Comprehensive Profiling of Tumor Immune Landscape

Total RNA was isolated from 2 to 4 5-µm FFPE sections with a Qiagen total RNA kit (Cat# 75144), and quantified by Bioanalyser. The NanoString assay was performed using the PanCancer IO 360 Gene Expression Panel with an additional 30 DNA repair genes as spike-ins (Supplemental Table 1). Gene expression was normalized to 20 housekeeping genes. The data was analyzed using the NanoString NSolver Advanced Analysis platform. Pathway- and cell-type scores were calculated as the first principal component of pathway gene normalized^{29 (link),30 (link)}. Immune score positivity was calculated from the pathway and cell-type scores using a cutoff of the ≥25% as positive for the score, and below that as negative. In the chemo-naive samples IS was called positive if the sample presented with a score of ≥25% of any of the three interferon pathways due to the overlap of the genes in the three pathways. In the chemo-exposed samples, a positive IS was assigned to a sample if the exhausted CD8 + T-cell/CD8T-cell score was among the ≥25%. A combined score was assigned using Sig3 and IS data so that tumors positive for IS, Sig3, or both being positive, and tumors, which were negative for both IS and Sig3 being negative for the combined score.