Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, ...macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68
, median 32%; M2 type CD163
, median 22%), whereas the proportion of TAMs expressing IDO-1 is lower (CD68
, median 5.5%; CD163
, median 1.4%). A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68
), or from CD163
TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1
and IDO-1
TAMs remain independent prognostic factors for freedom from treatment failure (PD-L1
CD68
/CD68
, HR = 2.63, 95% CI 1.17-5.88,
= 0.019; IDO-1
CD68
/CD68
, HR = 2.48, 95% CI 1.03-5.95,
= 0.042). In contrast, proportions of PD-L1
tumor cells, all TAMs or PD-L1
and IDO-1
TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Introduction: Cancer-associated fibroblasts (CAFs) constitute an important stromal component of the tumor microenvironment (TME). They are a heterogeneous population of cells, which can modulate the ...immune system and have both pro-tumorigenic and anti-tumorigenic effects in a context-dependent manner. In solid tumors, the impact of CAFs in shaping the TME is well recognized, although challenges, including lack of specific CAF markers, still exist. In classical Hodgkin lymphoma (cHL), the role of CAFs has remained largely undefined. Here, we aimed to characterize distinct CAF subsets as well as their interactions with other TME cells and associate the findings with clinical characteristics and outcome of patients with previously untreated cHL. Methods: We used a tissue microarray (TMA) containing formalin-fixed paraffin-embedded samples from 115 diagnostic cHL tumors. The TMA was stained with a 7-plex immunohistochemistry panel to characterize CAFs platelet-derived growth factor (PDGFR) -alpha and -beta, fibroblast-activating protein (FAP), secreted protein acidic and rich in cysteine (SPARC), macrophages (CD68), Hodgkin-Reed Sternberg (HRS) cells (CD30), and leukocytes (CD45). Image processing and quality control were performed by combining Ilastik and CellProfiler softwares, and nuclei segmented by a pretrained deep learning segmentation model. Single cell features from the images were extracted using HistoCAT software. We utilized the Phenograph clustering algorithm for cell phenotyping and a permutation test by HistoCAT for neighborhood analysis. Results: We identified a total of 952,099 single cells, which were split into 25 distinct phenotype subsets by the Phenograph clustering spanning CAFs, macrophages, leukocytes, and HRS cells. CAFs were classified into seven distinct subsets based on the status of the different CAF markers. Median proportion of all CAFs was 28% (range 2-80%), the proportion being higher in the nodular sclerosis subtype compared to the others. In general, higher proportions of CAFs associated with favorable freedom from treatment failure (FFTF) independently of the subtype, age, and stage (P<0.01). On the contrary, a subcluster of CD45+ immune cells with strong FAP-positivity, which were characterized as macrophages, was enriched in other than nodular sclerosis subtype (P<0.001) and associated with worse FFTF independently of age, stage, and subtype (P=0.01). The neighborhood analysis allowed identification of colocalization and statistically significant interaction or avoidance between pairs of cell phenotypes. For instance, higher proportions of FAP+PDGFRalpha+ CAFs interacting with other cells predicted better FFTF (P<0.01), whereas interactions of FAP+ macrophages with other cells were associated with worse FFTF. Despite the positive impact of CAF proportions on cHL outcome, patients with CD30+ HRS cells surrounded by PDGFRbeta+ CAFs had worse FFTF (P<0.001). Conclusions: We have characterized distinct CAF subsets in cHL and demonstrate their favorable clinical impact on cHL outcome. We also identified a novel subset of FAP-positive macrophages, the proportions and interactions of which translated to poor outcome. Our data highlight that not only the cell proportions, but the cell-cell interactions and spatial context play a crucial role. Further validation of the findings is ongoing.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction: Malignant classical Hodgkin lymphoma (cHL) cells express numerous immunoregulatory proteins that can shape their microenvironment and allow tumor cells to escape immune surveillance. ...Herein we aimed to characterize the immunological profile of cHL, and associate the findings with disease characteristics and outcome.
Patients and methods: We used NanoString digital gene expression profiling with the PanCancer Immune panel to assess the expression of 770 immune response genes in the tumor samples of 88 patients with primary cHL.
Results: The male/female ratio was 49%/51%, and the median age 32 years (range 16-85). Thirty-four (39%) patients were > 45 years, 66 (75%) had nodular sclerosis subtype and 46 (53%) stage III-IV disease. At the median follow-up of 42 months (range 12 to 164), 25 (28%) patients had relapsed and 10 (11%) died. Recurrence free survival (RFS) and overall survival (OS) rates at three years were 73% and 93%, respectively.
Unsupervised hierarchical clustering revealed five clusters of genes with differential expression between the patients. The clusters were enriched for the genes related to focal adhesion and extracellular matrix (ECM) interactions, Toll-like receptor signaling pathway, T-cell signaling, JAK-STAT signaling, and antigen processing and presentation. Interestingly, focal adhesion/ECM interaction and T-cell signaling signatures correlated with outcome. According to Kaplan-Meier estimates, the group of patients (n=14) with high expression of focal adhesion and ECM interaction signature genes had a 3-year RFS of 93% in comparison to 69% for those (n=74) with lower expression (p = 0.073). In addition, a significant unfavorable impact of low expression of T-cell signature on RFS was observed among the subgroup of patients over 45 years (44% vs 87%, p=0.003). Baseline characteristics, including gender, age, histological subtype and stage were equally distributed in all subgroups.
