Simultaneous, single cell, flow cytometric quantification of PD-L1/TILs/cell cycle in non-small cell lung cancer tissue biopsies: Concordance of PD-L1 expression detection between flow cytometry and immunohistochemistry.

Abstract

159 Background: Tumor cell expression of PD-L1 leads to the inhibition of immune responses specifically inactivation of cytotoxic T-cells. PD-L1 expression on tumor cells by immunohistochemistry does not provide the complete picture of therapeutic (PD-L1) and prognostic (TILs, aneuploidy) markers. Here, we report a clinical assay that quantifies tumor infiltrating lymphocytes (TILs), cell cycle/DNA content as well as PD-L1 expression on tumor and aneuploid tumor cell populations. Methods: Punch biopsies were taken from 19 fresh tissues specimens and were processed into single cell suspensions using the IVD/CE-IVD IncellPREP Single Cell Preparation Kit. Cells were fixed and permeabilized in 1 mL IncellMax. Cells were tested with the OncoTect iO Lung Assay which contains antibodies directed against PD-L1 (clone 28-8), CD45, CD3, and CD8, and a cell cycle dye. Concordance to IHC was tested by the Dako PD-L1 IHC 28-8 PharmDx Kit. Immune cell populations were quantified and aneuploidy was determined using a DNA index > 1.05. Results: The number of CD8+ CTL were significantly increased (P = 0.02) in tumor compared to normal lung tissue (37.4% to 28.2%). The number of CTL in aneuploid tumors was twice the number of CTL in diploid tumors. The percentage of antigen presenting cells (CD45+, high SSC) was decreased in the tumor cell samples relative to the normal lung tissue (P = 0.01). PD-L1 expression varies in cells depending on the cell cycle. Conclusions: In this study, the concordance between Oncotect iO and IHC was 95% (NPA-97%, PPA-89%). PD-L1 expression varies with DNA content, PD-L1 expression decreases with increasing DNA content. Though CTL are increased in aneuploid tumors, PD-L1 expression decreasing with DNA content may contribute to the association of aneuploidy with distant metastases. PD-L1 expression is a powerful determinant of treatment and aneuploidy is a powerful prognostic factor that combined yield important clinical information.