Abstract B23: P1 glycosphingolipid is expressed on ovarian cancer cells recognized by naturally occurring anti-P1 antibodies
Authors: Jacob Francis , Anugraham Merrina , Shahidul Alam , Bovin Nicolai , Packer Nicole , Hacker Neville , Heinzelmann-Schwarz Viola 
Published: August 2014

Abstract
Using printed glycan array we have recently demonstrated that natural occurring anti-glycan antibodies to chemically synthesized P1 trisaccharide and Pk are significantly reduced in ovarian cancer patients (1). These findings were successfully validated with two in-house developed independent glycan-based immunoassays (ELISA and suspension array) (2). Both candidates are glycosphingolipids (GSL) and share similar structural motifs. P1 naturally occurs as a pentasaccharide belonging to the neolacto series. Here we aim to a) validate our findings in an independent validation cohort, b) identify the immunoglobulin subtype causing this observation, c) identify the corresponding naturally expressed P1 antigen in cancer cells, and c) purify human anti-P1 antibodies from cancer ascites.
A cohort of 155 blood plasma samples comprising healthy controls and patients with cancer of the ovary, tube and peritoneum was profiled for P1 antibodies using monoplex suspension array. GSL isolated from fresh cancer tissue were PVDF blotted and extracted enzymatically following purification of glycan alditols. Glycan structures were identified by negative-mode LC-ESI-MS. P1-expressing cancer cell lines were detected using flow cytometry. Human anti-P1 antibodies were affinity purified from cancer ascites and studied using glycan-based immunoassays.
Glycan-based immunoassay profiling of validation cohort confirmed our recent findings, reduced anti-P1 antibody levels in cancer patients compared to healthy controls (p=0.0002). We could also demonstrate that this observation was caused by IgM subtypes and not IgG. Corresponding P blood group- related antigens (GSL) including P1 and Pk were identified in cancer tissue samples. Flow cytometry revealed that a serous ovarian cancer cell line is positive for P1 (up to 46%). We further demonstrate that affinity purified anti-P1 antibodies bind naturally expressed P1 GSL on ovarian cancer cell surface. Monoclonal as well as affinity purified anti-P1 antibodies applied to identify P1-expressing cancer cell lines revealed high specificity to P1 shown by inhibition assay and glycan-based immunoassays.
In this study we demonstrated anti-P1 IgM levels are decreased in a significant number of cancer patients and that the GSL P1 is present on ovarian/ peritoneal cancer cells, both in tissue and immortalized cell lines. We can therefore conclude that P1 is an epitope for human circulating anti-glycan antibodies. These results are in concordance with our previous findings and support the presence of a new antigenic tumor-associated GSL in ovarian cancer. The study of the molecular profile leading to the presence of P1 in ovarian cancer cells is presently under investigation.
1. Jacob F, et al. (2012) Serum antiglycan antibody detection of nonmucinous ovarian cancers by using a printed glycan array. International Journal of Cancer 130(1):138-146.
2. Pochechueva T, et al. (2011) Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies. Glycoconjugate journal 28(8-9):507-517.
Citation Format: Francis Jacob, Merrina Anugraham, Alam Shahidul, Nicolai Bovin, Nicole Packer, Neville Hacker, Viola Heinzelmann-Schwarz. P1 glycosphingolipid is expressed on ovarian cancer cells recognized by naturally occurring anti-P1 antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B23.