High-plex predictive marker discovery for melanoma immunotherapy treated patients using Digital Spatial Profiling

Background: Protein expression in formalin-fixed, paraffin-embedded (FFPE) tissue is routinely measured by immunohistochemistry (IHC) or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital Spatial Profiling (DSP) allows spatially-informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma. Methods: The NanoString GeoMx™ DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in Non-Small Cell Lung Cancer(NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 immunotherapy (ITx) treated melanoma patients on a TMA using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte and melanocyte) generating 132 quantitative variables. Results:The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared to QIF for stromal CD3, CD4, CD8, CD20 and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68 positive cells (macrophages) and not in tumor cells, was a predictive marker for PFS, OS and response. Conclusion: DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity we found a series of expression patterns associated with outcome including that the expression of PD-L1 in macrophages is associated with response.