| Objective:The diagnosis of AFP-negative hepatocellular carcinoma(ANHC)remains a clinically important issue,and the search for effective diagnostic markers for ANHC is of great importance.Aptamers are oligonucleotide sequences that can specifically bind to biological targets and are obtained from a synthetic random single-stranded oligonucleotide library by systematic evolution of ligands by exponential enrichment(SELEX)technology,which have promising applications in oncology diagnosis and treatment.Our group previously selected a group of ANHC serum aptamers and identified dozens of ANHC serum candidate markers utilized aptamer-based serum proteomics.In this study,we measured the serum levels of eight ANHC candidate markers using enzyme-linked immunosorbent assay(ELISA)and evaluated their diagnostic performance for ANHC to identify novel and valuable serum markers for ANHC.Methods:1.collection of study subjects and their serum specimens: serum specimens of patients with hepatocellular carcinoma,cirrhosis,chronic hepatitis and gastric cancer who were hospitalized in the First Affiliated Hospital of Nanchang University from September 2020 to December 2022 and normal individuals with normal physical examination were collected,and relevant clinical data,including general information,medical history,laboratory tests,imaging examinations,pathological examinations and other relevant information.2.Determination of serum levels of ANHC candidate markers: Commercial ELISA kits were used to detect serum levels of 8 candidate markers in patients with ANHC,AFP-positive hepatocellular carcinoma(APHC),liver cirrhosis(LC),chronic hepatitis(CH),gastric cancer(GC)and normal controls.8 candidate serum markers were leucine-rich alpha 2 glycoprotein 1(LRG1),serum amyloid A1 protein(SAA1),serum amyloid A2 protein(SAA2),complement C4b-binding protein alpha chain(C4BPA),haptoglobin(HPT),haptoglobin-related protein(HPR),α1-acid glycoprotein(A1AGP)and heparin cofactor 2(HC Ⅱ).Serum levels of abnormal prothrombin(DCP)were measured simultaneously for comparative studies.3.Evaluation of the diagnostic value of candidate markers for ANHC alone and in combination: the area under the subject operating characteristic(ROC)curve(AUC)was used to evaluate the value of candidate serum markers alone or in combination to discriminate ANHC from controls.Combined analysis of candidate markers was achieved by binary logistic regression analysis modeling.Determine the optimal positive cut-off value based on ROC curve analysis and calculate the diagnostic efficiency(sensitivity,specificity,accuracy,positive and negative predictive value,positive and negative likelihood ratio)of each candidate marker.4.Evaluate the value of candidate serum markers to discriminate ANHC from benign liver disease: ANHC was used as the case group and benign liver disease(chronic hepatitis + cirrhosis)as the control group,and evaluate the value of each candidate marker to discriminate ANHC from benign liver disease,and calculate the diagnostic efficiency.Results:1.Serum levels of candidate markers and their diagnostic value: in this study,the levels of 8 candidate markers were measured in 221-617 serum specimens,and the results were as follows:A1AGP: The serum concentrations of A1 AGP were measured in 52 cases of ANHC and 292 controls,and the serum A1 AGP levels of ANHC were lower than those of controls,among which the differences were statistically significant(P<0.001)with cirrhosis,chronic hepatitis and normal controls,and had moderate value in discriminating ANHC from cirrhosis,chronic hepatitis and normal controls(AUC of0.735-0.778)and better than DCP.SAA2: SAA2 concentrations were measured in 50 ANHC and 306 controls,and serum SAA2 levels were higher in ANHC than in controls,with statistically significant differences(P<0.001)between ANHC and all controls,cirrhosis,chronic hepatitis and normal controls,with moderate to good value in differentiating ANHC from all controls,cirrhosis,chronic hepatitis and normal controls(AUC 0.760-0.001).AUC was 0.760-0.845)and was better than or comparable to DCP.C4BPA: C4 BPA concentrations were measured in 55 ANHC and 448 controls,and serum C4 BPA levels were higher in ANHC than in controls,with statistically significant differences between groups with cirrhosis and normal controls(P<0.001),with moderate and good values in differentiating ANHC from cirrhosis and normal controls(AUC 0.710,0.855,respectively),and better than DCP.HC Ⅱ: HC Ⅱ serum concentrations were measured in 49 ANHC and 420 controls,and serum HC Ⅱ levels were higher in ANHC than in controls,with statistically significant differences between them and chronic hepatitis and normal controls(P<0.001),and had good value in discriminating AFP-negative hepatocellular carcinoma from chronic hepatitis and normal controls(AUC 0.832 and 0.843,respectively),and superior to DCP.HPR: HPR serum concentrations were measured in 27 cases of ANHC and 161 controls,and the serum HPR levels were lower in ANHC than in controls,with statistically significant differences between ANHC and gastric cancer and normal controls(P<0.001),and HPR was superior to DCP and had excellent diagnostic value in distinguishing ANHC from normal controls,but the number of cases was small.HPT: HPT serum concentrations were measured in 47 cases of ANHC and 293 controls,and serum HPT levels were higher in ANHC than in controls,with statistically significant differences between them and cirrhosis,chronic hepatitis and normal controls(P<0.001),with moderate value in differentiating ANHC from cirrhosis and all controls,and good value in differentiating chronic hepatitis from normal controls(AUC of 0.763-0.883),and better than or comparable to DCP.LRG1: LRG1 concentrations were measured in 47 ANHC and 324 controls,and serum LRG1 levels were lower in ANHC than in controls,with statistically significant differences between ANHC and all controls,cirrhosis and normal controls(P<0.001),and of moderate to good value in identifying ANHC from all controls,cirrhosis and normal controls(AUC 0.703-0.847)and better than or comparable to DCP.SAA1: SAA1 concentrations were measured in 45 ANHC and 198 controls,and serum SAA1 levels were higher in ANHC than in controls,but the difference was not statistically significant between ANHC and each control(P>0.05)and was not valuable in identifying AFP-negative hepatocellular carcinoma from each control(all AUCs were less than 0.6).2.Diagnostic value of the combination of candidate serum markers: the combination of two markers and three markers analysis can effectively enhance the diagnostic value.Among the two-marker combination,the best value of A1 AGP combined with SAA1 for the diagnosis of ANHC,with an AUC of 0.893,sensitivity,specificity and accuracy of 77.4%,93.7% and 91.4%,respectively;among the threemarker combination,the best value of DCP,HPT and A1 AGP combined for the diagnosis of ANHC,with an AUC of 0.918,sensitivity,specificity and accuracy of The sensitivity,specificity and accuracy were 85.2%,90.3% and 89.9%,respectively.3.The value of candidate serum markers in distinguishing ANHC from benign liver disease: in distinguishing ANHC from benign liver disease,both SAA2 and HPT had good value with AUC of 0.829 and 0.815,respectively,A1 AGP had moderate value with AUC of 0.744,both better than DCP(AUC=0.616),and the rest of candidate markers had no significant value.Conclusions:1.several candidate serum markers with diagnostic value for ANHC were identified,among which SAA2 was superior to DCP in diagnosing ANHC,and HPT and LRG1 were comparable to DCP and had the potential to become new diagnostic markers for AFP-negative hepatocellular carcinoma;2.the combination of candidate markers can effectively enhance the diagnostic ability of ANHC,among which the combination of two markers,A1 AGP and SAA1,and the combination of three markers,HPT,A1 AGP and DCP,has a more desirable diagnostic value for ANHC;3.some candidate markers are better than DCP in distinguishing ANHC from benign liver disease,among which SAA2 and HPT have good value in distinguishing ANHC from benign liver disease;... |
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