Conclusion: Our data reveal biologically relevant immunological profiles, and association of focal adhesion/ECM interaction and T-cell signaling signatures with treatment resistance in patients with primary cHL. A detailed characterization of immune cell composition in the cHL microenvironment and its impact on survival is ongoing.
Karihtala:Sanofi-Genzyme: Other: Travel expenses; Janssen-Cilag: Other: Travel expenses. Leppa:Janssen Cilag: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; Bayer: Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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 -noninflamed 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 (programmed cell death protein 1, PD-1 ligands, indoleamine 2,3 dioxygenase 1, lymphocyte-activation gene 3, and T-cell immunoglobulin and mucin domain containing protein 3) positive immune cells translated to unfavorable overall survival (OS) (5-year OS 76% vs 96%; P = .010) and remained an independent prognostic factor for OS in multivariable analysis (HR, 4.34; 95% CI, 1.05-17.91; P = .043). cHL samples 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 < .001) and in an expansion cohort of 84 cHL relapse samples (P = .048). Our findings demonstrate the impact of T cell- and macrophage-mediated checkpoint system on the survival of patients with cHL.
•Composition of the cHL microenvironment is heterogeneous and differs in the proportions of T cells and macrophages.•High T cell and macrophage-mediated checkpoint protein expression in the cHL microenvironment associates with inferior OS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Background: CCR7 is overexpressed in lymphoid malignancies. JBH492 is a first-in-class CCR7-targeting ADC, comprising a humanized, Fc-silenced anti-CCR7 IgG1 antibody conjugated to ...maytansinoid microtubule inhibitor DM4 via a cleavable linker. Preclinical studies support avidity-driven differential binding in CCR7-high tumor cells compared to CCR7-low healthy immune cells, and in vivo animal studies demonstrated robust and durable efficacy in multiple clinically relevant lymphoma models (Dang et al. 2022). Methods: This is an ongoing first-in-human, open-label, phase I/Ib, multi-center study in patients (pts) with relapsed/refractory (r/r) chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (NCT04240704), which completed enrollment for dose-escalation phase. Five dose levels of JBH492 monotherapy ranging from 0.4 to 3.6 mg/kg (intravenous administration every 3 weeks q3W) were tested with 3 - 7 pts enrolled per dose level. Preclinical data from xenografts predicted tumor stasis at 1.6 mg/kg q3W. The objective of dose escalation was to assess safety, tolerability, pharmacokinetics (PK), immunogenicity, and preliminary efficacy of JBH492 and to determine maximum tolerated and recommended doses. Pts with r/r CLL and NHL, who failed standard of care therapy (at least 2 prior regimens) and who were intolerant/ineligible to approved therapies, were included; CCR7 expression confirmation was not required prior enrollment. Results: Total 25 pts with r/r stage II-IV NHL (n = 24, adult T cell leukemia-lymphoma, 3; cutaneous T cell lymphoma, 4; diffuse large B cell lymphoma, 7; follicular lymphoma, 1; Mantle cell lymphoma, 1; marginal zone lymphoma, 2; peripheral T cell lymphoma, 3; others, 3) and CLL (n = 1) with median age 69.0 y (43 - 85) were enrolled. Mean duration of exposure was 13.5 wks. One pt had dose-limiting toxicity — grade (G) 3 thrombocytopenia lasting for 13 d. Overall, 23 (92%) pts had at least 1 adverse event (AE) (G ≥ 3, n = 11). Treatment-related AEs (TRAEs) were reported in 15 (60%) pts (G ≥ 3, n = 3). Of 6 (24%) pts who had serious AEs (SAEs) (G ≥ 3, n = 3), 2 (8%) had TRSAE. The most frequent AEs (≥ 20%) included anemia (32%), thrombocytopenia (28%), neutropenia and dry eye (20% each). Two deaths due to underlying disease were reported. PK for total ADC was roughly dose proportional. Four NHL pts (2 T-NHL and 2 B-NHL) had partial response (PR) and 2 had stable disease, with PRs observed at doses between 2.4 and 3.6 mg/kg q3W. Post data-cut-off, 1 pt with T-NHL achieved CR following 4 cycles of treatment. No correlation between CCR7 expression and response could be drawn (CCR7 expression was observed in all samples from 14 pts). Conclusions: The data from this phase I study suggest that targeting CCR7 with JBH492 is a promising therapeutic strategy for lymphoid malignancies with manageable hematological and non-hematological safety profile. K.I. and S.L. contributed equally to this study. Citation Format: Pau Abrisqueta, Reinhard Marks, Irit Avivi, Martin Wermke, Francesca Lim, Tae Min Kim, Alba Cabirta Touzon, Kristiina Karihtala, Shinichi Makita, Lillian Werner, Chiaki Tanaka, Anhthu Dang, Anwesha Chaudhury, Seth Rice, Rowshan Chowdhury, Deborah Knee, Belén Gomezcarrillo, Elissa Furutani, Koji Izutsu, Sirpa Leppä. A phase 1 study of JBH492, an anti C-C chemokine receptor 7 antibody-drug conjugate (anti-CCR7 ADC), assessing safety and efficacy in lymphoid malignancies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT174.
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