Abstract A113: Harnessing lymphoid organ neogenesis as a novel prognostic biomarker and therapeutic target
Authors: Silina Karina , Soltermann Alex , Burkhardt Chiara , Attar Farkhondeh Movahedian , Casanova Ruben , Curioni-Fontecedro Alessandra , Moch Holger , Posch Florian , Winder Thomas , Dijk Nick van , Voskuilen Charlotte , Heijden Michiel van der , Broek Maries van den 
Published: February 2019

Abstract
Lymphoid organ neogenesis takes place in chronically inflamed tissues including cancer and yields the development of tertiary lymphoid structures (TLS). TLS are ectopic lymphoid organs that activate antigen specific T-cells and B cells in infection and autoimmunity and correlate with prolonged survival in various cancer types. This suggests that TLS contribute to protective anti-tumor immunity. Therefore, promoting the development of tumor-associated TLS could be a novel immunotherapeutic approach. However, the molecular and cellular mechanisms of TLS development in human cancer or how TLS contribute to survival are largely not understood. Here we used multiparameter immunofluorescence and digital pathology to quantify TLS and to characterize their cellular composition and tissue context in cohorts of lung squamous cell carcinoma (LSCC, n=138), colorectal cancer (CRC, n=111), clear cell renal cell carcinoma (ccRCC, n=50) and bladder cancer (BC, n=33) patients. Furthermore, we established an experimental model to characterize TLS development and its impact on tumor-specific immunity. We discovered that TLS development and maturation followed the same steps in all analyzed tumor types as well as in the lungs of mice in our experimental model. First, B and T lymphocytes accumulated around blood vessels. Second, a network of follicular dendritic cells developed within the lymphocytic aggregate, and third, a germinal center (GC) reaction was activated. Additionally, we identified a niche of CXCL13+ perivascular stroma and CXCL12+LTB+ and PD-L1+ epithelial cells that were associated with TLS in LSCC. We found that the number of tumor-associated TLS was an independent prognostic factor for prolonged survival in untreated LSCC, CRC and BC, but not in ccRCC patients or in LSCC and BC patients who were treated with neoadjuvant chemotherapy. By comparing the chemotherapy-treated and untreated cohorts we observed that the number of TLS was not changed but TLS maturation (i.e. GC formation) was significantly impaired after chemotherapy. This difference was at least partially dictated by corticosteroids, which are commonly used to treat the side effects of chemotherapy of LSCC patients. We further studied the mechanisms underlying TLS development using the experimental model. We identified a combination of stimuli that induces the development of mature TLS in the lungs of mice. Besides inflammatory stimuli, a foreign antigen was necessary to achieve a significant increase in TLS numbers and maturation stage, suggesting that cognate interactions are crucial for lymphoid organ neogenesis. This is further supported by our observation that CRC patients with microsatellite instability, which presumably results in more neoantigens, had an increased proportion of mature TLS. The negative impact of corticosteroids on TLS development was confirmed in this model. In summary, we propose that GC+ TLS represent the relevant TLS phenotype contributing to survival in different tumor types. Lymphoid organ neogenesis is negatively affected by corticosteroids, which might impair the spontaneous as well as therapy-induced anti-tumor immunity. The established experimental model will allow investigation of the mechanisms of TLS development and function in cancer and assessment of their therapeutic potential.
Citation Format: Karina Silina, Alex Soltermann, Chiara Burkhardt, Farkhondeh Movahedian Attar, Ruben Casanova, Alessandra Curioni-Fontecedro, Holger Moch, Florian Posch, Thomas Winder, Nick van Dijk, Charlotte Voskuilen, Michiel van der Heijden, Maries van den Broek. Harnessing lymphoid organ neogenesis as a novel prognostic biomarker and therapeutic target [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A113.