Abstract 1705: Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay
Authors: Nunns Harry , Parnell Erinn A. , Lazare Michael , Kuo Judy , Leones Eric , Sahafi Flora , William Josette , Au Qingyan 
Published: June 2022

Abstract
Immune checkpoint blockade (ICB) therapy has revolutionized the landscape of cancer therapy in multiple tumor types since Ipilimumab, the first ICB agent, was approved for the treatment of metastatic melanoma is 2011. Current predictive biomarkers for therapy response include PD-L1 expression, tumor mutational burden (TMB) and microsatellite instability (MSI) status. However, responses to ICB vary widely and these predictive markers have demonstrated imperfect predictive power to ICB therapy. For instance, half of microsatellite instability-high (MSI-H) colorectal carcinoma (CRC) patients do not respond to ICB immunotherapy. There is still a crucial need to identify and develop biomarkers predictive of outcome to ICB therapy. Tertiary lymphoid structures (TLS) have been observed in a variety of solid tumors in humans, and their presence is a favorable prognostic indicator for survival in a variety of solid cancers including CRC. Further, it was recently shown that the presence of mature TLS was associated with improved response rate to ICB and overall survival in patients with advanced tumors in a large retrospective study. In this study, we used a 17-plex MultiOmyx panel to detect TLS, explore TLS maturation stage and characterize the tumor microenvironment (TME) in 40 CRC patients with known MSI status. The 17-plex includes CD3, CD4, CD8, CD45RO, FOXP3, CD20, CD56, CD68, CTLA-4, PD1, PD-L1, CXCL13, PNAd, CD21, CD23, DC-LAMP and PanCK. Using this panel, we successfully detected different stages of TLS in these CRC samples. Using proprietary deep-learning-based image analysis algorithm (NeoLYTX), we quantified TLS’s in the CRC samples and classified them by maturation stage based on biomarker expression and spatial organization of immune cells. While there was no difference in the number of early TLS (E-TLS; characterized by CD3, CD20 and pNAd) between MSI-H and microsatellite-stable (MSS) CRC samples, more primary follicle-like TLS (P-TLS; defined by CD3, CD20 and CD21) and secondary follicle-like TLS (S-TLS; defined by CD3, CD20 and CD23) were observed in the MSI-H group than the MSS group. In particular, the abundance of mature S-TLS was significantly higher in MSI-H CRC samples. This 17-plex MultiOmyx assay provides a powerful tool to characterize the cellular composition and spatial organization of the tumor microenvironment. The panel enables quantification of TLS, PD-L1 expression and abundance of tumor infiltrating lymphocytes from one single FFPE slide. The rich datasets generated by the MultiOmyx assay can provide greater understanding of the immune contexture within the TME and deeper insights into the correlations between biomarkers. These findings may have broad application and help identify biomarker signatures with improved predictive performance to immune checkpoint inhibition efficacy in solid tumors.
Citation Format: Harry Nunns, Erinn A. Parnell, Michael Lazare, Judy Kuo, Eric Leones, Flora Sahafi, Josette William, Qingyan Au. Spatial interrogation of tertiary lymphoid structures (TLS) in colorectal carcinoma (CRC) tumor microenvironment (TME) using the MultiOmyx࣪ assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1705.