The previously described T cell–inflamed GEP was derived by using a stepwise derivation process of discovery, validation, and refinement of candidate gene sets across a wide variety of solid tumors (15 (link)). The GEP was composed of 18 inflammatory genes related to antigen presentation, chemokine expression, cytolytic activity, and adaptive immune resistance, including CCL5, CD27, CD274 (PD-L1), CD276 (B7-H3), CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, IDO1, LAG3, NKG7, PDCD1LG2 (PDL2), PSMB10, STAT1, and TIGTT. For GEP analysis, total RNA was isolated from 5-μm-thick FFPE sections of tumor tissue fixed on positively charged slides (Ambion RecoverAll total nucleic acid isolation kit for FFPE; catalog no. AM1975) at ALMAC, United Kingdom. Total RNA concentrations were measured using the NanoDrop ND1000 (Thermo Fisher Scientific) in 1.5 μl of test sample.
Gene expression analysis was conducted on the NanoString nCounter gene expression platform (NanoString Technologies, Seattle, WA) as described previously (15 (link)). Per sample, 50 ng of total RNA was mixed in a final volume of 5 to 7 μl with a 3′-biotinylated capture probe and 5′-reporter probe tagged with a fluorescent barcode, from the desired custom gene expression codeset (HUIMR680_V2_C2406+PLS_SPI-KE80_C2765 for Batch 1 and HUIMR800_C3176 for Batch 2), containing probes designed to function as positive and negative hybridization controls. Probes and target transcripts were hybridized overnight at 65°C for 14 to 18 hours as per manufacturers’ recommendations. Hybridized samples were run on the NanoString nCounter preparation station by using a high-sensitivity protocol where excess capture and reporter probes were removed and transcript-specific ternary complexes were immobilized on a streptavidin-coated cartridge. The cartridge samples were scanned at maximum resolution by using the nCounter digital analyzer. GEP scores were calculated as a weighted sum of normalized expression values for the 18 genes. Quality control of the gene expression data followed an approach similar to that of the NanoString clinical-grade assay, with the use of joint criteria that assessed the relationships between housekeeping genes and the negative control probes plus a weighted score evaluating the GEP gene counts versus background-subtracted counts. For housekeeping normalization, raw counts for the individual genes were log10 transformed and then normalized by subtracting the arithmetric mean of the log10 counts for a set of 11 housekeeping genes.
Gene expression analysis was conducted on the NanoString nCounter gene expression platform (NanoString Technologies, Seattle, WA) as described previously (15 (link)). Per sample, 50 ng of total RNA was mixed in a final volume of 5 to 7 μl with a 3′-biotinylated capture probe and 5′-reporter probe tagged with a fluorescent barcode, from the desired custom gene expression codeset (HUIMR680_V2_C2406+PLS_SPI-KE80_C2765 for Batch 1 and HUIMR800_C3176 for Batch 2), containing probes designed to function as positive and negative hybridization controls. Probes and target transcripts were hybridized overnight at 65°C for 14 to 18 hours as per manufacturers’ recommendations. Hybridized samples were run on the NanoString nCounter preparation station by using a high-sensitivity protocol where excess capture and reporter probes were removed and transcript-specific ternary complexes were immobilized on a streptavidin-coated cartridge. The cartridge samples were scanned at maximum resolution by using the nCounter digital analyzer. GEP scores were calculated as a weighted sum of normalized expression values for the 18 genes. Quality control of the gene expression data followed an approach similar to that of the NanoString clinical-grade assay, with the use of joint criteria that assessed the relationships between housekeeping genes and the negative control probes plus a weighted score evaluating the GEP gene counts versus background-subtracted counts. For housekeeping normalization, raw counts for the individual genes were log10 transformed and then normalized by subtracting the arithmetric mean of the log10 counts for a set of 11 housekeeping genes.
