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Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

ABSTRACT: Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient`s tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Michal Bassani-Sternberg

PROVIDER: MSV000080517 | MassIVE | Mon Jan 30 08:10:00 GMT 2017

SECONDARY ACCESSION(S): PXD004894

REPOSITORIES: MassIVE

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Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry.

Bassani-Sternberg Michal M Bräunlein Eva E Klar Richard R Engleitner Thomas T Sinitcyn Pavel P Audehm Stefan S Straub Melanie M Weber Julia J Slotta-Huspenina Julia J Specht Katja K Martignoni Marc E ME Werner Angelika A Hein Rüdiger R H Busch Dirk D Peschel Christian C Rad Roland R Cox Jürgen J Mann Matthias M Krackhardt Angela M AM

Nature communications 20161121

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and pho ...[more]

PMID: 27869121

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Project description:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the presentation and immunogenicity of nonC antigens across different cancer types to better understand their contribution to cancer immunosurveillance and to address whether they could be exploited therapeutically. To this end, we employed a proteogenomics pipeline to identify nonC HLA-I ligands derived from off-frame translation of coding sequences and non-coding regions (UTR, ncRNA, intronic and intergenic) in patient-derived tumor cell lines (TCL) of different histological types (4 gynecological cancer, 3 melanoma and 2 head and neck cancer patients). First, peptides bound to HLA-I were isolated and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using state-of-the-art procedures. Amino acid (Aa) sequences were identified through the previously described pipeline Peptide-PRISM, with some modifications. Briefly, for each MS spectrum, the top 10 candidates were first identified by de novo sequencing and later mapped to a database including the 3-frame transcriptome and 6-frame genome. Additionally, whole-exome sequencing (WES) information of each TCL was included to interrogate the presentation of mutated peptides derived cancer-specific NSM. The false-discovery rate (FDR) was calculated independently for each category considering the search space and peptide length in a stratified mixture model as previously described. Next, in order to select nonC peptides preferentially presented by tumor cells, immunopeptidomics data from several healthy tissues and samples available from the HLA ligand atlas was used to filter out peptides presented in healthy tissues. To overcome a potential bias toward frequent alleles, the peptides were excluded at the ORF level rather than the Aa sequence. As a result, we found that from a total of 839 unique nonC peptides detected in our tumor samples, 38.5% were predicted to derive from ORFs also present in healthy tissue (nonC-HL). Hence, 61.6% (n=517) were considered preferentially presented on tumor HLA-I, referred to as non-canonical tumor ligands (nonC-TL). Out results showed that, although nonC-TL constitued the most abundant source of candidate tumor antigens, as compared to neoantigens, cancer-germline or melanoma-associated antigens, pre-existing antitumor T cells in cancer patients preferentially recognized neoantigens rather than nonC-TL. Nonetehless, nonC-TL elicited de novo T-cell responses via in vitro sensitization of donor lymphocytes. We identified TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of the C5orf22C. These immunogenic nonC-TL were expressed across tumor types but were barely or not detected in healthy cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they are attractive novel targets for widely applicable immunotherapies.