Project Summary/Abstract Therapies against immunologic checkpoint proteins, such as PD-L1, have revolutionized the treatment of multiple malignancies, such as malignant melanoma, lung cancer, bladder cancer, or gastroesophageal cancer. All of these therapies depend on predictive biomarker assays for PD-L1 protein expression in tumor tissue to identify patients who will most likely respond. PD-L1 assays face two significant challenges: (i) they often need to be performed on small tissue biopsies where tumor tissue is limited and (ii) the threshold for positivity by immunohistochemistry (1%) is very small, highlighting crucial dependence on utmost precision and pre-analytical sample validity. To address the issue of small and limited samples in biopsies, we have developed a chromogenic multiplexed immunohistochemical assay that combines PD-L1 assessment with tissue and cell type-specific markers to provide a combined diagnostic and predictive assay on a single histologic slide. To move multiplexed assays around PD-L1 into the clinic, there exists an important knowledge gap: what are the patient-specific factors and specimen-related pre-analytical variables that can influence the readout of PD-L1 positivity? Very limited knowledge is available about these variables. Importantly, because the PD-L1 positivity threshold is so low and requires the reliable separation of two very small numbers (<1% vs. ≥1%), even minute pre-analytical variabilities would be expected to have significant negative impact on assay validity. In that respect, PD-L1 testing in tissue is particularly in need of extensive characterization and control of pre-analytical variability, even more so than other assays whose cut-off points lie in more favorable ranges. Our proposal is based on the hypothesis that both patient-specific factors (such as molecular features of the cancer, current immune status, prior drug therapy, etc.) and specimen-related factors (such as timing of biopsy, size of tissue, ischemic time, fixation protocol, etc.) can significantly influence subsequent biomarker measurements. We further hypothesize that solid knowledge about these influences will allow controlling for and mitigating patient-specific and specimen-related effects and will lead to more accurate and valid biomarker assessment. Aim 1: We will create a cohort to test the influence of patient-specific context factors. We will make use of our extensive immuno-oncology database and biobank of >5,000 patients. Aim 2: We will test how specimen-related pre-analytical variables affect the assay using a wide variety of fresh, frozen, and formalin fixed tissue types and sizes. Aim 3: Once we have defined optimal patient- specific and specimen-related procedures, we will validate our multiplex assay in a prospective cohort of immunotherapy patients at MSKCC. Significance: This project will yield abundant data about the pre-analytical variables that influence a PD-L1 multiplex immunohistochemistry assay. These data will inform optimal specimen acquisition and handling and strategies for avoiding or mitigating inaccurate assay results. Innovation: This will be the first study to systematically explore both patient context factors and specimen pre-analytics in multiplexed immunohistochemical testing for immuno-oncology. Our close collaboration with commercial test developers as part of our research team will accelerate the translation of our scientific findings into optimized assays for the direct benefit of patients.

Project Narrative Understanding the pre-analytical variables that influence tissue-based biomarker tests is of utmost importance for validity and precision of the measurements. This project will systematically and comprehensively evaluate patient factors and tissue specimen pre-analytics for their effects on the validity, sensitivity, and specificity of singleplexed and multiplexed immunohistochemical predictive PD-L1 biomarker assays. This project will define the best pre- analytical conditions for specimen acquisition and inform a biomarker testing paradigm for the next generation of clinical tissue-based immune checkpoint protein assays for the benefit of cancer patients.