Abstract 5364: A targeted NGS solution to evaluate gene expression signature of the tumor microenvironment from 40 NSCLC FFPE and matched fresh frozen samples

Cancer cells and their surrounding non-malignant cells, including immune cells, signaling molecules, stromal and extracellular matrix, create the tumor microenvironment (TME). The composition of this TME plays important roles in tumor progression, evading growth suppressors and activating metastasis. However, the regulatory mechanism and function of each constituent remains poorly understood. With several checkpoint blockade therapy studies, the presence of PD-L1 has been reported to be a promising marker to predict positive response. Current IHC methods to measure PD-L1 are subjective and highly variable. A higher-throughput and standardized solution that can systematically measure gene expression of cells present in the TME has emerged to be a more desirable alternative. Here, we applied the OncomineTM Immune Response Research Assay to measure the expression of 395 genes in non-small cell lung cancer (NSCLC) samples from 40 matched FFPE and fresh frozen sample types. This assay leverages NGS technology to sequence and count reads derived from the original transcript. With an input requirement of 10 ng of total RNA, libraries were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM System. Results showed that, despite small input amount, the expression profiles of FFPE and fresh frozen samples are highly correlated with an average correlation greater than 0.9. We selected 22 genes out of the panel to validate expression with qPCR using FFPE samples. These genes were selected to cover a range of low, medium, and high expressors per our NGS data. Again, we observed a strong correlation (R ~ 0.9) between NGS and qPCR data. Approximately 80% of the 40 samples show moderate to high expression of CD8+ T cell cytokines, IFNG and TNFa. We further found that the expression of CD8A and CD8B are highly correlated with CD4, suggesting the co-presence of both cytotoxic and helper T cells. High correlation among CD4, FOXP3, TGFB1, and IL2RA (CD25) also suggests that their expression can be used as markers for the presence of Treg cells. We conducted a differential expression analysis between a group of samples (n=8) with high percentage of surrounding and infiltrating lymphocytes and another group (n=5) with low stromal content but devoid of infiltrating lymphocytes. Interestingly, we found a large number of genes which annotated as markers for infiltrating lymphocytes (CTSS, CXCR4, CD37, SRGN, FCER1G, SAMHD1, and GZMA) are significantly up-regulated i