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Tumor-associated macrophages in PTL
with outcome have been described in primary, mainly nodal DLBCL patients treated with immunochemothera- py, forming the backbone for our study.12 We have recent- ly demonstrated that tumor-associated macrophages (TAMs) have a favorable prognostic impact on survival in DLBCL patients after immunochemotherapy,13 whereas other groups have investigated the role of programmed cell death-1 (PD-1) pathway in DLBCL.14-18 While PD-1 protein is expressed predominantly by activated tumor- infiltrating lymphocytes (TILs), its ligands (PD-L1 and PD- L2) have been shown to be expressed both by the tumor
15,19-21
cells and the tumor microenvironment. An unexpect-
ed feature has been that PD-L1 expression by the tumor- infiltrating myeloid and other immune cells can be more prevalent than PD-L1 expression by the tumor cells.15,19,20 Recently, it was also shown that the expression of PD-L1, not only by the tumor cells but also by the host cells, plays a critical role in mediating the immunosuppressive func- tion of the PD-1 pathway.21
In DLBCL, expression of PD-L1 by lymphoma cells has been associated with poor outcome.14 Interestingly, 9p24.1/PD-L1/PD-L2 copy number alterations and addi- tional translocations of these loci are frequent in PTLs (>50%), leading to increased expression of the PD-Ls,4 and possibly also to immune escape. Whether the expression of PD-1 and PD-Ls predict survival in PTL, and in which compartments, is unknown.
With the aim of resolving the relative expression of checkpoint molecules by the tumor and host immune cells in patients with PTL, we examined B cells, TAMs, TILs, and checkpoint molecules by using multiplex immunohis- tochemistry (mIHC),22 allowing simultaneous detection of CD68+ TAMs, CD163+ or c-Maf+ M2-polarized TAMs, CD4+ and CD8+ T cells, CD20+ B cells, and the checkpoint molecules PD-L1, PD-L2 and PD-1. The findings were cor- related with clinical parameters and survival.
Methods
Patients
We identified 74 PTL patients with DLBCL histology diagnosed between the years 1987 and 2013 from the pathology databases of the University Hospitals in Southern Finland. Histological diagno- sis was established from surgical pretreatment tumor tissue according to current criteria of the World Health Organization (WHO) classification.23 The majority of the patients were treated with anthracycline-based chemotherapy. About half of the patients received rituximab as a part of their treatment. Contralateral testis was treated with surgical excision or irradia- tion for a minority of the patients. Patients were divided into three equal tertiles, based on the content of different immune cell sub- types (high, intermediate, low). The patient characteristics are described in more detail in Table 1. The protocol and sampling were approved by the Institutional Review Boards, Ethics Committees and the Finnish National Supervisory Authority for Welfare and Health.
Multiplex immunohistochemistry (mIHC)
Formalin-fixed, paraffin-embedded (FFPE) primary tumor tis- sues were collected from the local biobanks and reviewed to match the latest WHO classification.23 Selection of the cores on the tissue microarray (TMA) was based on the evaluation of a hematopathologist. TMA was constructed and the sections (3.5 μm) stained with 4-plex primary antibody panels (PD-L1, PD-L2,
CD68, c-MAF; CD3, CD4, CD8, PD-1; CD20, CD163, PD1, PD- L1; Online Supplementary Table S1), followed by fluorescently labelled secondary antibodies and DAPI counterstain (nuclear stain). A more detailed description of the stainings is provided in the Online Supplementary Methods. Fluorescent images were acquired with AxioImager.Z2 (Zeiss, Germany). Machine-learning platform CellProfiler24 2.1.2 was used for cell segmentation, inten- sity measurements (upper quartile intensity) and immune cell clas- sification. Different cell types were quantified as proportion to all cells (e.g., PD-L1+CD68+ implying the number of PD-L1+CD68+ TAMs from all cells in a TMA spot) or as a proportion to a specific cell subtype (e.g., PD-L1+CD68+/CD68+ implying the number of PD-L1+CD68+ cells from all CD68+ TAMs). Spots with less than 5000 cells were excluded from the analysis, and data from dupli- cate spots from the same patient were merged.
Gene expression analysis
CD68, CD163, MAF, MS4A1 (CD20), CD274 (PD-L1), PDCD1LG2 (PD-L2), and PDCD1 (PD-1) mRNA levels were measured from 60 PTL samples using digital gene expression analysis with NanoString nCounter (Nanostring Technologies, Seattle, WA, USA).25
Survival definitions and statistical analyses
Overall survival (OS) was defined as time between diagnosis and death from any cause, disease specific survival (DSS) as time between diagnosis and lymphoma related death, and progression free survival (PFS) as time between diagnosis and lymphoma pro- gression or death from any cause.
Statistical analyses were performed with IBM SPSS v.24.0 (IBM, Armonk, NY, USA). Differences in the frequency of prognostic factors between three patient groups were analyzed by Kruskal- Wallis test. Correlations between gene expression values and cell counts as well as between different immune cell subpopulations were tested with Spearman's rank correlation.
Survival rates were estimated using the Kaplan–Meier method. Univariate and multivariate analyses were performed according to the Cox proportional hazards regression model. The potential bias due to duration of follow up was assessed by Schoenfeld residual. Probability values below 0.05 were considered statistically signifi- cant. All comparisons and all comparative tests were two-tailed.
Results
Patient characteristics
Patient and treatment characteristics of the study cohort are shown in Table 1. The majority of the patients repre- sented non-GCB phenotype, low stage, and had low/intermediate International Prognostic Index (IPI). Altogether, 34 deaths, 24 relapses and 24 lymphoma-asso- ciated deaths occurred during the median follow up of 67 months (range from 6.7 to 120 months). Five-year OS, DSS and PFS rates were 56%, 68%, and 53%, respectively.
Association of CD68, PD-L1 and PD-L2 encoding gene expression with survival
First, we determined the gene expression of the macrophage markers (CD68, CD163 and MAF), check- point molecules CD274 (PD-L1), PDCD1LG2 (PD-L2) and PDCD1 (PD-1), and the B-cell marker MS4A1 (CD20). CD68 expression correlated positively with CD274 (rs=0.654, P<0.001), PDCD1LG2 (rs=0.636, P<0.001), CD163 (rs=0.602, P<0.001), and MAF (rs=0.425, P=0.001) levels, and to a lesser extent with PDCD1 (rs=0.300, P=0.020), whereas no correlation between CD68 and
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