PROJECT SUMMARY PD-L1 membranous (cell surface) expression in pretreatment biopsies is the most commonly used correlate of the likelihood of response to anti-PD-1 therapy. The general finding of an association of PD-L1 expression with tumor response to anti-PD-1 therapy has been substantiated across tens of thousands of patients with numerous tumor types treated with anti- PD-(L)1. However, while PD-L1 expression enriches for response to anti-PD-(L)1, it is not sufficient. Additionally, while pathologists demonstrate good reproducibility for scoring tumor cell (TC) PD-L1 expression by chromogenic IHC and light microscopy, they have poor reproducibility for scoring PD-L1 expression on immune cells (IC). Other related features which have been shown to improve on the PD-L1 biomarker include the proximity of PD-1 to PD-L1, the density of CD8+FoxP3+ cells, and CD68 and tumor marker immunostains, the latter of which help identify co-expression of PD-L1 on ICs and TCs, respectively. A quantitative multiplex immunofluorescence assay which captures all of these features has been developed and includes PD-L1, PD-1, CD8, FoxP3, CD68, a tumor marker (cytokeratin AE1/3 for non-small cell lung carcinoma, NSCLC), a pan-membrane marker and DAPI. Through this assay, it is possible to enumerate key cellular subsets and their co-expression profiles. It is also possible to include spatial parameters, including the distance between PD-1 and PD-L1, which have not previously been included in predictive or prognostic surgical pathology specimen-based assays. This mIF assay has increased sensitivity and specificity for response to anti-PD1 therapy when compared to the assessment of PD-L1 expression alone in multiple tumor types, including melanoma, NSCLC, and Merkel cell carcinoma, amongst others. The purpose of this proposal is to perform inter-site validation of the mIF staining assay and associated lock-down algorithm amongst four major academic sites (Johns Hopkins, MD Anderson, Yale University, and Providence Portland Medical Center). Following analytical validation, discovery and validation cohorts from 250 patients with advanced NSCLC will be used to establish final assay parameters (including thresholds), linked to clinical outcomes following anti-PD-1-based therapy. The deliverable of the study is a refined, multiplex biomarker assay for response/resistance to anti-PD-1 that has been validated across multiple academic sites. The result will be a multiplex IF assay that is suitably staged for advanced development aimed at clinical implementation. While NSCLC is the focus of the current grant proposal, preliminary results suggest that this assay will also have great value in numerous other solid tumor types.