The Clinical Diagnostic Study On Alpha 2 Macroglobulin And Its Core Fucosylation In Hepatocellular Carcinoma

Primary liver cancer(PLC)is one of the major global health problems,with high mortality.It is one of the most common malignant tumors in China and even in the world,ranking the second in the cause of cancer death in China.HCC is the most common type of primary liver cancer.The main cause of HCC in China is related to hepatitis B virus.The adverse outcome of hepatitis includes liver cirrhosis and liver cancer,but the onset of liver cancer is relatively hidden,and the patients have been found in the middle and late stage.About 50.5%of new patients and 51.4%of cancer-related deaths occur in China every year.Currently,alpha fetoprotein(AFP)is widely used as a diagnostic marker of liver cancer,but it will also increase in some benign liver diseases,which can not meet the needs of clinical diagnosis of liver cancer.In mang human diseases,such as:the occurrence of tumor and autoimmune disease development process with abnormal modification of protein glycosylation.In recent years,the study of glycomics has attracted much attention.The research at home and abroad including our laboratory,glycosylation techniques including mass spectrometry and capillary electrophoresis were used to find the differential proteins of liver cancer and cirrhosis from the glycosylation level.Matrix assisted laser desorption mass spectrometry(MALDI-MS)was used to detect the serum of patients with different liver diseases.The glycochain structure of single protein was analyzed to explore its clinical application value to liver cancer,and to reveal that these differential glycoproteins have certain application prospects as liver cancer markers.At present,most of the serum markers of tumor screening are glycoproteins,especially some glycoproteins with glycosylation changes have been used in the diagnosis and prognosis evaluation of human diseases,such as AFP and core fucosylated AFP(AFP-L3).Alpha 2 macroglobulin(AMG)is the highest molecular weight protein in plasma,which is mainly synthesized by liver cells and mononuclear macrophage system.AMG is a 720 kDa homotetramer,it plays an important role in maintaining the homeostasis of cytokines and growth factors.As an important acute phase response protein,it participates in the pathophysiology of various diseases.AMG can be activated by intracellular proteases.The activated AMG binds to the corresponding cell surface receptors.As a regulator of many signaling pathways,participates in cell regulation,AMG plays an important role in the development of tumors.The results of Poon et al suggest that the expression of AMG in liver cancer was significantly higher than that in normal liver tissue.The results showed that the expression of AMG in liver cancer was significantly higher than that in normal liver tissue.Glucose-regulated protein 78(GRP78)is a cell surface receptor specifically binding to AMG.AMG combined with GRP78 on the cell surface can promote the interaction between EGFR and c-Src,phosphorylate EGFR at Y1101 and Y845,and promote the invasion and metastasis of liver cancer.On the basis of previous studies on N-glycan marker of liver diseases in our laboratory,we focused on AMG,one of the specific proteins in serum,as the research target molecule,to explore the changes of AMG in the occurrence and development of liver cancer,elaborate the correlation between the changes and the occurrence and development of liver cancer,and on this basis,to establish the detection method of core fucosylated AMG(LCA-AMG)by using lens culinaris agglutinin(LCA),which can specifically combine with core fucosylated AMG,to detect the specific protein of abnormal glycosylated modification.To study the changes of protein level and fucosylation level in chronic hepatitis B(CHB),liver cirrhosis(LC)and HCC and their potential clinical diagnostic value.Part 1:Study on the clinical diagnosis of serum alpha 2 macroglobulin in Hepatocellular carcinomaIn this study,serum AMG levels of HCC,LC and CHB were analyzed and their clinical significance was evaluated.We collected 402 cases of HCC,151 patients with liver disease without HCC(including 135 patients with