title: T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes.
abstract: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.
  
  
  
  We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.
  
  
  
  Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.
  
  
  
  These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Mennonna
authors.affiliation: Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Maccalli
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Michele C
authors.initials: MC
authors.lastname: Romano
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Garavaglia
authors.affiliation: Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Capocefalo
authors.affiliation: Institute for Biomedical Technologies, National Research Council, Segrate, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Bordoni
authors.affiliation: Institute for Biomedical Technologies, National Research Council, Segrate, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Severgnini
authors.affiliation: Institute for Biomedical Technologies, National Research Council, Segrate, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
authors.affiliation: La Jolla Institute for Allergy & Immunology, La Jolla, California, USA.
authors.forename: John
authors.initials: J
authors.lastname: Sidney
authors.affiliation: La Jolla Institute for Allergy & Immunology, La Jolla, California, USA.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sette
authors.affiliation: Institute of Molecular Recognition Chemistry, National Research Council, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Gori
authors.affiliation: Institute of Molecular Recognition Chemistry, National Research Council, Milan, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Longhi
authors.affiliation: Department of Surgery, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Braga
authors.affiliation: Department of Surgery, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Ghirardelli
authors.affiliation: Department of Surgery, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Ludovica
authors.initials: L
authors.lastname: Baldari
authors.affiliation: Department of Surgery, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Orsenigo
authors.affiliation: Department of Pathology, San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Albarello
authors.affiliation: Unit of Molecular and Functional Immunogenetics, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Zino
authors.affiliation: Unit of Molecular and Functional Immunogenetics, San Raffaele Scientific Institute, Milan, Italy.|Institute for Experimental Cellular Therapy, University Hospital Essen, Essen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Fleischhauer
authors.affiliation: Department of Medical Sciences, Center for Transplantation Biology and Immunogenetics, University of Turin, Turin, Italy.
authors.forename: Gina
authors.initials: G
authors.lastname: Mazzola
authors.affiliation: Department of Medical Sciences, Center for Transplantation Biology and Immunogenetics, University of Turin, Turin, Italy.
authors.forename: Norma
authors.initials: N
authors.lastname: Ferrero
authors.affiliation: Department of Medical Sciences, Center for Transplantation Biology and Immunogenetics, University of Turin, Turin, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Amoroso
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Casorati
authors.affiliation: Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Parmiani
authors.affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Dellabona
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D018797:Cell Cycle Proteins / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000276:immunology*; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000032:analysis*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics* / Q000276:immunology; D044783:F-Box Proteins / Q000235:genetics; D000073979:F-Box-WD Repeat-Containing Protein 7; D015870:Gene Expression; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology; D057166:High-Throughput Screening Assays; D006801:Humans; D014411:Neoplastic Stem Cells / Q000276:immunology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D051901:Smad4 Protein / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D018984:Epitopes, T-Lymphocyte; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; D006680:HLA Antigens; C117307:KRAS protein, human; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D016159:Tumor Suppressor Protein p53; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: ANTIGENS; CANCER IMMUNOBIOLOGY; COLORECTAL CANCER; GENE MUTATION; IMMUNE RESPONSE