Expression of PD-1, PD-L1 and PD-L2 in Lymphomas in Patients with Pre-Existing Rheumatic Diseases-A Possible Association with High Rheumatoid Arthritis Disease Activity

Abstract

Current research seeks to identify subgroups of non-Hodgkin lymphoma (NHL) patients responsive to PD-1 blocking agents. Whether patients with pre-existing rheumatic diseases might constitute such a subgroup is unknown. We determined intratumoral expression of PD-1 and its ligands in lymphoma patients with pre-existing rheumatic diseases. We included 215 patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or Sjögren's syndrome with subsequent lymphoma and 74 diffuse large B-cell lymphoma (DLBCL) controls without rheumatic disease. PD-1 and PD-ligand immunohistochemical markers were applied on tumor tissue microarrays. The number of PD-1+ tumor infiltrating leukocytes (TILs) and proportions of PD-L1+ and PD-L2+ tumor cells and TILs were calculated and correlated with clinical data. Expression of PD-L1 in tumor cells and TILs was highest in classical Hodgkin lymphoma and DLBCL. In DLBCLs, expression of PD-1 in TILs and PD-L1 in tumor cells was similar in RA, SLE and controls. In RA-DLBCL, high expression of PD-L1 in tumor cells was significantly more common in patients with the most severe RA disease and was associated with inferior overall survival in multivariable analysis.

Keywords: PD-1; PD-L1; immunohistochemistry; lymphoma; rheumatic disease.

Figure 1

Flowchart of patients with pre-existing rheumatoid arthritis, systemic lupus erythematosus or Sjögren’s syndrome and subsequent lymphoma development included in the study with complete clinical information and with sufficient tumor tissue for tissue microarray construction. * Of 85 originally identified DLBCL patients without any rheumatic disease, specimens suitable for TMA construction remained in 74 patients used as controls for comparison with the rheumatoid arthritis- and systemic lupus erythematosus-DLBCL cases.

Figure 2

Patients with rheumatoid arthritis with diffuse large B-cell lymphoma stained with PD-1, PD-L1/PAX5 and PD-L2/PAX5 (antibody NAT105/ab52587 (Abcam, Cambridge, UK) for PD-1, antibody E1L3N/13684 (Cell Signaling Technology, Danvers, MA, USA) for PD-L1, antibody D7U8C/82723 (Cell Signaling Technology) for PD-L2 and antibody M7307/DAK-Pax5 (Dako, Santa Clara, CA, USA) for PAX5). (A): High amounts of PD-L1+ tumor cells and TILs. (B): A high amount of PD-L1+ TILs and a low amount of PD-L1+ tumor cells. (C): Low amounts of PD-L1+ tumor cells and TILs. (D): High amounts of PD-L2+ tumor cells and TILs. (E): A high amount of PD-L2+ TILs and a low amount of PD-L2+ tumor cells. (F): Low amounts of PD-L2+ tumor cells and TILs. (G): A high amount of PD-1+ TILs and no PD-1+ tumor cells. (H): PD-1+ tumor cells and a few scattered PD-1+ TILs. Magnification: 400× in all images. Legends: PD-1+/PD-L1+/PD-L2+ tumor cells = black arrow, PD-1+/PD-L1+/PD-L2+ TILs = black arrowhead, PD-1-/PD-L1-/PD-L2- tumor cells = green arrow, PD-1-/PD-L1-/PD-L2- TILs = green arrowhead.

Figure 3

Boxplots of distribution of proportions of PD-L1+ tumor cells (A) and TILs (B) and number of PD-1+ TILs/HPF (C) in cHL, DLBCL and FL. Comparison of median values with the group with the highest value, p-values according to Wilcoxon signed rank-test. TILs, tumor infiltrating leukocytes; cHL, classical Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma.

Figure 3

Boxplots of distribution of proportions of PD-L1+ tumor cells (A) and TILs (B) and number of PD-1+ TILs/HPF (C) in cHL, DLBCL and FL. Comparison of median values with the group with the highest value, p-values according to Wilcoxon signed rank-test. TILs, tumor infiltrating leukocytes; cHL, classical Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma.

Figure 4

Kaplan–Meier curve according to ≥17% (yellow line) and <17% (blue line) PD-L1+ tumor cells in patients with rheumatoid arthritis and diffuse large B-cell lymphoma.