Abstract 2802: In situ detection of PD-1+CXCL13+CD8+ T cells in non-small cell lung cancer

Abstract

Abstract Recent reports identified one subset of intratumoral CD8+ cytotoxic T lymphocytes (CTLs) in non-small cell lung cancer (NSCLC) that are PD-1high with distinct molecular and functional properties. Strikingly, these cells produce very high levels of CXCL13 mRNA and protein, which may mediate immune recruitment. Furthermore, the presence of PD-1high CD8+ T lymphocytes are strongly predictive for both response and survival in NSCLC patients treated with PD-1 blockade. Thus, it is of great value to develop a practical biomarker assay to specifically detect these cells in formalin-fixed paraffin-embedded (FFPE) tumor biopsies. In this study, we combined the highly sensitive and specific RNAscope multiplex fluorescent RNA in situ hybridization (ISH) assay detecting CXCL13 and PDCD1 mRNAs with immunohistochemistry (IHC) detecting CD8 protein in a single tissue section to directly visualize PD-1+CXCL13+CD8+ T lymphocytes in NSCLC tissues. Two NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE samples were stained with full automation using the Leica BOND RX instrument. The resulting slides were scanned, and the images were analyzed using the Perkin Elmer Phenochart software. 57 of the 63 TMA cores were available for image analysis. Each tissue core was first examined under 4X magnification, then snapshot images of three independent 40X fields with enriched CD8+ cells (if present) were taken. CD8+ cells, CXCL13+ cells, and PD-1+ cells in each snapshot were counted. Every snapshot contained both stromal and tumor regions. 43 samples contained high (>20) CD8+ CTLs whereas 14 samples contained low (≤20) CD8+ CTLs across the three snapshots. Interestingly, PD-1+CXCL13+CD8+ cells were detected in both high and low CTL tumors. Five of 57 tumors carried high percentages of PD-1+CXCL13+CD8+ cells (>10% of CD8+ CTLs), with three from high CTL tumors and two from low CTL tumors. These results demonstrate that this fully automated multiplexed RNAscope dual ISH/IHC assay allows for co-localization of RNA and protein biomarkers in single cells with morphological context. The ability to detect RNA and protein in a single slide-based assay enables immune profiling applications to include biomarkers such as secreted proteins and non-coding RNAs that are difficult or impossible to detect by IHC. Citation Format: Na Li, Bingqing Zhang, Niyati Jhaveri, Zhifu Zhang, Xin Wang, Hongzhe Sun, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. In situ detection of PD-1+CXCL13+CD8+ T cells in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2802.