Quantification of PD-L1 and PD-1 expression on tumor and immune cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry

Abstract

Objective: We report a truly quantitative technology for PD-L1 expression in non-small cell lung cancer (NSCLC). In addition, we present a non-enzymatic technology that creates a cell suspension from fresh tumor tissue so that either fine-needle aspiration (FNA) or fresh tissue can be used in this assay.

Methods: Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, California) was performed on 4-mm punch biopsies. An FNA was taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, and PD-L1 and then stained with DAPI to identify intact, single cells, and to analyze cell cycle.

Results: Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r ² - 0.8) for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1 % cutoff for positivity in the tumor cell population and a 1 % cutoff of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive. Ten of 12 lung tumor samples were concordant while 2 were discordant. PD-L1 expression by flow cytometry varied widely (1.2-89.4 %) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population. Fine, unequivocal quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue.

Keywords: Cell cycle; Immune cells; Non-small cell lung cancer; PD-1; PD-L1.

Fig. 1

PD-L1 quantification of IncellPREP-prepared cell suspension by flow cytometry. a Immune cells were electronically separated from tumor cells by CD45 antibody staining and side scatter (ss). Tumor cells (red box) were gated for PD-L1 expression in b, c. PD-L1 expression in tumor cells varied from low (b) to high (c) as listed in Table 2

Fig. 2

Comparison of matched FNA and IncellPREP-prepared cells for PD-L1 demonstrated strong correlation (r ² − 0.8) independent of the starting sample type. Aneuploid tumors are indicated with red circles

Fig. 3

a Immunohistochemical staining for PD-L1 in tissue blocks from matched flow cytometry samples. PD-L1-positive and PD-L1-negative tissues are shown (×200). Positive staining is indicated by a brown precipitate. A positive control tonsil sample and an isotype control (without primary antibody) are also shown. b High power view of characteristic rim pattern PD-L1 staining in tissues shown in a