Unique Alterations in the Immunopeptidome of Colorectal Cancer Reflect Specific Transformations in Cancer-Associated Signaling Pathways and Reveal Tumor-Specific HLA-Ligand Modulations

Background: Immunotherapy with checkpoint-inhibitors has shown spectacular results in the treatment of certain cancer types including microsatellite instable colorectal cancer (CRC). Applicability is believed to be dependent on the number of potential neo-epitopes derived from genetic mutations that are presented on cancer cells. In case of most microsatellite-stable CRC however, clinical responses to immune checkpoint blockade are so far disappointing. Therefore, we analyzed the non-mutant HLA immunopeptidome of CRC in order to provide an extensive dataset for the development of immunotherapeutic strategies in this very common malignancy.

Methods: Tissue specimens from 35 primary CRC and corresponding non-malignant colon were analyzed after HLA immunoprecipitation by uHPLC tandem mass spectrometry. Maximally attainable quantities of source proteins (MAQS) expectable in HLA-ligandomes were estimated by regression analyses. Identified peptides and source proteins were annotated for their pathway association using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the protein analysis through evolutionary relationships (PANTHER). HLA-ligands and source proteins were further analyzed semi-quantitatively assessing significant modulations on CRC tissue compared to adjacent benign colon, with particular focus on specific alterations observed exclusively in CRC tissue. Immunogenicity of the identified HLA-ligands (prioritized ligands for the 8 most frequent HLA-alleles HLA A*01, A*02, A*03, A*24, B*07, B*08, B*44, C*07) was evaluated in peripheral blood mononuclear cells (PBMC) from 50 additional CRC patients and 120 healthy controls (15 each for the 8 most frequent HLA-types) using ELISpot and flow cytometry.

Results: For MHC class I, peptides from 7684 source proteins (81% MAQS) were identified on CRC, as well as peptides from 6312 source proteins on (non-malignant) colon tissue (79% MAQS). For MHC class II, peptides from 1602 source proteins (63% MAQS) were identified, as well as peptides from 3835 source proteins on non-malignant colon tissue (75% MAQS). HLA-ligands and their respective source proteins were compared on tissue level (CRC vs. adjacent benign tissue), as well as against a database of 100 non-malignant human tissues of different origin in order to identify ligands and source proteins exclusively presented on CRC tissue. Implementing KEGG and PANTHER pathway analysis, overrepresented source proteins within MHC class I and II restricted ligands could be assigned to classical tumorigenesis pathways like WNT- and integrin signaling, as well as to the p53 signaling pathway. HLA ligands were further semi-quantitatively analyzed comparing tumor and autologous adjacent colon tissue leading to the exclusive identification of 1364 up-modulated and 1070 down-modulated source proteins in CRC tissue. Notably, 3 source proteins (represented by 10 HLA-ligands) showing significant up-modulation and frequent tumor-exclusive detection of derived HLA-ligands (in ≥3 CRC) were identified (LAMC-2, SLC52A2, SULF-1). For MHC class II, a single up-modulated source protein with exclusive detection in ≥2 CRC tissues (IL6R) was identified. Further analyses of HLA class I restricted peptides (n=359) derived from simultaneously up- and down-modulated source proteins revealed that the respective modulation was mainly a peptide sequence specific feature (31/359 (8.6%) peptides with up- and down-modulation). Preexisting T cell responses were observed against one tumor-exclusive up-modulated peptide (SULF-1) in CRC patients and 3 HLA restricted peptides established as immunogenic epitopes in CRC patients (TACC2, TNS4, IGHG2), as well as 2 additional HLA-restricted peptides confirmed as epitopes in healthy controls (GLA, ESRRA).

Conclusions: We provide the first comprehensive analysis of the HLA immunopeptidome in a solid cancer (CRC). We observed that the presented ligandome can reflect tumor-specific alterations in protein metabolism. Moreover, tumor-exclusive up- and down-modulation of HLA-peptides was mainly sequence-specific, suggesting a differential posttranslational regulation of HLA-restricted peptides in CRC. The described approach for identification of relevant antigens might also enable patient-specific immunotherapeutic approaches in CRC patients.