Elevated Level Of Serum Glycoprotein Bifucosylation And Prognostic Value In Chinese Breast Cancer

Objective: Aberrant glycosylation is highly associated with cancer progression and has been explored as an established hallmark of cancer. The aim of this study is to detect the expression of N-glycans and find out the differences in serum of breast cancer patients and healthy controls by glycomics method, and identify the the aberrant N-glycan structures specific to breast cancer. We compare the N-glycans in serum between healthy controls and breast cancer patients with two different stages, with the goal of providing a potential indicator for monitoring the recurrent and metastasis of breast cancer.Methods:(1) N-glycans were relased by PNGase. Various N-glycans were purified by Sep-Pak C18 cartridges and porous graphitized carbon(PGC) column.(2) Permethylated N-glycans of the glycoproteins extracted from 91 breast cancer patients and 43 healthy controls were detected using LTQ-ESI-MS which appeared to be a highly sensitive and informative approach to detect and identify N-glycans.(3) In order to further confirm the structure of all N-glycans, we used sequential multistage MS technology to compare their linkage and branching spectra details.(4) ELISA test proved there was higher expression level of Lewis X and UEA-1 in breast cancer patients, compared to healthy control. The expression level of Lewis A and AAL was no significant difference between two groups.(5)Various statistical analysis tools were applied, including t test, partial least squares discriminant analysis(PLS-DA) and receiver operating characteristic curve(ROC), to evaluate the MS profiles.Results:(1) The expression level of five bifucosylated N-glycans(peaks m/z 1127, 1228, 1352, 1409 and 1453) were increased significantly(P < 0.001) in the serum of breast cancer patients.(2) The structural information of N-glycans were clear by MSn analysis.(3) ROC analysis of the data about MS1 pattern respectively from healthy controls and breast cancer samples showed that the AUC value of the combination of five bifucosylatied N-glycans was 0.913.(4) In addition, The combination of the bifucosylation degree and CEA had an AUC of 0.963 to distinguish the healthy controls and breast cancer patients, showing a distinctively improved diagnostic performance compared to that of CEA alone(AUC=0.794).Conclusion: In our study, we have described a relative quantitative mass spectrometry-based approach to determine fucosylation alterations of serum proteins in 91 breast cancer patients. The results showed that the bifucosylation degree was clearly elevated in recurrent breast cancer patients, comparing healthy controls and initial diagnostic breast cancer patients. The increased N-glycans may be considered as potential targets for drugs in tumor therapy. Meanwhile, knowledge of these specific N-glycans may help to understand the mechanisms of progression and metastasis of breast cancer.