Radiofrequency Ablation of Liver Metastases from Colorectal Cancer Induces a Cancer-Specific T Cell Repertoire in Humans
Authors: Loeffler Markus W. , Nussbaum Bianca , Jurmeister Philipp S. , Budczies Jan , Kowalewski Daniel Johannes , Clasen Stephan , Koenigsrainer Ingmar , Beckert Stefan , Wagner Silvia , Sipos Bence , Bullinger Florian , Riess Olaf H. , Pereira Philippe , Koenigsrainer Alfred , Kanz Lothar , Stevanovic Stefan , Denkert Carsten , Rammensee Hans-Georg , Gouttefangeas Cecile , Haen Sebastian 
Published: October 2019

Abstract
Introduction: For patients with otherwise non-resectable primary and secondary cancer manifestations within the liver or contraindications against surgery, radiofrequency ablation (RFA) is a potentially curative treatment option. Heat causes coagulative necrosis and breaks local immune tolerance with subsequent release of cellular content like soluble danger signals which may reshape adaptive anti-tumor immune responses. The induction of effective functional tumor-specific T cells and in situ tumor-recognition remain a major challenge for immunotherapy, frequently limiting therapeutic effects. In this study, the induction of antigen-specific T cells against patient-individual, naturally presented tumor antigens and the infiltration of immune cells into distant liver metastasis after RFA were evaluated.
Methods: Six patients suffering from at least two metastatic sites in different liver lobes derived from colorectal carcinoma (CRC) were enrolled. As an initial step, RFA was performed on those manifestations that were inaccessible by surgery. Patients subsequently underwent liver surgery of the residual metastases. These specimen were used for HLA-ligandome analyses of tumor and corresponding non-malignant liver (NML) tissues by uHPLC-coupled tandem mass spectrometry (MS) following HLA-immunoprecipitation. The identified naturally presented HLA-ligands were screening against an extensive HLA-ligand database containing data from non-malignant tissues from different origins to assess tumor specificity of single peptides. Further, whole transcriptome sequencing (WTS) was performed and correlated with MS data selecting HLA-ligands of interest. Functional T cell reactivity against selected antigens was analyzed by intracellular cytokine staining (ICS). Immune cell infiltrates of surgically resected liver metastases were assessed by immunohistochemistry staining (IHC including antibodies specific for CD3, CD4, CD8, CD56, HLA class I and II, and heat shock protein 70) in 11 and 5 patients undergoing hemihepatectomy with or without previous RFA, respectively.
Results: 32 tumor-exclusive T cell epitope candidates were selected based on WTS and ligandome analyses. Antigen-specific T cells were detected in all included patients at least at one timepoint (including boosting of preexisting immune responses and de novo induction). Of note, one patient exhibited RFA-mediated induction of antigen-specific CD4+ T cells against one mutation-derived HLA-class II peptide. This epitope was predicted on the basis of the WST data, but could not be corroborated in MS data as a naturally presented HLA-ligand. Immunohistochemistry of immune cell infiltrates in the lesions resected after RFA did not reveal any significantly increased immune infiltrates in RFA pre-treated patients as compared to the control group.
Conclusions: Specific T cell responses directed against tumor-antigens were detected in every patient investigated. Most notably, T cell responses were boosted or induced in 3 out of 6 patients following RFA, including in one patient against a tumor-specific neoantigen. Still, this tumor-specific immunity per se is probably insufficient to eradicate established tumors. This is underlined by the observation that no increased immune infiltrates in distant metastases were shown. Therefore, our findings support the clinical development of combinatorial therapies, combining RFA with suitable immune stimulatory agents.

Disclosures
Kowalewski: Immatics Biotechnologies GmbH: Employment.