N-glycomic Monitoring Of Liver Fibrosis And Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related death in the world. In China, hepatitis B Virus (HBV) infection is a well-recognized risk factor for chronic liver diseases, such as cirrhosis and liver cancer. Hepatitis-liver fibrosis/cirrhosis-HCC is the typical pathological progress pathway of chronic hepatitis B. The early diagnosis of liver fibrosis and HCC are of great clinical desirable due to the reversibility of liver fibrosis and the improved prognosis of HCC if the patients could get surgical treatment early. Serum alpha-fetoprotein (AFP) and ultrasonography examination are commonly used in HCC screen from high risk population. But neither of them is sensitive enough to identify HCC in the very early stage. So, a more sensitive and specific non-invasive serological marker is required for the early diagnosis of HCC and for monitoring treatment.Abnormal glycosylation is reported to be associated with malignant transformation of cells. Most serum N-linked glycoproteins are synthesized by the liver or B-lymphocytes, any changes in serum total N-glycans could reflect an alteration of liver or B-lymphocyte physiology. So changes in the quantity and type of N-glycans in serum could be exploited for the non-invasive diagnosis of liver diseases. DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) was proved in the previous study to be a rapid, highly sensitive and high throughput method for N-glycome profiling, making it is available to apply N-glycomics to clinical diagnosis and prediction of HCC. The present study is to assess the diagnostic value of N-glycan in both large sample clicinal case control study and diethylinitrosamine (DENA)-induced HCC rat model. Part 1:N-glycomic study in HBV-related liver fibrosis and HCC patientsThis study aimed to assess the diagnostic value of N-glycan based diagnostic model in HCC identification and follow-up. A total of 393 subjects including HBV-related HCC, liver fibrosis and healthy controls were recruited. Follow-up was carried out before and after surgical treatment in HCC. N-glycome of serum glycoprotein was profiled by DSA-FACE. Multiparameters diagnostic models were constructed based on N-glycan markers. The result showed that two of N-glycan structures (NG1A2F, Peak 4; NA3Fb, Peak 9) were useful as N-glycan HCC markers. The diagnostic efficacy of the log ratio [log(p9/p4)] was similar to that of AFP in differentiating HCC from fibrosis. The accuracy and sensitivity of the diagnostic model combining AFP and N-glycan markers (Cscore B) were increased by 7-10% compared with that of AFP. Log(p9/p4) was more efficient in monitoring the progression of HCC with regarding to vascular invasion at improved specificity (16%) and accuracy (8%) compared with that of AFP. The N-glycan markers were found to be changed significantly after surgical resection in HCC follow-up. We conclude that two N-glycans (NG1A2F and NA3Fb) are promising as N-glycan markers. The diagnostic models based on the N-glycan markers and AFP improve the efficacy in HCC diagnosis and progression monitoring.Part 2:Studies on N-glycome in DENA-induced HCC rat modelWe studied glycomics during development of rat HCC induced by DENA. Both serum and liver protein N-glycans were profiled using the DSA-FACE technique. In comparison with control rats, the abundance of two fucosylated structures (R5a and R5b) in serum total N-glycans of DENA rats increased gradually but significantly during the progression of liver cirrhosis and cancer, whereas a biantennary glycan (P5) decreased. The log of the ratio of R5a to P1 (NGA2F) and R5b to P1, [log(R5a/P1) and log(R5b/Pl)], were significantly (p<0.0001) elevated in HCC rats, but not in cirrhosis, fibrosis compared to control animals. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid (FTS), ananticancer drug, GlycoTest markers [P5, R5a, R5b, log(R5a/P1), log(R5b/P1)] reverted towards non-DENA levels, indicating that FTS prevented progression to HCC. Serum immunoglobulin and liver protein N-glycan profiling showed that GlycoTest marker R5b derived from hepatocyte. Moreover, we found an increase in core-a-1,6-fucosylated glycoproteins in serum and liver tissue of HCC rats, demonstrating altered fucosylation during progression of HCC. The change of RNA lever of glycosylglycosyltransferases in liver tissue showed the mechanism of abnormalglycosylation in liver diseases. In conclusion, our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat.From the present study, we concluded that abnormal fucosylation is closely associated with HCC development, N-glycan showed great value in early diagnosis, monitoring progression and evaluating of drug efficacy. The improvements of diagnostic efficacies confirm again that multiparameters models are more powerful, and promising to be a noninvasive serum maker in early HCC diagnosis.