Background Tertiary lymphoid structures (TLS) have been described in close proximity to tumor areas in a variety of cancer types. Abundance of TLS is related to cancer-specific survival and susceptibility to immune checkpoint inhibition. TLS in the tumor microenvironment are assumed to represent hotspots for T cell and B-cell activation leading to tumor-specific cellular and humoral immune responses.

Materials and Methods To identify shared and TLS/secondary lymphoid organ (SLO)-specific features, we performed comprehensive analyses of 110 treatment-naïve pancreatic ductal adenocarcinoma (PDAC) samples. Five-color immunofluorescence microscopy and immunohistochemical staining were performed for identification of structural characteristics of TLS and SLO in PDAC patients. Tissue extraction by laser microdissection and NanoString-based RNA expression analysis was conducted to compare gene expression in TLS, PDAC, SLO and normal pancreatic tissue. Moreover, we performed B-cell receptor sequencing of microdissected TLS and SLO.

Results Abundance of TLS was variable and patients with a high density of TLS inside and surrounding the tumor showed a higher overall survival. Moreover, we observed a correlation between abundance of TLS and infiltration by T cells in the tumor. We revealed structural homologies between TLS and SLO with immune cells in TLS expressing Ki67, Pax5, AID, IgG and IgA as essential markers of germinal center formation. TLS-high patients showed increased abundance of activated tumor-draining lymph nodes and we detected an overlap of expanded clonotypes between TLS and SLO. Moreover, we found largely overlapping expression patterns of a variety of immune related gene clusters, but distinct expression patterns of T-cell and complement-associated genes.

Conclusions Our results indicate a role of TLS in cancer immunosurveillance of PDAC, which may be susceptible to therapeutic targeting in this highly aggressive and immunotherapy-resistant disease.

K. Wennhold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Else Kröner-Fresenius-Stiftung. J. Lehmann: None. M. Thelen: None. C. Kreer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; German Research Foundation. M. Garcia-Marquez: None. P. Lohneis: None. S. Boeck: None. S. Kruger: None. S. Ormanns: None. M. Rudelius: None. J. Werner: None. F. Popp: None. F. Klein: None. M. von Bergwelt-Baildon: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; BMS. Other; Significant; Astellas, Roche, MSD. C. Bruns: None. A. Quaas: None. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; German Research Foundation, German Cancer Aid. Other; Significant; Astellas.