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R.D. Jachimowicz et al.
Figure 1. Flowchart of the study cohort and validation cohort. Average values of percentage of the respective cell types are indicated. For calculation of cell counts of macrophages (CD68) and Hodgkin and Reed-Sternberg cells (CD30) see the Online Supplementary Methods.
deviation [SD]: 80.84 mm2) (Online Supplementary Figure S1). Since we included the entire lymph node in the analysis, any hetero- geneity of cell distribution did not influence our data. Cutting arti- facts, and overstained or unstained areas were manually excluded from the analysis. Adjusting the threshold based on several repre- sentative locations on the sample also ensured that the setting for analysis for each sample had been selected to cover the specific staining of the lymph node. See the Online Supplementary Methods for a detailed description and statistics.
Results
Mapping the microenvironment in classical Hodgkin lymphoma through whole-slide image analysis in the study cohort
Within the study cohort, WSI detected a mean total cell count of 1,550,980 (SD: 949,004) for all analyzed lymph node specimens. The main cellular components of the tumor tissue (CD3+ T cells, CD20+ B cells, CD30+ HRSC and CD68+ macrophages) reflect the variable cell propor- tions dependent on the histological subtype (Figure 2). Overall, T cells, B cells, HRSC and macrophages account- ed for a mean of 80% of all cells within the tumor tissue, highlighting the strength of WSI for comprehensively assessing cHL tumors. As expected, lymphocyte-rich cHL (n=9) showed high counts of B cells and T cells with con- comitant low levels of HRSC and macrophages. Conversely, lymphocyte-depleted cHL (n=4) displayed the lowest counts of B cells and T cells while CD30 and CD68 levels were markedly elevated. The T-cell content dis- played a moderate positive correlation with B-cell content
(ρ=0.42; P<0.001), as well as a weak correlation with macrophage content (ρ=0.15; P=0.0049) and CD30 con- tent (ρ=0.15; P=0.0063) (Table 2). We also observed a weak relationship between macrophage and HRSC con- tent (ρ=0.21; P=0.0001). Of note, we did not detect a cor- relation between B-cell and macrophage content.
B-cell content was associated with risk of progression and relapse in the study cohort
We analyzed whether CD3, CD20, CD30 and CD68 differed between patients with lasting complete remission and patients with subsequent progression or relapse. While we did not observe any association of T-cell count, macrophage content or CD30-positivity with long-term remission status in our study cohort, B-cell content was significantly lower at the time of diagnosis in patients with later progression or relapse (mean 13.5%) than in patients with long-lasting complete remission (mean: 13.5% vs. 17.7%, respectively; P=0.0079) (Table 3). Further subgroup analyses revealed that patients with mixed cellularity and nodular sclerosis cHL had signifi- cantly lower B-cell counts when at risk for relapse and a similar trend was observed for patients with lymphocyte- depleted and lymphocyte-rich cHL (data not shown).
To further assess the association between B-cell content and progression-free survival, we performed explorative receiver operating characteristic (ROC) analyses in which a B-cell content of 21% or less was the best predictor of progression or relapse in the study cohort (ROC estimate – chance = 0.00959, P<0.0001). Utilizing this cut-off, both progression-free survival (P=0.0004, hazard ratio [HR]=2.479, 95% CI: 1.479-4.157) and overall survival
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