| Background and objective:AFP and medical imaging are currently two main methods for screening of primary hepatic cancer(PHC), and they are not ideal clinical practice. About 30%~40% of patients with PHC and most small PHC are AFP-negative, and it is also not easy for medical imaging to detect of small PHC. Some studies showed that GPC3 was specifically expressed in PHC tissue and could release into blood as a potential biomarker for the diagnosis of PHC. Serum GPC3 is generally assayed by immunological method, but it is difficult for clinical application due to the poor sensitivity of method. Aptamers, artificial nucleic acid ligands of biological molecules, are capable of binding to targets with high specificity and affinity and therefore valuable in diagnostic application. In our previous work, we selected a group of aptamers against GPC3 by SELEX. In this study, we will evaluate their value in the diagnosis of PHC by a fluorescence-based method which we previously developed. Methods:1. Collection of serum specimen and clinical data: The leftover serum specimens were collected from the patients with PHC, liver cirrhosis(LC), chronic hepatitis and normal people for health checkup(normal control) hospitalized in the First Affiliated Hospital of Nanchang University from 2013 to 2014 and refrigerated at-80 oC. Clinical data, including gender, age, hepatic function, serum tumor markers, imaging examination and pathologic examination were also collected.2. Selection of GPC3 aptamers valuable for PHC diagnosis: Twenty GPC3 aptamers were synthesized and incubated with sera of PHC and LC, and the fluorescence values were then detected before and after incubation. The diagnostic significance of single and combined fluorescence values was evaluated by the ROC curve. Two aptamers with the largest AUROC were selected for further study.3. Optimization of experimental conditions: The two aptamers selected were incubated with 32 serum specimens of PHC and LC cases under 6 conditions(different amounts of aptamers and EvaGreen), and the fluorescence values were detected and analyzed by the same method above. The condition with the highest AUROC was considered the optimum experimental condition.4. Evaluation of diagnostic value of aptamers for PHC: The diagnostic values of two selected aptamers for PHC were firstly evaluated in a small sample(80 cases of PHC and LC) with the same method. The aptamer(s) with the AUROC more than 0.8 in single fluorescent value and 0.9 in multiple fluorescent values were validated in a larger sample(more than 100 cases of PHC, LC, chronic hepatitis and normal control, respectively). The diagnostic performance were calculated, including specificity, sensitivity, accuracy, positive and negative predictive value, positive and negative likelihood ratio.5.Detection of serum GPC3 level by ELISA and evaluation of diagnostic value: The serum GPC3 levels of small sample(80 cases of PHC and LC) were measured by using a commercial ELISA kit and its diagnostic value for PHC was evaluated.6.Value of serum tumor markers for the diagnosis of PHC: The serum levels of AFP, CEA, CA125 and CA19-9 of the specimens were collect and their diagnostic values were evaluated through the ROC curve. Results:1.Selection of GPC3 aptamers and optimization of conditions for the diagnosis of PHC: There were 9 aptamers with the highest AUROC of single fluorescence value more than 0.8 and 16 aptamers with AUROC of multiple fluorescence value more than 0.9 in diagnosis of PHC. Aptamers of AP-GPC3-13 and AP-GPC3-27 were two aptamers with the largest AUROC. The optimum experimental conditions were aptamer 0.3pmol/Eva Green 2.5μL and 0.5pmol/2.5μL, respectively, for the two aptamers.2.The diagnostic value of GPC3 aptamers for PHC: In the small sample(80 cases of PHC and LC, respectively) analysis, the highest AUROC of the two aptamers were 0.852, 0.884 in single fluorescence values and more than 0.9 in multiple fluorescence values, which were superior to AFP in the diagnosis of PHC. In the larger sample(144 cases of PHC, 158 cases of LC, 126 cases of hepatitis and 100 cases of normal control) validation of their diagnostic value, the AUROC in diagnosis of PHC and non-PHC was about 0.75 in single fluorescence values and 0.9 in multiple fluorescence values. The combination of AP-GPC3-13 with AP-GPC3-27 showed more powerful for diagnosing PHC and non-PHC, with AUROC, sensitivity, specificity and accuracy all more than 0.9. In 55 cases of small liver cancer(≤3cm), the positive rates of the two aptamers single and combination were 63.6%, 49.1% and 74.5%, respectively, higher than that of AFP(43.6%). In 68 cases of AFP negative liver cancer, the positive rates of the two aptamers single and combination were 70.6%, 69.1% and 83.8%, respectively.3.The diagnostic value of serum GPC3 detection by ELISA: The serum GPC3 levels were 1.22±3.07 ng/m L in PHC and 1.36±3.35 ng/m L in LC(P>0.05), and the AUROC was 0.575.4.Value of serum tumor markers in diagnosis of PHC: Only AFP were valuable in the diagnosis of PHC, with the AUROC 0.761. Other tumor markers(CEA, CA125, CA19-9) were not significant in diagnosis. The AUROC of 4 biomarker combined analysis were same as AFP. Conclusions:1.GPC3 aptamers are valuable for the diagnosis of PHC and superior to serum GPC3 and AFP.2.GPC3 aptamers are also valuable for the diagnosis of small and AFP negative PHC.3.Combined analysis of aptamers can further improve the diagnosis value for PHC, including small and AFP negative PHC. |
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