Page 183 - 2021_06-Haematologica-web

B-cell content in advanced-stage cHL
Introduction
Classical Hodgkin lymphoma (cHL) is a B-cell-derived lymphoid malignancy, affecting 2.5-3/100,000 people per year. Depending on the clinical stage and associated risk factors, first-line treatment consists of poly-chemotherapy with or without consolidating radiotherapy, and results in long-term tumor control in 70-80% and overall survival of 80-90% of patients.1,2 Crucially, all patients with advanced-stage cHL currently continue to receive aggres- sive poly-chemotherapy within the German Hodgkin Study Group (GHSG) clinical trials. This treatment regi- men is common practice because, to date, there is no reli- able tool to distinguish, a priori, the subset of patients at high risk of relapse or progressive disease from those with lower risk. As a result, two groups of patients may be underserved by current treatment approaches: those who could be treated with less toxic treatment regimens; and those who are likely to relapse or progress after standard chemotherapy, as their prognosis is often very poor with less than 50% becoming long-term survivors.3 In contrast to non-Hodgkin lymphoma, clinical risk indices for cHL such as the International Prognostic Score (IPS) have not been successfully applied in treatment decision-making within the subgroup of patients with advanced stage cHL.4
Similarly, the commonly defined histological subtypes of cHL lack prognostic significance under the currently used standard treatment protocols. Four microenviron- mental patterns reflect the basis of the histological sub- types in the current World Health Organization classifica- tion, namely nodular sclerosis, mixed cellularity, lympho- cyte-rich and lymphocyte-depleted. In recent research, however, the tumor microenvironment of cHL has attract- ed interest as a predictor of disease outcome.5 cHL is char- acterized by the presence of neoplastic Hodgkin and Reed-Sternberg cells (HRSC) that constitute only a minor- ity of cells within the affected lymph nodes. The tumor is mainly composed of non-neoplastic stromal and immune cells, which form a tumor microenvironment around HRSC. Depending on the cHL subtype, the cellular part of the tumor microenvironment is made up of variable pro- portions of macrophages, neutrophils, eosinophils, T cells, B cells, fibroblasts and plasma cells. The neoplastic HRSC is dependent on both endogenous- and exogenous signals, the latter stemming from crosstalk with the microenviron- ment, e.g., by the interaction between macrophages and tumor cells.6
The prognostic value of HRSC CD20 expression is con- troversial.7-10 Numerous studies utilizing quantitative assessment of mRNA or immunohistochemistry have sug- gested an adverse effect of increased macrophage infiltra- tion11-15 and a favorable effect for markers of normal B cells in the tumor microenvironment of cHL.16-18 However, the immunohistochemistry-based approaches used to assess the microenvironmental composition and the prognostic impact suffer from limitations hampering their integration into routine diagnostic procedures and clinical decision- making for cHL. Unlike gene expression analysis, which usually analyzes bulk tumor tissue, including the non- malignant microenvironment, classical immunohisto- chemical studies analyze a limited size of tumor regions and may be biased by intratumoral heterogeneity and observer-dependent selection of the areas analyzed. Moreover, validation of microenvironmental biomarkers has rarely been performed in large clinical trial cohorts. To
overcome these limitations and to test the prognostic role of the tumor microenvironment in advanced-stage cHL, we performed whole-slide-image analysis (WSI) of T cells (CD3), B cells (CD20), HRSC (CD30) and macrophages (CD68) in a study cohort and an independent validation cohort consisting of hundreds of patients treated within trials of the German Hodgkin Study Group (GHSG).
Methods
Study design and patients’ samples
Between January 1999 and July 2017, 5,801 adult patients with primarily diagnosed and histologically confirmed advanced-stage cHL were randomized to receive first-line treatment with the BEACOPP regimen, containing bleomycin, etoposide, doxoru- bicin, cyclophosphamide, vincristine, procarbazine, and pred- nisone in standard or escalated (eBEACOPP) doses within the ran- domized GHSG clinical trials HD12, HD15, and HD18.19-21 Our cohort comprised trial patients with (cases) and without (controls) progression or relapse at the ratio of 1:2.
Inclusion criteria for the case-control cohort were documenta- tion of the reference histology result and reference center and availability of a formalin-fixed paraffin-embedded lymph-node specimen obtained at first diagnosis as well as complete documen- tation of the presence or absence of prognostic factors. Inter- observer agreement of WSI was tested using randomly selected WSI slides analyzed by a second observer and a randomly selected cohort stained and analyzed in a second center (Online Supplementary Methods).
The WSI findings of the study cohort of patients (n=340) from HD12 and HD15 (Figure 1A) were matched with clinical data and analyzed for prognosis. The performance was subsequently tested using an independent validation cohort comprising HD18 patients (n=147) (Figure 1B). Median follow-up times were 66 months (95% confidence interval [95% CI]: 63- 71) in the study cohort and 62 months (95% CI: 57-65) in the validation cohort.
Table 1 summarizes the clinical characteristics of the study and validation cohorts in relation to all randomized HD12 and HD15 patients or all randomized HD18 patients, respectively. Patients included in the study cohort were slightly younger compared to all patients from the HD12 and HD15 randomized cohorts (median age: 31 years [range, 24-40] vs. 33 years [25-42], respectively; P=0.0093). There were no differences in prognostically relevant laboratory parameters nor the IPS scores; however, more patients in the study cohort had ≥3 nodal areas affected and stage IV dis- ease at diagnosis (Table 1A). The validation cohort did not differ from the HD18 randomized cohort regarding demographic and prognostic factors (Table 1B). The study was conducted in accor- dance with the recommendations of the ethics board of the Medical Faculty, University of Kiel.
Whole-slide image analysis
For each patient, the whole available tissue specimen/block was cut and subsequent slides were stained in the Department of Pathology at Kiel University for CD3 (clone SP7, Waltham, MA, USA), CD20 (clone L26, Dako, Glostrup, Denmark), CD30 (clone BerH2, maintained at the Department of Pathology, Kiel, Germany) and CD68 (clone PG-M1, Dako, Glostrup, Denmark), using a Leica-Bond-Max stainer (Leica Microsystems, Wetzlar, Germany). The slides were scanned (Hamamatsu Nanozoomer, Hamamatsu Photonics, Ammersee, Germany) and the resulting images were processed by TissueStudio 64, according to the man- ufacturer’s recommendations (Definiens AG, Munich, Germany). The area ranged between 4-455 mm2 (mean: 133.81 mm2, standard
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