The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Background: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P-1 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 P-1 antigen recognised by circulating anti-P-1 antibodies and to shed light into the possible function of this glycosphingolipid. Methods: An independent Australian cohort was assessed for the presence of anti-P-1 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 P-1 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-P-1 antibodies (P=0.0002). The presence of corresponding antigen P-1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P-1) by showing that human naturally circulating and affinity-purified anti-P-1 IgM isolated from patients ascites can bind to naturally expressed P-1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P-1-high, 66.1%; and P-1-low, 33.3%) and observed that cells expressing high P-1-levels migrate significantly faster than those with low P-1-levels. Conclusions: This is the first report showing that P-1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P-1 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-P-1 IgM antibodies present with a more aggressive phenotype and earlier relapse.