Diagnostic Evaluation Of Aptamers Against AFP-negative Hepatoma Serum For Primary Hepatic Carcinoma

Background and objective:Primary hepatic cancer(PHC)is one of the common malignant tumors.Most of the patients were in the middle or late stage at diagnosis.Imaging examination is one of the main clinical diagnostic methods,but it is not satisfactory for the diagnosis of small carcinoma.Although the detection of PHC by serum AFP is simple and convenient,nearly 40% of PHC patients were AFP negative.Aptamers,artificial nucleic acid ligands of biological molecules,are selected by the SELEX technique from a synthetic library of random single-stranded oligonucleotides,which has a good prospect in the diagnosis of diseases.In previous work,a group of aptamers against AFP-negative hepatoma serum was selected and showed valuable in the diagnosis of AFP negative PHC,but their diagnostic value is unknown in real clinical settings.In this study,we evaluated the diagnostic values of these aptamers for primary hepatic carcinoma in real clinical settings.Methods:1.Collection of serum specimens and clinical data: Serum specimens were collected from the hospitalized patients with PHC,liver cirrhosis,chronic hepatitis and normal controls in the First Affiliated Hospital of Nanchang University from 2015 to 2016 and refrigerated at-80 oC.Their clinical data available were also collected,including blood cell analysis,liver and kidney function tests,blood lipids,blood glucose,electrolytes,coagulation function and imaging and pathology.2.Optimal selection of aptamers against AFP-negative hepatoma serum by a small sample: The fluorescence intensities before and after adding Eva Green and aptamers were measured in 32 serum samples of PHC and liver cirrhosis each by the triple fluorescent analysis method established in our previous work.The area under receiver operational characteristic(ROC)curve(AUROC)was used to evaluate the diagnostic values of single fluorescence indicators and multiple fluorescence indicators(by Logistic regression model)for PHC.The aptamers with good diagnostic value were used for next analysis.3.Optimal selection of aptamers against AFP-negative hepatoma serum by an enlarged sample: The same method was used to measure the triple fluorescence of the optimal aptamers in 64 specimens of PHC and cirrhosis each,and the results were merged with the small sample analysis data(totally,96 cases of PHC and liver cirrhosis each)for diagnostic evaluation by AUROC.The aptamers with good diagnostic value of liver cancer was used for next analysis.4.Optimization of the experimental conditions: The triple fluorescence of optimal aptamers were measured under 6 experimental conditions generated according to different dosages of aptamer with Eva Green in 64 serum specimens of PHC and liver cirrhosis each.ROC curve was used to evaluate the diagnostic value of various conditions for PHC.The condition with maximum AUROC was selected as the optimal experimental condition for the next analysis.5.Diagnostic evaluation of the optimal aptamers for PHC: The triple fluorescence intensities of the selected aptamers were measured under the optimized experimental condition in 160 serum samples of PHC,160 serum samples of cirrhosis,113 serum samples of chronic hepatitis,97 serum samples of normal control.ROC curve were used to evaluate the diagnostic values of fluorescence indicators single and combined(Logistic regression modeling)for diagnosing PHC,and then determined the cut-off value to calculate the diagnostic performance,including sensitivity,specificity,accuracy,predictive value and likelihood ratio.6.The diagnostic performance of serum tumor markers for PHC: The results of conventional tumor markers(AFP?CEA?CA125 and CA19-9)of the serum samples were collected and their diagnostic value was analyzed.7.Combination of aptamers and serum tumor markers for the diagnosis of PHC: The triple fluorescent indicators of aptamers were combined with serum tumor markers to establish diagnostic models and their diagnostic value for PHC were evaluated by ROC curve.Results:1.Optimal selection of aptamers against AFP-negative hepatoma serum by a small sample: Fifty five AFP-negative hepatoma serum aptamers were synthesized and analyzed in the small sample size and the results showed various aptamers had different values for PHC diagnosis,with the largest AUROCs 0.687-0.918 in single fluorescence indicators,and 0.794-0.979 in diagnostic models.Eighteen aptamers with AUROC more than 0.8 in single fluorescence indicator or 0.950 in multiple fluorescence indicators model were selected for further analysis.2.Optimal selection of aptamers against AFP-negative hepatoma serum by an enlarged sample: The 18 preferred aptamers were evaluated in 64 serum samples of PHC and liver cirrhosis each.The results showed that AUROCs for diagnosing PHC were 0.652-0.813 in single fluorescence indicators and 0.713-0.937 in the multiple fluorescence indicators.Two test results were merged for further evaluation,and finally we selected two aptamers AP-ANHC-8-1-7 and AP-ANHC-8-2-7 for next diagnostic evaluation.3.Optimization of the experimental conditions: Experimental conditions were optimized for two aptamers.The results showed that AP-ANHC-8-1-7 and AP-ANHC-8-2-7 had the same optimal experimental condition,that is,aptamer 0.5pmol and 2 × Eva Green 0.5?L,under which the AUROCs of multiple fluorescence indicators were 0.895 and 0.886.4.Diagnostic evaluation of the two aptamers for PHC: Aptamer AP-ANHC-8-1-7 distinguished PHC from non-PHC by multiple indicators with AUROCs 0.872,and the sensitivity,specificity and accuracy were 58.8%,91.4% and 81.5%,respectively;Aptamer AP-ANHC-8-2-7 distinguished PHC from non-PHC by multiple indicators with AUROCs 0.860,and the sensitivity,specificity and accuracy were 51.3%,87.8% and 76.8%,respectively;The combination of AP-ANHC-8-1-7 with AP-ANHC-8-2-7 showed more powerful for distinguished PHC from non-PHC by multiple indicators with AUROCs 0.898,and the sensitivity,specificity and accuracy were 61.3%,89.7% and 81.1%,respectively.5.Combination of aptamers and serum tumor markers for the diagnosis of PHC: The combination of 4 serum tumor markers for distinguished PHC from non-PHC by multiple indicators with AUROCs 0.820,and the sensitivity,specificity and accuracy were 51.0%,95.8% and 81.0%,respectively;The combination of AP-ANHC-8-1-7 with 4 serum tumor markers for distinguished PHC from non-PHC by multiple indicators with AUROCs 0.920,and the sensitivity,specificity and accuracy were 70.6%,94.5% and 86.6%,respectively;The combination of AP-ANHC-8-2-7 with 4 serum tumor markers for distinguished PHC from non-PHC by multiple indicators with AUROCs 0.909,and the sensitivity,specificity and accuracy were 65.4%,92.9% and 83.8%,respectively;The combination of two aptamers with 4 serum tumor marker for distinguished PHC from non-PHC by multiple indicators with AUROCs 0.944,and the sensitivity,specificity and accuracy were 77.1%,94.5% and 88.7%,respectively.Conclusions:1.AFP-negative serum aptamers have different diagnostic value for primary hepatic carcinoma,and through stepwise selection,we successfully selected aptamers valuable for diagnosis.2.Aptamers against AFP-negative serum have different diagnostic values for primary hepatic carcinoma under different experimental conditions,indicating the experimental condition should be optimized before diagnostic evaluation.3.The prefered aptamers against AFP-negative liver cancer serum are valuable for the diagnosis of PHC,superior to traditional tumor markers.4.The combination of aptamers with serum tumor markers can improve diagnostic performance,indicating that aptamers can be complementary to AFP.