| Background and objective: Small primary hepatic carcinoma(s PHC) lacks of obvious symptoms, and serum AFP is mostly negative, therefore, its diagnosis mainly depends on ultrasound B, CT, MRI and/or other imaging techniques. Meta analysis showed that the sensitivities and specificities of these imaging techniques in the diagnosis of all PHC are only 77% ~ 84%, and diagnostic performance is worse in s PHC. Serum marker detection is a simple method for tumor diagnosis, but so far there are no markers with clinical value available for the diagnosis of s PHC. Aptamers are artificial nucleic acid ligands of biological molecules, which are selected by SELEX technology from a synthetic random single-stranded oligonucleotide library. Aptamers are capable of binding to targets with high specificity and affinity and therefore are prospective for diagnostic application. In previous work, we selected a group of aptamers by using pooled serum of primary hepatic carcinoma as a targets and part of these aptamers are specific in binding to serum specimens of primary hepatic carcinoma. In the present study, we evaluated the diagnostic value of two aptamers, AP-HCS-9-74 and AP-HCS-9-132, for s PHC, and compared with AFP and other classic tumor markers, and two newly reported markers of primary hepatic carcinoma, DKK1 and MDK, in order to provide data to establish a new diagnostic method for s PHC screening.Methods: 1. Collection of serum specimens and clinical data: the subjects of this study were the patients with s PHC, liver cirrhosis(LC) and chronic hepatitis(CH) hospitalized in the First Affiliated Hospital of Nanchang University and the normal controls(NC) received healthy check-up in this hospital from 2014 to 2015. The leftover serum specimens of all subjects were collected and frozen at-80℃ before test. The clinical data of these subjects were collected.2. Aptamer-based detection for the diagnosis of s PHC: fluorescence analysis method was used for aptamer-based detection of the serum specimens. Firstly, a small sample of s PHC and LC serum specimens were assayed under different quantities of aptamers and Eva Green to determine the optimal condition, and then more specimens of s PHC, LC, CH and NC were assayed. AUROC was applied to evaluate the diagnostic values of single fluorescent indicators, Logistic regression models derived by multiple fluorescent indicators and combination of two aptamers for s PHC, and the diagnostic performance(sensitivity, specificity, accuracy, predictive value and likelihood ratio) were also calculated. 3. Detection of new serum markers of primary hepatic carcinoma for the diagnosis of s PHC: commercial ELISA kits were utilized to detect DKK1 and MDK levels in the serum specimens of s PHC, LC, CH and NC, and AUROC was used to evaluate their diagnostic values for s PHC individually and combinedly. 4. Evaluation of classic serum tumor markers in the diagnosis of s PHC: the test results of serum AFP, CEA, CA125 and CA199 were collected. The values of AFP alone and combined with other markers were evaluated by AUROC in the diagnosis of s PHC, and their diagnostic performance(sensitivity, specificity, accuracy, predictive value and likelihood ratio) were calculated. 5. Combination of aptamers and conventional laboratory tests for the diagnosis of s PHC: Logistic regression models were developed with aptamers, tumor markers and liver function as variables, and their diagnostic values for s PHC were evaluated by AUROC and diagnostic performance(sensitivity, specificity, accuracy, predictive value and likelihood ratio).Results: 1. Demographic data and clinical characteristics of subjects: four hundred and thirty two subjects were collected in this study, including 102 s PHC(male 83, female 19, age 55.9±12.4 years), 110 LC(male 88, female 22, age 52.0±11.6 years), 110 CH(male 86, female 24, age 36.8±12.6 years), 110 NC(male 77, female 33, age 55.5±6.2 years). Hepatitis B virus infection is the principle cause of liver diseases inthree groups(69.1% ~ 88.0%). Most patients of each group are Child-Pugh grade A(49.1% ~ 54.9%). 2. The diagnostic values of aptamers for s PHC: the highest AUROCs of single indicators were 0.748 in aptamer AP-HCS-9-74 and 0.713 in AP-HCS-9-132 for the diagnosis of s PHC. The AUROCs of Logistic regression models with multiple indicators were 0.819 in aptamer AP-HCS-9-74 and 0.880 in AP-HCS-9-132 for the diagnosis of s PHC. The AUROC of both aptamers combined by a Logistic regression model was 0.931 for the diagnosis of s PHC, with sensitivity 90.2%, specificity 81.2%, and accuracy 83.3%. 3. The diagnostic values of DKK1 and MDK for s PHC: concentrations of DKK1 in s PHC and LC serum specimens(both n=32) were 331.3±141.6 pg/ml and 339.5±71.0 pg/ml(P=0.774). Concentrations of MDK in s PHC and LC serum specimens(both n=32) were 1043.5±447.5pg/ml and 967.6±942.8pg/ml(P=0.643). The AUROCs of DKK1, MDK and combination of both were 0.538, 0.734 and 0.540 in the diagnosis of s PHC. 4. The diagnostic values of classic tumor markers for s PHC: the AUROCs of AFP alone and combined with CEA, CA125 and CA199 were 0.684 and 0.744. 5. The diagnostic values of aptamers and routine laboratory results combined for s PHC: the AUROC of Logistic regression model established by aptamers, tumor markers and liver function was 0.965 in the diagnosis of s PHC, with sensitivity 90.2%, specificity 90.3%, accuracy 90.3%, positive predictive value 74.2%, negative predictive value 96.8%, positive likelihood ratio 9.30, and negative likelihood ratio 0.11.Conclusions: 1. Aptamer AP-HCS-9-74 and AP-HCS-9-132 are valuable for the diagnosis of small primary hepatic carcinoma, especially both aptamers combined. 2. Small sample analysis failed to confirm serum DKK1 and MDK levels significantly different between small primary hepatic carcinoma and liver cirrhosis and their diagnostic value for small primary hepatic carcinoma.3. The diagnostic value of AFP for the diagnosis of small primary hepatic carcinoma is limited, but combination with the aptamers can significantly improve their diagnostic value. |
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