title: The glycosphingolipid P₁ is an ovarian cancer-associated carbohydrate antigen involved in migration.
abstract: BACKGROUND
  The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid.
  
  
  METHODS
  An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system.
  
  
  RESULTS
  Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels.
  
  
  CONCLUSIONS
  This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.
authors.affiliation: 1] Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland [2] Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Prince of Wales Clinical School, Building C25 Kensington Campus, Sydney, NSW 2052, Australia.
authors.forename: F
authors.initials: F
authors.lastname: Jacob
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Balaclava Road, North Ryde, Sydney, NSW 2109, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Anugraham
authors.affiliation: Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Pochechueva
authors.affiliation: 1] Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Prince of Wales Clinical School, Building C25 Kensington Campus, Sydney, NSW 2052, Australia [2] Australian Prostate Cancer Research Centre Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Brisbane, QLD 4102, Australia.
authors.forename: B W C
authors.initials: BW
authors.lastname: Tse
authors.affiliation: Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Alam
authors.affiliation: 1] Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland [2] Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Prince of Wales Clinical School, Building C25 Kensington Campus, Sydney, NSW 2052, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Guertler
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, Moscow 117997, Russian Federation.
authors.forename: N V
authors.initials: NV
authors.lastname: Bovin
authors.affiliation: Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Fedier
authors.affiliation: Gynaecological Cancer Centre, Royal Hospital for Women, School of Women's and Children's Health, Barker Street, Randwick, NSW 2031, Australia.
authors.forename: N F
authors.initials: NF
authors.lastname: Hacker
authors.affiliation: Division of Thoracic Surgery and Thoracic Oncology, Department of Cardiothoracic Surgery, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Huflejt
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Balaclava Road, North Ryde, Sydney, NSW 2109, Australia.
authors.forename: N
authors.initials: N
authors.lastname: Packer
authors.affiliation: 1] Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland [2] Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Prince of Wales Clinical School, Building C25 Kensington Campus, Sydney, NSW 2052, Australia [3] Gynaecological Cancer Centre, Royal Hospital for Women, School of Women's and Children's Health, Barker Street, Randwick, NSW 2031, Australia.
authors.forename: V A
authors.initials: VA
authors.lastname: Heinzelmann-Schwarz
mesh_terms: D000912:Antibodies, Neoplasm / Q000276:immunology / Q000302:isolation & purification; D000951:Antigens, Neoplasm / Q000276:immunology*; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D005260:Female; D005434:Flow Cytometry; D006028:Glycosphingolipids / Q000276:immunology*; D006801:Humans; D009362:Neoplasm Metastasis / Q000276:immunology*; D010051:Ovarian Neoplasms / Q000276:immunology* / Q000473:pathology
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D006028:Glycosphingolipids