Abstract 399: Tertiary lymphoid structures features associate with outcome in non-small cell lung carcinoma
Authors: Segura Rossana Natalia Lazcano , Catao Andre , Solis Luisa M. , Jiang Mei , Tamegnon Auriole , Fujimoto Junya , Rodriguez-Canales Jaime , Chow Chi-Wan B. , Behrens Carmen , Kalhor Neda , Weissferdt Annika , Heymach John , Swisher Stephen , Sepesi Boris , Lee Jack , Moran Cesar , Futreal Andrew , Zhang Jianjun , Parra Edwin R. , Wistuba Ignacio I. , Tetzlaff Michael T. , Francisco-Cruz Alejandro 
Published: September 2020

Abstract
Introduction: Tertiary lymphoid structures (TLS) are ectopic lymphoid structures organized in a nodular pattern secondary to chronic inflammation. The presence of TLS has been associated with efficacious response to immune checkpoint blockade in a wide array of tumor types, including Non-Small Cell Lung Carcinoma (NSCLC). At early stages of NSCLC, TLS are observed in up to 70% of primary tumors where they are also associated with effective anti-tumor immune-responses and response to anti-PD-1 therapy. Despite complete surgical resection, however, up to 50% of patients with early stage NSCLC will eventually relapse. A systematic histomorphologic analysis of TLS and their association with relapse has not been well characterized.
Design: Serial sections from Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from 33 patients with stage I NSCLC were obtained (23 adenocarcinomas and 10 squamous cell carcinomas). Slides were stained with hematoxylin and eosin (H&E), immunohistochemistry for Vimentin, and 2 multiplex immunofluorescence (mIF) panels (Panel 1: cytokeratin (CK), CD3, CD8, CD68, PD-1, PD-L1 and DAPI Panel 2: CK, CD3 CD8, CD45RO, FOXP3, Granzyme B and DAPI). TLS were classified based on the H&E and Vimentin analysis into lymphoid aggregates (LA), immature TLS (iTLS), and mature TLS (mTLS). Morphometric analysis (number, area, and distance to nearest malignant cell) of intratumoral (IT) and peritumoral (PT) TLS was performed. mIF slides were scanned and IT representative areas were selected for cell densities and percentage quantification of immune-phenotypes. Morphometric analysis of TLSs was correlated with clinical outcomes, and tumor infiltrating immune cells.
Results: Patients who recurred had fewer IT mTLS (1.8 vs 6; p=0.02) and smaller area of mTLS (64541.7μm2 vs 149870.5 μm2; p=0.004) compared with patients who did not recurr.The cell density of IT antigen-experienced cytotoxic T-lymphocytes (CTLs), regulatory T-cells, memory CTLs, and memory CTLs expressing FOXP3 were inversely correlated with the mTLS area (r=-0.6, p≤0.02). Antigen-experienced CTLs (r=-0.61; p≤0.05) and non-CTLs (r=-0.63; p≤0.05) were inversely correlated with the number of IT mTLS.
Conclusion: Detailed morphometric analysis of mTLS offers relevant prognostic information for recurrence at stage I of NSCLC. mTLSs are associated with reduced IT infiltration by PD-1+ TILs. Taken together, germinal center development in mTLSs might convey a protective immune response due to immuno-dominant neoantigen presentation. Supported in part by CPRIT RP160668 grant and UT Lung SPORE.
Citation Format: Rossana Natalia Lazcano Segura, Andre Catao, Luisa M. Solis, Mei Jiang, Auriole Tamegnon, Junya Fujimoto, Jaime Rodriguez-Canales, Chi-Wan B. Chow, Carmen Behrens, Neda Kalhor, Annika Weissferdt, John Heymach, Stephen Swisher, Boris Sepesi, Jack Lee, Cesar Moran, Andrew Futreal, Jianjun Zhang, Edwin R. Parra, Ignacio I. Wistuba, Michael T. Tetzlaff, Alejandro Francisco-Cruz. Tertiary lymphoid structures features associate with outcome in non-small cell lung carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 399.