Optimization for better Immunohistochemistry assay and more comprehensive pathological interpretation for PD-L1 expression in Classical Hodgkin lymphoma

Abstract Bckgoround:Treating the relapsed or refractory Classical Hodgkin Lymphomas were still challenging, previous studies indicated they can benefit from immune checkpoint inhibitors, over expression of PD-L1 can also be predictive marker for anti -PD -1 therapeutic efficacy, however, harmonization with different IHC assay s and explanation of the PD -L1 immunostaining results remains controversial in CHL. In this study, we sought to optimize the PD-L1 Immunohistochemistry (IHC) assay in classical Hodgkin lymphoma (CHL). Methods: All tests were performed on tumor tissue microarray established from 54 CHLs. Three IHC assays (405.9A11, SP142, 22C3) for PD-L1 expression were compared semi-quantitatively with RNAscope assay (No. 310035, ACD), and the difference for their expression on background immune cells (ICs), their associations with densities of TIL/TAM makers and their implication on survival were also analyzed. Results: 405.9A11 assay demonstrated best specificity on HRS cells and sensitivity on ICs. Thus, positive expression of PD-L1 was more frequent on ICs (85.2%) than on HRS cells (48.1%). Different subgroups of background IC cells including tumor associated macrophages (TAMs) were assessed and scored by CD4, CD8, FOXP3, and CD163. PD-L1 expression on ICs were most associated with elevated densities of TAMs, and in concordance with TAMs(P=0.026), PD-L1 expression on ICs ( P=0.067)indicated a negative effect on survival.Conclusions: 405.9A11 assay was proved to be most convincible for demonstrating PD-L1 expression，pathologists should report PD-L1 expression in a combined manner including both positive status of HRS cells and positive percentage of ICs.