have analyzed the possible correlation between EBV-status, a number of tumor microenvironment parameters and outcome in a large retrospective series of newly diagnosed cHL patients. Design and Methods: A tissue microarray was constructed from paraffin embedded pre-therapeutic tumor tissue biopsies obtained from 288 newly diagnosed cHL cases. The expression in the tumor microenvironment of the macrophage markers CD68 and CD163, the regulatory T-cell marker FoxP3 and the cytotoxic T-cell marker Granzyme-B (GrB) was assessed by immunohistochemistry (IHC) using a previously described semi-automated stereological counting approach (Haematologica 2011;96:269–276). The presence of EBV in HRS cells was investigated using 'in situ' hybridization for EBV-encoded RNAs 1 and 2 and LMP-1 IHC. Clinical data were obtained from clinical records. The correlation between clinic-pathological features and EBV was assessed using the rank-sum or Kruskal-Wallis test. The impact of clinico-pathological parameters on event-free (EFS) and overall survival (OS) was evaluated using the log rank test. Results: The 288 patients had a median age of 37 yrs (range: 6–86 yrs). The M:F ratio was 1.3. One third (33%) of the patients were positive for EBV in the HRS cells. EBV-positive cases exhibited higher numbers of CD68 (p=0.001), CD163 (p=0.0002), GrB (p<0.0001), and FoxP3 (0.0009)-positive cells. Excluding cases of mixed cellularity from the analysis, the significant correlation between EBV and CD163 (p=0.03), GrB (p=0.003), FoxP3 (p=0.006) remained, whereas the correlation for CD68 was slightly weakened (p=0.06). In the entire cohort (n=288), a high expression of CD68, CD 163 and GrB were found to correlate with significantly lower OS and EFS (high vs. low CD68: 5-year OS, 73% vs. 87% p=0.002, 5-year EFS, 58% vs. 70% p=0.03; high vs. low CD163: 5-year OS, 78% vs. 87%, p=0.03, 5-year EFS, 62% vs. 69%, p= 0.04) and high GrB: 5-year OS, 77% vs 88 %, p=0.004, 5-year EFS, 61% vs. 69%, p= 0.02). Interestingly, the influence of tumor microenviromental parameters on outcome was more pronounced in EBV-negative cases (n=193) than in EBV-positive ones (n=95). In the former, significantly lower OS and EFS values were associated with a high expression of CD68 (high vs. low CD68: 5-year OS, 60% vs. 92%, 5-year EFS, 43% vs. 71%, both p<0.001), high CD163 (5-year OS, 72% vs. 89%, p<0.001, 5-year EFS, 58% vs. 69%, p= 0.03) and high GrB (5-year OS, 61% vs. 90%, 5-year EFS, 43%vs. 71%, both p<0.001). Among the EBV-positive cohort, the corresponding OS and EFS values were high CD68 (5-year OS, 85% vs. 82%, p=0.69, 5-year EFS, 72%vs. 66%, p=0.43), high CD163 (5-year OS, 86% vs. 84%, p=0.34, 5-year EFS, 67%vs. 71%, p=0.48) and high GrB (5-year OS, 88% vs. 81%, p=0.34, 5-year EFS, 73%vs. 65%, p= 0.63).The number of FoxP3-cells was not found to affect the prognosis in neither EBV-negative nor EBV-positive cases. Conclusions: The present study confirms that the EBV-status in cHL is associated with distinct features of the tumor microenvironment. As a novel finding, our results suggest that the prognostic impact of intratumoral reactive non-neoplastic cell subsets is EBV-status dependent, i.e. a significantly adverse impact of an increased amount of certain bystander cell subsets on outcome was only found in EBV-negative cases. Disclosures: No relevant conflicts of interest to declare.

