An Automated 5-Plex Fluorescent Immunohistochemistry Enabled Characterization Of Pd-L1 Expression And Tumor Infiltrating Immune Cells In Lung And Bladder Cancer Specimens

Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L19s complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies. Citation Format: Wenjun Zhang, Antony Hubbard, Adriana Racolta, Nick Cummins, Mehrnoush Khojasteh, Liping Zhang, Karl Garsha, Joerg Bredno, Dustin Harshman, Srabani Bhaumik, Tobin Jones, Marcin Kowanetz, Sanjeev Mariathasan, Ian McCaffery, Dustin Smith, J Andrew Williams, Lidija Pestic-Dragovich, Larry Morrison, Lei Tang. An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5117.