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.