MSACL 2019 EU Abstract

Site-Specific Haptoglobin N-Glycosylation Changes in Colorectal Cancer

Jelena Simunovic (1), Viktoria Dotz (2), Irena Trbojevic-Akmacic (1), Mislav Novokmet (1), Kathrin Stavenhagen (2), Lisa de Neef (2), Rob A. Tollenaar (2), Wilma E. Mesker (2), Iva Kirac (3), Katarina Vucic (4), Marija Pezer (1), Manfred Wuhrer (2), Gordan Lauc (1)
(1) Genos Glycoscience Research Laboratory, Zagreb, Croatia (2) Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands (3) Department of Surgical Oncology, University Hospital for Tumours, Sestre milosrdnice University Hospital Centre, Zagreb, Croatia (4) Department for Safety and Efficacy Assessment of Medicinal Products, Agency for Medicinal Products and Medical Devices, Zagreb, Croatia

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Jelena Simunovic (Presenter) Genos Glycoscience Research Laboratory

Presenter Bio: Jelena Simunovic is a PhD student at University of Zagreb, Croatia. She is doing her PhD at Genos Glycoscience Research Laboratory, Zagreb, Croatia from February 2016. After she graduated with a Master΄s degree from the Faculty of Pharmacy at the University of Sarajevo, Bosnia and Herzegovina, she started her PhD as an early stage researcher within Marie Curie European Training Network GlyCoCan. The main focus of her PhD is haptoglobin glycosylation in colorectal cancer. Part of the research on this she did at Center for Proteomics and Metabolomics in Leiden University Medical Center, Netherlands.

Relevant Financial Disclosures (within past 24 months) Salary Genos Glycoscience Research Laboratory, Zagreb, Croatia Conflict mitigation NOT REQUIRED.

INTRODUCTION: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Europe in both men and women. Discovering less invasive diagnostic tools is essential for clinical practice, in particular for the early stage detection, but also for patient stratification. Previous studies showed significant changes of total plasma N-glycome in CRC. Assessing the site-specific glycosylation of an isolated carrier protein can provide valuable insights into CRC-associated glycosylation signatures. Previously, it was reported that elevated difucosylation of haptoglobin (Hp) tetraantennary glycans can be used to discriminate between early stage of hepatocellular carcinoma and cirrhosis. Hp is an acute-phase protein which is synthesized in the liver and is one of the major serum glycoproteins. We here present the first large-scale study of Hp glycosylation in CRC.
METHODS: Two different methods were applied on two independent clinical cohorts. A nano-LC-ESI-MS method for glycopeptide analysis of the four glycosylation sites was developed and applied on serum samples from a clinical cohort consisting of 186 CRC cases and 186 presumably healthy controls, in addition to 86 post-surgery CRC patients. To this end, Hp was captured from serum using affinity purification, and digested with trypsin, resulting in the detection of 75 different glycopeptide compositions in total. Data processing and quantification were performed in a semi-automated manner using the in-house built software LaCyTools. The second cohort consisted of 224 CRC cases and 268 healthy controls for which Hp was isolated from plasma followed by N-glycan release by PNGase F and measurement by UPLC with fluorescence detection. Using logistic regression, we tested for associations of glycopeptide features with case-control status.
RESULTS: Our preliminary data indicate that Hp fucosylation and sialylation increase in CRC compared to controls. Site-specific analysis revealed specific alteration in synthesis of tetra-antennary structures, i.e. on asparagine 241.
CONCLUSION: Moreover, the independent on-going replication setup will enable us to validate our findings and to compare two different analytical approaches for N-glycosylation analysis and further assess the potential of Hp glycosylation as a biomarker for patient stratification or disease outcomes in CRC.