liver cirrhosis and 16 cases of chronic hepatitis B).The serum level of AMG was measured by immunoturbidimetry.The serum AMG levels of HCC,LC and CHB groups were 2.32(1.90,2.74),2.02(1.66,2.48),1.89(1.58,2.77)[median(25%,75%)](unit:g/L),respectively.Compared with LC group,the serum AMG level of HCC group was significantly higher(P<0.001);HCC and CHB group there was no statistically significant difference(P>0.05).The serum AMG level of HCC group was significantly higher than that of Disease control group(including LC and CHB)[2.32(1.90,2.74)vs 1.98(1.65,2.48),P<0.001].Serum AMG level was positive correlated with the laboratory indexes such as HA(R=0.115,P=0.022),GLB(R=0.235,P<0.001),ALB(R=0.157,P=0.002),TP(R=0.292,P<0.001);and was negatively correlated with the laboratory indicators such as GGT(R=-0.105,P=0.037).There was no statistically significant difference of serum AMG level between the TNM stage of HCC and microvascular invasion(MVI).For the follow-up patients with HCC,through the analysis of survival curve,it was found that the patients with higher serum AMG had lower recurrence-free survival rate and lower survival rate(P<0.05).Part ?:Study on the clinical diagnosis of core fucosylated alpha 2 macroglobulin in Hepatocellular carcinomaIn this study,413 serum samples,including 193 HCC patients,104 LC patients,45 CHB patients and 71 healthy controls(HC),were collected to analyze the value of core fucosylated AMG in the differential diagnosis of HCC from non-HCC.The quantitative determination of core fucosylated AMG in serum was carried out by the combination of LCA affinity chromatography and immunonephelometry.The levels of core fucosylated AMG(LCA-AMG)of HCC,LC,CHB and HC groups were 0.74(0.55,0.94),0.56(0.42,0.72),0.54(0.45,0.93)and 0.35(0.28,0.44)[median(25%,75%)](g/L),respectively.The study found that the level of LCA-AMG in HCC group was significantly higher than LC group and HC group,and had significant statistical significance(P<0.001),HCC group and CHB group had no significant difference(P>0.05),LC group and CHB group were higher than HC group(P<0.001);The area under ROC curve of LCA-AMG for identifying HCC from non-HCC(LC,CHB and HC)was 0.751 and 95%confidence interval was 0.706-0.792.The levels of LCA-AMG/AMG�100(LCA-AMG%)of HCC,LC,CHB and HC groups were 31.25(26.61,35.42),26.00(22.30,30.64),26.23(23.86,31.89),20.29(17.35,22.60)[median(25%,75%)],respectively.It was statistically significantly higher in HCC group than that in LC group(P<0.001),CHB group(P<0.01)and HC group(P<0.001).The levels of LCA-AMG%in LC group and CHB group were significantly higher than HC group(P<0.001).The area under ROC curve of LCA-AMG%for identifying HCC from non-HCC(LC,CHB and HC)was 0.768 and 95%confidence interval was 0.725-0.808.Serum LCA-AMG level of HCC group was negatively correlated with the laboratory indicators such as ALP(R=-0.160,P=0.029),GGT(R=-0.214,P=0.003).Serum LCA-AMG%level was positive correlated with some indexes related to clinical liver function such as TBIL(R=0.157,P=0.030),DBIL(R=0.148,P=0.041);and was negatively correlated with some indexes related to clinical liver function such as GLB(R=-0.193,P=0.008),ALP(R=-0.151,P=0.040),LDH(R=-0.212,P=0.004).There was no statistically significant correlation between serum LCA-AMG%level with some laboratory indexes such as AST,AFP,ALT,CA199,CEA.To differentiate and diagnose HCC from non-HCC,a joint diagnosis model was established by logistic regression:LogitAMG1=-5.102+0.462�AMG+0.140�LCA-AMG%,the area under the curve was 0.776 and 95%confidence interval was 0.733-0.816.LogitAMG2=-4.025+0.007�AFP+0.163�LCA-AMG%-0.080�TBIL,the area under the curve was 0.918 and 95%confidence interval was 0.887?0.943.The area under ROC curve of LCA-AMG%for AFP negative HCC patients was 0.782,95%confidence interval:0.726?0.832,the sensitivity was 77.42%,and the specificity was 71.36%.With the increase of tumor diameter,serum LCA-AMG%decreased.There was no significant difference of serum LCA-AMG%level in the concentration of AFP,TNM,MVI and the presence or absence of capsule in HCC.This study suggested that LCA-AMG%may have a potential application prospect in the differential diagnosis of HCC and HCC with negative AFP.