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Using a multiplexed immunofluorescence approach to compare immune cell populations in subtypes of non-small cell lung cancer.

e20702 Background: Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancer cases, and is characterized by a poor response to chemotherapy and a low survival rate. While immunotherapy has recently emerged as a successful approach in the treatment of NSCLC patients only 20-25% of patients show a positive response to anti-PD/PDL1 therapy, stressing the urgent need for additional immunotherapy options. NSCLC is a very heterogeneous disease with the two most common types being adenocarcinoma (ADCA) and squamous cell carcinoma (SCC). While a study using flow cytometry to analyze NSCLC on protein level reported the immune cell composition to be fundamentally different in ADCA compared with SCC (Daraselia et al. Am J Cancer Res, 2012 2(1):93), to the best of our knowledge this study will be the first of its kind performing an extensive analysis of NSCLS immune cell composition in tissue. Methods: In order to perform this comprehensive immunoprofiling of the NSCLC subtypes ADCA and SCC we will be using MultiOmyx™, an immunofluorescence (IF) multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers can be interrogated from a single FFPE section. Proprietary cell segmentation algorithms generate unique IDs for every cell and deep learning based cell classification algorithms then identify positive cells for each biomarker. Results: Using a 16-marker panel consisting of CD3, CD4, CD8, FoxP3, CD20, CD68, CD163, CD15, PD1, PDL1, ICOS, OX40, LAG-3, TIM-3, and PanCK we will analyze the proportion of B cells, T cell subtypes, M1/M2-type tumor-associated macrophages, as well as the expression of PD1/PDL1, in addition to novel immunotherapy targets LAG3, TIM3, ICOS, and OX40 in 20 samples from patients with NSCLC (10 ADCA and 10 SCC). Finally, from the same FFPE blocks that we will use to profile the immune cell composition on protein level we will also be extracting DNA/RNA in order to perform T cell receptor (TCR) sequencing, which will provide additional information on the diversity of T cells present in these tumors. Conclusions: It is our hope that these data will help provide new insights into the biology of ADCA and SCC that can ultimately be used to explore novel immunotherapeutic interventions for lung cancer treatment.