Topological importance of CD8+ T-cell enrichment in the tumor microenvironment of classic Hodgkin lymphoma

Annals of Hematology https://doi.org/10.1007/s00277-025-06189-1 RESEARCH Topological importance of CD8+ T-cell enrichment in the tumor microenvironment of classic Hodgkin lymphoma Hiromichi Takahashi1 · Shun Ito1 · Yoko Nakanishi2 · Katsuhiro Miura1,7 · Haruna Nishimaki2 · Masaru Nakagawa1,8 · Shimon Otake1 · Takashi Hamada1 · Takashi Koike1 · Kazuhide Iizuka1,3 · Shinobu Masuda2 · Tomohiro Nakayama3 · Tetsuo Shimizu4 · Naoya Ishibashi5 · Hirofumi Kogure6 · Hideki Nakamura1 Received: 22 May 2024 / Accepted: 3 January 2025 © The Author(s) 2025 Abstract Classic Hodgkin lymphoma (CHL) histologically consists of Hodgkin Reed-Sternberg (HRS) cells and the tumor microenvironment (TME), but the relationship between TME characteristics and clinical features of CHL remains unclear. We aimed to investigate the effects of the TME structure on the outcomes of patients with CHL. We performed a highthroughput analysis of HRS cells and their topological relationship with the reactive immune cells in the TME. After multiplexed immunofluorescence labeling against CD4, CD8, CD30, CD68, CD163, PD-1, and PD-L1, visual images were analyzed. Phenotypes were assigned to all reactive cells, such as CD4+ and CD8+ T-cells and macrophages. Since the densities of PD1+/CD4+ T-cells, CD8+ T-cells, and PD-L1+ macrophages were significantly higher in the area < 60 μm than in the area < 120 μm from each HRS cell in 45 tissue samples from 34 patients with CHL, we further analyzed the TME-component cells by focusing on the 60 μm radius in the initial samples. TMEs containing > 15 CD8+ T-cells were associated with a significantly better 3-year progression-free survival than those with ≤ 15 CD8+ T-cells (100% vs. 53%, p = 0.006). In comparison with TMEs containing ≤ 15 CD8+ T-cells, TMEs containing > 15 CD8+ T-cells had significantly more PD-L1− macrophages (mean 3 vs. 1 cell, p = 0.015) and fewer PD-1+/CD4+ T-cells (mean 16 vs. 28 cells, p = 0.036). Epstein-Barr virus positivity in HRS cells was significantly associated with a higher number of macrophages in the 60 μm radius area. In conclusion, the TME structure in patients with CHL can differ, enabling precision therapies. Keywords Classic Hodgkin lymphoma · Tumor microenvironment · CD8+ T-cell · Hodgkin Reed-Sternberg cell · Multiplexed immunofluorescence Hiromichi Takahashi and Shun Ito contributed equally to this work as co-first authors. Katsuhiro Miura 1 2 3 Department of Medicine Division of Hematology and Rheumatology, Nihon University School of Medicine, Tokyo, Japan Department of Pathology and Microbiology Division of Oncologic Pathology, Nihon University School of Medicine, Tokyo, Japan Department of Pathology and Microbiology Division of Laboratory Medicine, Nihon University School of Medicine, Tokyo, Japan 4 Department of Medicine, Division of Respiratory Medicine, Nihon University School of Medicine, Tokyo, Japan 5 Department of Radiology, Nihon University School of Medicine, Tokyo, Japan 6 Department of Medicine Division of Gastroenterology and Hepatology, Nihon University School of Medicine, Tokyo, Japan 7 Department of Medicine, Division of Hematology and Rheumatology, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi City, Tokyo 173-8610, Japan 8 Present Address: Department of Hematology, Kasukabe Medical Center, Saitama, Japan 13 Annals of Hematology Introduction Classic Hodgkin lymphoma (CHL) is histologically composed of Hodgkin Reed-Sternberg (HRS) cells and the tumor microenvironment (TME) consisting of T-cells and macrophages [1, 2]. Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) expression plays a critical role in the immune response against CHL [3, 4]. Most patients with CHL are cured with multidrug chemotherapy (adriamycin, bleomycin, vinblastine, and dacarbazine [ABVD]), with or without radiation therapy [5]. Furthermore, combining brentuximab vedotin with AVD (BVAVD) improves the long-term prognosis of patients with advanced-stage CHL [6]. However, 5–10% of the patients who receive initial therapy still develop refractory disease, and 10–30% relapse [7]. Immune checkpoint inhibitors induce reconstitution of the immune response by targeting malignant cells and have changed the therapeutic landscape for patients with relapsed/refractory CHL [8, 9]. Therefore, CHL is a representative lymphoma wherein targeting immunoreactive cells can provide potential benefits, and a deeper understanding of the TME is expected to have a significant impact on treatment selection [10]. Although clinical prognostic factors such as the International Prognostic Score (IPS) are established for CHL [7], the pathologic prognostic factors are unclear, and identifying pathologic structures that are responsive to multi-agent chemotherapy or immune checkpoint inhibitors remains a challenge. Multiplexed immunofluorescence (MIF) staining and image analysis have enabled the evaluation of the TME in CHL [11, 12]. Consequently, a unique topology of CHL was defined, wherein PD-L1+ tumor-associated macrophages (TAMs) surround the HRS cells, and CD4+ T-cells are targets for PD-1 blockade [13]. However, the relationship between TME characteristics and clinical features of CHL is not fully understood. Additionally, the association between Epstein-Barr virus (EBV) positivity in HRS cells and TME status has been poorly understood. Therefore, this study analyzed the pathological features and clinical outcomes of patients with CHL using the MIF technology to understand the clinical features and variations in the TME in CHL, as well as the role of EBV in the pathological topology of CHL. Materials and methods Patient selection We retrospectively evaluated the clinical records and pathological samples of patients with primary or relapsed CHL treated at our institution between January 2010 and December 13 2021. Diagnostic biopsies were performed as part of the standard workup for suspected lymphoid neoplasms. Initial biopsies were conducted for diagnostic purposes in patients with suspected lymphoma, and recurrent biopsies were carried out in patients with previously treated CHL to diagnose CHL recurrence. All pathological diagnoses were reviewed by two pathologists (H. N. and S. M.) prior to this study to rule out other lymphoproliferative diseases and confirm CHL. Stored formalin-fixed, paraffin-embedded (FFPE) lymph node blocks were retrieved for analysis. Retrospective data, including disease characteristics, clinical prognostic factors, prior treatment, treatment response, and survival of patients with CHL, were extracted from the institutional medical records. Patients were excluded from the analysis if they did not have sufficient pathologic material for MIF assay or a history of ABVD-based curative chemotherapy. This study was approved by the Nihon University Itabashi Hospital Clinical Research Judging Committee in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Sub (...truncated)