Biomarker Discovery in Immunotherapy-treated Melanoma Patients with Imaging Mass Cytometry.

Purpose: Imaging Mass Cytometry (IMC) is among the first tools with the capacity for multiplex analysis of over 40 targets, which provides a novel approach to biomarker discovery. Here we used IMC to characterize the tumor microenvironment (TME) of patients with metastatic melanoma who received immunotherapy (ITx) in efforts to find indicative factors of treatment response. In spite of the new power of IMC, the image analysis aspects are still limited by the challenges of cell segmentation. Experimental Design: Here, rather than segment we performed image analysis using a newly designed version of the AQUA{trade mark, serif} software to measure marker intensity in molecularly defined compartments: tumor cells, stroma, T cells, B cells, and macrophages. IMC data were compared to quantitative immunofluorescence (QIF) and Digital Spatial Profiling (DSP). Results: Validation of IMC results for immune markers was confirmed by regression with additional multiplexing methods and outcome assessment. Multivariable analyses by each compartment revealed significant associations of 12 markers for progression-free survival (PFS), and 7 markers for overall survival (OS). The most compelling indicative biomarker, beta2-microglobulin (B2M), was confirmed by correlation with overall survival by QIF in the discovery cohort and validated it in an independent published cohort profiled by mRNA expression. Conclusions: Using digital image analysis based on pixel colocalization to assess IMC data allowed us to quantitively measure 25 markers simultaneously on FFPE tissue microarray samples. In addition to show high concordance with other multiplexing technologies, we identified a series of potentially indicative biomarkers for immunotherapy in metastatic melanoma including B2M.