Abstract 5252: 2D and 3D spatial characterisation of tertiary lymphoid structures in head and neck cancer patients receiving immune checkpoint blockade therapy

Tertiary lymphoid structures (TLS) play an important role in the tumor microenvironment (TME) of Head and Neck Cancer (HNC) and have been linked to improved response to immunotherapy. Moreover, HNC patients with human papillomavirus (HPV)+ tumors have better survival compared to those with HPV- tumors, although both groups have better clinical outcomes when the tumors are TLS-rich. How TLS composition, characteristics, and signatures differ between response to treatment in relation to HPV status is still unclear. To investigate this, whole-tissue samples from 55 HNC patients were collected across two separate sites in Brisbane, Australia (The Princess Alexandra Hospital and The Royal Brisbane and Women’s Hospital) prior to immune checkpoint therapy (ICI). Using a multi-pathway panel, covering the ‘hallmarks of cancer’ and ‘TLS-associated proteins,’ we profiled 53 spatially resolved biomarkers using both a PhenoCycler-Fusion panel and 7-plex PSP panel (Akoya Biosciences). Using spatial proteomics, the TLS were assessed for their composition, activity, maturity, and distance metrics from the tumor. Using spatial transcriptomics, we profiled 96 TLS using whole transcriptome profiling (Bruker Spatial Biology). In addition, in patient samples abundant with TLS, we profiled 50 serial sections to obtain and profile the volumetric 3D data. Our study identified various degrees of TLS maturity, proteomic, and transcriptomic profiles associated with TLS localisation within the TME and proximity to the tumor. In addition, we identified TLS signatures associated with clinical endpoints such as response and resistance to ICI therapy. Taken together, this research highlights the multifaceted role of TLS in modulating the immunogenic landscape of HNC, influencing immunotherapy efficacy, and potentially serving as a biomarker for patient stratification and therapeutic strategies. Citation Format: Meg L. Donovan, Habib Sadeghirad, Naomi Berrell, James Monkman, Vahid Yaghoubi Naei, Chin Wee Tan, Joseph Yunis, Zoe West, Majid E. Warkiani, Rahul Ladwa, Brett G. Hughes, Jessica Da Gama Duarte, Arutha Kulasinghe. 2D and 3D spatial characterisation of tertiary lymphoid structures in head and neck cancer patients receiving immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5252.