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The classical Hodgkin lymphoma tumor microenvironment: macrophages and gene expression-based modeling

Hematology ◽

10.1182/asheducation-2014.1.144 ◽

2014 ◽

Vol 2014 (1) ◽

pp. 144-150 ◽

Cited By ~ 22

Author(s):

David W. Scott ◽

Christian Steidl

Keyword(s):

Gene Expression ◽

Tumor Microenvironment ◽

Hodgkin Lymphoma ◽

New Technologies ◽

Biomarker Discovery ◽

Predictive Biomarkers ◽

Prognostic Models ◽

Classical Hodgkin Lymphoma ◽

Specific Patient ◽

Long Term Treatment

Abstract Despite the high cure rate in classical Hodgkin lymphoma (CHL), more accurate tailoring of upfront treatment is required to maximize cure while avoiding unnecessary short- and long-term treatment side effects. To this end, the unique tumor microenvironment of CHL has been searched extensively for prognostic biomarkers. Beyond targeted immunohistochemistry (IHC) studies, gene expression profiling (GEP) of diagnostic whole tissue biopsies has allowed a de novo approach to biomarker discovery. Among numerous candidate biomarkers, an association between the number of tumor-associated macrophages in the microenvironment and outcomes after ABVD (doxorubicin + bleomycin + vinblastine + dacarbazine) chemotherapy emerged, and multiple subsequent studies have validated this biological relationship using IHC. These studies have also defined key aspects for macrophage interrogation, including the characteristics of the CD68 and CD163 antibodies, appropriate scoring methodologies, and the identification of specific patient populations in which macrophage IHC may not be prognostic. The GEP studies also led to the development of gene expression-based prognostic models for advanced-stage CHL, with new technologies allowing reliable gene expression quantitation using RNA from routinely produced formalin-fixed paraffin-embedded biopsies. The bridge to predictive biomarkers that can be used reliably to inform upfront treatment selection requires further studies to demonstrate that these biomarkers can identify robustly, at diagnosis, patients at high risk of treatment failure with ABVD and that this risk may be overcome using alternative treatments.

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Tumor microenvironment composition in pediatric classical Hodgkin lymphoma is modulated by age and Epstein-Barr virus infection

International Journal of Cancer ◽

10.1002/ijc.27314 ◽

2011 ◽

Vol 131 (5) ◽

pp. 1142-1152 ◽

Cited By ~ 46

Author(s):

Mário Henrique M. Barros ◽

Gabriela Vera-Lozada ◽

Fernando A. Soares ◽

Gerald Niedobitek ◽

Rocio Hassan

Keyword(s):

Tumor Microenvironment ◽

Virus Infection ◽

Hodgkin Lymphoma ◽

Epstein Barr Virus ◽

Epstein Barr Virus Infection ◽

Classical Hodgkin Lymphoma ◽

Barr Virus ◽

Epstein Barr ◽

Barr Virus Infection

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Checkpoint protein expression in the tumor microenvironment defines the outcome of classical Hodgkin lymphoma patients

Blood Advances ◽

10.1182/bloodadvances.2021006189 ◽

2021 ◽

Author(s):

Kristiina Karihtala ◽

Suvi-Katri Leivonen ◽

Marja-Liisa Karjalainen-Lindsberg ◽

Fong Chun Chan ◽

Christian Steidl ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Tumor Microenvironment ◽

Hodgkin Lymphoma ◽

Immune Cell ◽

Immune Escape ◽

Multivariable Analysis ◽

Classical Hodgkin Lymphoma ◽

Checkpoint Protein ◽

The Impact

Emerging evidence indicates a major impact for the tumor microenvironment (TME) and immune escape in the pathogenesis and clinical course of classical Hodgkin lymphoma (cHL). We used gene expression profiling (n=88), CIBERSORT, and multiplex immunohistochemistry (n=131) to characterize the immunoprofile of cHL TME, and correlated the findings with survival. Gene expression analysis divided tumors into subgroups with T cell-inflamed and non-inflamed TME. Several macrophage-related genes were upregulated in samples with the non-T cell-inflamed TME, and based on the immune cell proportions, the samples clustered according to the content of T cells and macrophages. A cluster with high proportions of checkpoint protein (PD-1, PD-L1, IDO-1, LAG-3, and TIM-3) positive immune cells translated to unfavorable overall survival (OS) (5-year OS 76% vs. 96%, P=0.010), and remained as an independent prognostic factor for OS in multivariable analysis (HR 4.34, 95% CI 1.05-17.91, P=0.043). cHLs with high proportions of checkpoint proteins overexpressed genes coding for cytolytic factors, proposing paradoxically that they were immunologically active. This checkpoint molecule gene signature translated to inferior survival in a validation cohort of 290 diagnostic cHL samples (P<0.001) and in an expansion cohort of 84 cHL relapse samples (P=0.048). Our findings demonstrate the impact of T cell- and macrophage-mediated checkpoint system on the survival of patients with cHL.

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