NanoString analysis
Surgically resected post-treatment formalin-fixed paraffin-embedded (FFPE) tissue samples from 19 Nivo+CT-treated and 19 Ipi+Nivo+CT-treated patients were cut into 4-µm-thick sections and shipped to the Immunotherapy Platform at our institution for NanoString analysis. The analysis was performed as per the umbrella protocol PA13-0291. Tissue sections were dewaxed using deparaffinization solution (Qiagen), and total RNA was extracted using the RecoverALL Total Nucleic Acid Isolation Kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were assessed using the NanoDrop spectrometer (NanoDrop ND-1000, Thermo Fisher Scientific). For the assay, 100 ng of RNA was used to detect immune gene expression using the nCounter PanCancer Immune Profiling panel along with custom CodeSet. nCounter Digital Analyzer was used to tabulate the counts of the reporter probes, and, for further analysis, raw data output was imported into nSolver analysis software (version 4.0.70) (