| Background and objectives:Alpha-fetoprotein(AFP)-negative primary hepatic carcinoma(ANHC)is the main problem in the diagnosis of hepatic carcinomar at present.It is necessary to find new effective markers of ANHC.The aptamer is an artificial nucleic acid ligand of biological molecule,which is similar to antibody in function,but has more advantages in application.It is a new way to discover new tumor markers.In the previous work,our team screened a group of serum adapters with good diagnostic value for ANHC by targeting mixed serum of ANHC captured and identified their serum target proteins by mass spectrometry,and found a group of deferentially expressed target proteins.In this study,we will screen some differentially expressed proteins and detect their serum expression levels by ELISA to evaluate their diagnostic value for ANHC in order to find new serum markers for ANHC.Methods:1.Collection of serum samples: serum samples collected from hospitalized ANHC,AFP-positive liver cancer,liver cirrhosis,chronic hepatitis patients,other patients with digestive system,and normal physical examinations at the First Affiliated Hospital of Nanchang University from 2016 to 2018.Store at-80°C for later use.At the same time,relevant clinical data were collected,including blood cell analysis,liver and kidney function,blood lipids,blood glucose,electrolytes,coagulation function,and imaging and histopathological data.2.Screening of candidate serum markers for ANHC: To evaluate the diagnostic value of a batch of differentially expressed target proteins detected by multiple reaction monitoring mass spectrometry(MRM)technique in the early stage of the project,and screen out the specific or expressed proteins of ANHC which are more than two times higher than those of the control group.At the same time,to consult the literature to find out the target proteins of hepatocellular carcinoma or tumor-related proteins,and to select the diagnostic price The target protein with higher value was identified as a candidate serum marker for ANHC for further analysis.3.Small sample to verify the diagnostic value of candidate serum markers for ANHC: 16 cases of AFP-negative hepatocellular carcinoma,16 cases of AFP-positive hepatocellular carcinoma,12 cases of cirrhosis,12 cases of chronic hepatitis,12 cases of other tumors of digestive system,12 cases of normal control,80 cases of serum samples were collected.The confirmed candidate markers and serum DCP were detected by ELISA,and their serum samples were analyzed quantitatively in 80 cases of different diseases mentioned above.The level of expression in specimens.ROC curve was used to evaluate the diagnostic value of candidate markers for ANHC.Sensitivity,specificity,accuracy,positive and negative predictive values,positive and negative likelihood ratios were calculated.Candidate markers with better diagnostic value than DCP or complementary diagnostic value to DCP or complementary diagnostic value between markers were identified as new serum markers of ANHC for further analysis.4.Expanding the sample to further evaluate the diagnostic value of candidate serum markers for ANHC: 80 cases of AFP-negative hepatocellular carcinoma,80 cases of AFP-positive hepatocellular carcinoma,80 cases of cirrhosis,80 cases of chronic hepatitis,40 cases of other tumors of digestive system,40 cases of normal control,a total of 400 new serum samples were taken to detect the candidate markers and serum DCP with good diagnostic value in the aforementioned small samples by ELISA.Quantitative analysis of their expression levels in serum samples of 480 patients with different diseases was performed.ROC curves were used to evaluate the diagnostic value of ANHC alone,and the sensitivity,specificity,accuracy,positive and negative predictive values,positive and negative likelihood ratios were calculated.5.The diagnostic value of candidate serum markers and DCP for ANHC was analyzed jointly.The diagnostic value of candidate serum markers and DCP was evaluated.ROC curve was used to evaluate the diagnostic value of their combination(logistic regression modeling)for ANHC.Sensitivity,specificity,accuracy,positive and negative predictive values,positive and negative likelihood ratios were calculated.Results:1.Candidate serum markers of ANHC preliminary screening results: among the 20 serum proteins of,13 serum protein single indicators distinguish ANHC from cirrhosis with AUROC of 0.7 or more;4 serum protein multi-indicators combined analysis and diagnosis The AUROC of ANHC was above 0.8,and the AUROC of A4 negative liver cancer combined with C4 BPA and SAA1 was 0.928,and the expression levels of two target proteins in ANHC and benign liver disease were 1.5 times higher than that of the control.the above.The literature found that serum protein SAA2 is a liver cancer or tumor associated protein.Preferably,the three AFP negative liver cancer serum proteins are candidate serum protein markers.2.Diagnostic value of small sample to verify ANHC candidate serum protein markers: small sample clinical serum samples(16 cases of AFP negative liver cancer,16 cases of AFP positive liver cancer,12 cases of liver cirrhosis,12 cases of chronic hepatitis,12 other tumors of digestive system 12 For example,12 cases of normal control),the diagnostic value of the above three candidate serum protein markers for ANHC was analyzed.The AUROC of serum protein SAA1 and serum protein C4 BPA were 0.921 and 0.977,respectively.The multi-index combined analysis AUROC was above 0.9,which was obviously excellent.At DCP.The diagnostic value of SAA2 serum protein was poor,and the AUROC for identifying ANHC was only 0.540.Comprehensive analysis,preferably serum protein SAA1,serum protein C4 BPA for expanded sample detection.3.Expanding the sample to evaluate the diagnostic value of candidate markers for ANHC: 400 clinical serum samples(80 cases of AFP-negative hepatocellular carcinoma,80 cases of AFP-positive hepatocellular carcinoma,80 cases of cirrhosis,80 cases of chronic hepatitis,40 cases of other tumors of digestive system,40 cases of normal control)were detected,and the results of small sample test showed that the diagnosis of ANHC was effective.C4 BPA showed a good diagnostic value for AFP-negative hepatocellular carcinoma.AUROC was 0.839,similar to DCP(AUROC=0.845).C4 BPA showed a good diagnostic value.AUROC could also be more than 0.8 in distinguishing ANHC from non-hepatocellular carcinoma.The sensitivity and specificity of SAA1 for single diagnosis of ANHC were only 94.8% and 46.2%,which was not superior to DCP.Most noteworthy is that C4 BPA is superior to DCP in differentiating ANHC from cirrhosis,with AUROC of 0.846 and 0.759,respectively.4.Diagnostic value of combined ANHC serum markers and DCP in hepatocellular carcinoma: Candidate serum markers and DCP were analyzed to evaluate their diagnostic value.The results showed that the combination of SAA1 and CB4 PA had a sensitivity of 80.2% and specificity of 78.8% for AUROC,a sensitivity of 0.846,a sensitivity of 80.2%,and a specificity of 65.6%,a specificity of 92.8% for SAA1 and DCP,and a sensitivity of 71.9% and a specificity of 91.1% for the combination of C4 BPA and DCP.AUROC was 0.883,sensitivity 72.0% and specificity 91.0% for combined diagnosis of ANHC.Conclusions: 1.Serum target proteins captured by serum adapters of hepatocellular carcinoma have different diagnostic value for ANHC.The candidate markers SAA1,SAA2 and CB4 PA of ANHC were successfully selected.2.The expression levels of SAA1 and CB4 PA in ANHC group were higher than those in control group.SAA1 has certain diagnostic value for ANHC,while C4 BPA has good diagnostic value.Combined analysis with DCP can improve the diagnostic ability and can be used as a potential new serum marker for ANHC. |
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