Unexpected identification of tumor-infiltrating H. pylori or human papillomavirus (HPV) 16 in three exceptional responders in immunochemotherapy-treated esophageal cancer

Background: Understanding why only few patients achieve durable responses to therapy remains challenging. Of 38 metastatic esophageal cancer (EC) patients treated on the LUD2015-005 trial (NCT02735239), three achieved >7 year ongoing survival following anti-PDL1/CTLA4 immunochemotherapy, in contrast to the median survival of 9-12 months under then standard of care. While microbial pathogens are increasingly recognized for their role in modulating tumor immunogenicity, their role in EC remains controversial. Methods: Bulk and single-cell RNA sequencing, including B and T cell receptor (TCR) analysis, were performed on serial tumor biopsies. Humoral responses to HPV16 were assessed via multiplex serology, using patient plasma and recombinantly expressed tumor-infiltrating antibodies. ELISpot assays were performed against HPV16-derived peptides. Subsequent TCR sequencing of peptide-stimulated immune cells was used to identify antigen-specific clones for recombinant expression and validation. Results: Transcriptomic profiling of all three long-term survivors unexpectedly revealed intratumoral presence of H. pylori (n=1) and HPV16 (n=2), alongside a highly pro-inflammatory tumor microenvironment. Based on available material, the contribution of HPV16 in one patient's durable response was further investigated. Upon 4 weeks of immunotherapy, complete tumor clearance was accompanied by an HPV-targeted host response: serological analysis identified a systemic response against the E6 oncoprotein as well as an intratumoral B cell clone targeting E2. ELISpot assays revealed broad virus-specific T cell reactivity and led to the identification of nine novel HLA-A*02:01-restricted TCRs specific for E2 (2 epitopes) and E6 (1 epitope), each demonstrating high avidity (nM EC 50 ). Notably, clonally expanded T cells expressing these TCRs were also identified within the tumor where they exhibited a tissue-resident memory phenotype, consistent with antigen-driven activation and long-term immune surveillance. Conclusions: Our findings demonstrate that H. pylori and HPV16 can be detected in EC. We show that HPV16-derived antigens are targeted by both peripheral and tumor-resident immune cells and, alongside the striking response to immunotherapy, suggest that virus-specific immunity may play a role in tumor clearance and long-term disease control. The naturally derived, HPV16-specific TCRs identified here represent promising candidates for adoptive immunotherapy across a range of HPV-driven cancers. More broadly, detection of microbial signatures alongside a robustly activated tumor microenvironment in all three exceptional responders supports the exploration of pathogen-directed therapies in cancers not typically linked to infection, opening new avenues to enhance immunotherapy outcomes.