| Primary hepatic carcinoma(PHC)is one of the most common malignant tumours,whose early detection and diagnosis are very important for the treatment and prognosis of patients,especially for those who suffer from cirrhosis.At present,clinicians tend to take serological diagnosis as the primary screening method for PHC.However,there are few reports about discovering and diagnosing PHC value through applying serum tumour markers of hepatocellular carcinoma in cirrhosis groups.In the first part of this research,it aims at observing and comparing the expression of AFP,AFP-L3%,GP73 and IL-6 in the serum of cirrhosis patients,with or without PHC.To discuss about its function of discovering and diagnosing PHC among cirrhosis patients,and try to find out the most suitable serological indicators to diagnose PHC among cirrhosis people.From January 2014 to December,2015,521 cases of previously untreated liver cirrhosis patients with PHC were enrolled in a prospective parallel control study.And 274 cirrhosis patients without PHC were randomly recruited in our hospital.AFP,AFP-L3,GP73,and IL-6 in the venous blood serum from the patients who were made a definite diagnose before further treatment were detected.Meanwhile,AFP-L3%(AFP-L3/AFP)was calculated.At the same time,venous blood was taken to test liver function,hepadnaviruses copies and other related indicators.And also general data,reports and examination results of patients were gathered and analysed.Results:(1)The expression levels of the serum AFP(Z=10.77,P<0.05),IL-6(Z=15.62,P<0.05)for patients suffering from cirrhosis with PHC were significantly higher than those of cirrhosis patients without PHC,while the expression level of serum GP73(Z=2.45,P<0.05)was significantly lower.The difference of serum AFP-L3% for these two groups of patients did not reach statistical significance(Z=1.92,P=0.055).To further characterize the dynamic fluctuation of all the serum tumour markers during the progress of disease,we classified the patients into the group of compensatory cirrhosis,the group of decompensated cirrhosis,the group with cirrhosis and early HCC,and the group of cirrhosis and liver cancer at middle and later periods,finding out that the expression levels of serum AFP(Z=11.67,P<0.05),AFP-L3%(Z=5.56,P<0.05),and IL-6(Z=20.64,P<0.05)gradually increased with the progress of diseases.Nevertheless,the expression level of GP73 was the highest in the decompensated cirrhosis group,followed by the group of cirrhosis with mid/late stage of liver cancer and the group of compensatory cirrhosis.The differences among different groups also reached statistical significance(Z=9.48,P<0.05).(2)The expression level of AFP was related to the Child-Pugh Score of liver functions.Higher Child-Pugh score was associated with higher expression level with statistical significance(P<0.05);the expression level of AFP-L3% did not have obvious correlation with patients' general situations,liver function,and other factors(P>0.05).The expression level of GP73 was positively correlated to the complications of cirrhosis,including peritonitis,electrolyte disturbances,anemia,hypoproteinemia,ascites,splenomegaly,esophagogastric varices,and portal hypertension,as well as the Child-Pugh Score of liver functions(P<0.05),indicating that GP73 was related to the severity of cirrhosis and its complications.The expression level of IL-6 was positively related to peritonitis,pleural effusion and ascites,and the Child-Pugh score of liver function(P<0.05).The serum AFP,GP73,and IL-6 of patients suffering from viral hepatitis cirrhosis and liver cancer were associated with the titer of virus.(3)In cirrhotic patients with liver cancer,the expression level of serum AFP,AFP-L3%,GP73,and IL-6 was positively correlated with the size of tumour,number of tumour lesion,and the presence of remote metastasis.The inter-group difference had statistical significance(P<0.05).Meanwhile,the expression level of serum AFP,AFP-L3%,and IL-6 but not that of GP73 was positively related to the local vascular invasion.The expression level of IL-6 was positively correlated with lymphatic metastasis.The expression level of GP73 was the highest in patients at BCLC stage B,followed by those at BCLC stage C,and BCLC stage A.(4)In patients with cirrhosis,IL-6 had the highest sensitivity of diagnosing liver cancer(99.8%),and then GP73(75.8%),AFP(45.7%),and AFP-L3%(28.2%).AFP-L3% had the highest specificity(97.4%),followed by AFP(87.2%),IL-6(35.4%),and GP73(23.4%).In diagnosing the early HCC among the persons with cirrhosis,IL-6 had the highest sensitivity(98.6%)and AFP-L3%(97.4%)achieved highest specificity.Compared with AFP,which is commonly used in daily practice,IL-6 and GP73 had higher sensitivity,but lower specificity,while AFP-L3% was more specific but less sensitive.Sequential employment of all kinds of serum tumour markers can enhance the diagnosis accuracy of liver cancer and early HCC among cirrhotic patients.To summarize,we have found in the first part of our research that AFP and AFP-L3% can be effectively used to diagnose liver cancer among persons with cirrhosis.Meanwhile,AFP-L3% is more superior,GP73 and IL-6 cannot be used to diagnose the liver cancer of cirrhosis persons,but they could serve as warning signal for liver cancer.Sequential employment of these tests could improve diagnostic sensitivity and specificity.Moreover,GP73 could have a role in the evaluation of severity of decompensated cirrhosis.Another part of this research is to establish the cGAMP rapid test method.The first part of this research showed that IL-6 had the highest sensitivity in diagnosing liver cancer among cirrhotic patients,which might be suitable for screening patients with liver cancer,but its specificity was poor.During the development of tumour,IL-6 is controlled by NF-?B,which is influenced by the cGAS-STING pathway,which is associated with inflammation-related tumours.Therefore,we can increase the diagnostic specificity of IL-6 by evaluating the activation level of cGAS-STING pathway.In other words,the activation level of this pathway might act as a candidate predictor of early HCC.Since the level of cGAMP can reflect the activation level of cGAS-STING pathway,we choose to measure cGAMP level and endeavoured to establish a rapid method in achieving this goal,as well as to provide technical support for the follow-up study.First,we stimulated the cells of liver cancer(BEL-7402cells)with different concentrations of HT-DNA,and determined that 2?g/ml was the appropriate concentration for following experiments by measuring the mRNA level of IFN? through Real-Time PCR.Second,we repeated the measurement at 2 hour and 4 hour intervals after stimulating the cells of liver cancer by HT-DNA or saline control,obtaining the cGAMP level of cell supernatant by LC-MS/MS,and performing Real-Time PCR and Western blot to measure the activity of cGAS-STING pathway.The results revealed that with the lengthening of the stimulation time of HT-DNA,the cytosolic cGAMP increased remarkably,and the expression level of mRNA for IFN?,as well as the phosphorylation of protein IRF3 also increased significantly,indicating that cGAMP level dose characterize the activation level of cGAS-STING pathway.Moreover,LC-MS/MS could be an effective and rapid method to measure the cGAMP level in cells of liver cancer. |
PDF Full Text Request