| Background and objectives: Early diagnosis and treatment is the key to improving the prognosis of patients with primary liver cancer.Regular screening of patients with chronic liver disease is the main strategy for early detection of liver cancer.At present,alpha-fetoprotein(AFP)and B ultrasound are mainly used for screening liver cancer,but the effect is not satisfactory,and it is urgent to create a simple and practical new screening method.Aptamers are called "chemical antibodies" of biomolecules,but compared with antibodies,they have their unique advantages and a good prospect in clinical diagnosis.Previously,our research group selected 245 aptamers against liver cancer serum through systematic evolution of ligands by exponential enrichment(SELEX)technology and large sample analysis showed that the sensitivity,specificity and accuracy of three united aptamers in the diagnosis of liver cancer were more than 90%,indicating that between aptamers it is complementary to the diagnosis of liver cancer.We speculate that these aptamers may target different target molecules in liver cancer serum.Using them to construct aptamer arrays may have a good diagnostic value for liver cancer.For this purpose,this study will select high-specificity liver aptamers from 245 aptamers to construct aptamer arrays,which use serum as a test material.We would develop a new detection technology of liver cancer based on liquid aptamer arrays and evaluate the value of diagnosing and screening among patients with chronic liver disease through detection of a large sample.Eventually,we would open up a new way for opportunistic screening of liver cancer.Methods:1.Collection of serum specimens and clinical data: The serum specimens of patients with chronic hepatitis,and cirrhosis and liver cancer who were hospitalized in the First Affiliated Hospital of Nanchang University from 2010 to 2018 were collected and stored in a refrigerator at-80 ?.The medical records of patients were reviewed,and the clinical data of patients were collected.2.Small sample screening of liver cancer serum aptamers with high diagnostic value: According to the previous results of sequence homology analysis of the selected liver cancer serum aptamers and using the double-dye and triple fluorescence analysis method established earlier in this research group to screen 31 aptamers and analyze their diagnostic value for liver cancer.The serum aptamers with better diagnostic value were selected into the analysis of secondary structure.3.Selection of aptamers of liver cancer to construct the array based on the sequence and secondary structure of aptamers: Using the RNA Structure 4.6 software to perform secondary structure analysis of those selected aptamers with good diagnostic value,and combination with the result of small sample screening of liver serum aptamers with high diagnostic value to select the aptamers for constructing arrays.4.Expanding sample evaluation of the diagnostic value of candidate aptamers for liver cancer: Double-dye and triple fluorescence analysis methods are used to analyze the larger serum samples,and the fluorescence value of reaction between aptamers for constructing the array and chronic hepatitis,cirrhosis and liver cancer,respectively was detected.Then we evaluate its diagnostic value for liver cancer.5.Construction of liver cancer liquid aptamer array and evaluation of the diagnostic value of liver cancer: We perform correlation and cluster analysis of the selected aptamers and select the aptamers that can complement each other in the diagnosis of liver cancer to construct the array of aptamer.Using the diagnosis of samples as dependent variable and fluorescence indicators which were detected after reacting with aptamer as independent variable,we combine with least absolute shrinkage and selection operator(LASSO)regression and stepwise backward method based on akaike information criterion(AIC)to construct a liquid aptamer array.The diagnostic value of aptamer array for liver cancer was evaluated by receiver operating characteristic(ROC)curve.6.Evaluation of clinical serological indicators and AFP combined with liquid aptamer array for liver cancer of the diagnostic value: Analysis of the diagnostic value of clinical common serological indicators for liver cancer,and the AUROC,net reclassification index(NRI)and integrated discrimination improvement (IDI)were used to evaluate the diagnostic value of the liver cancer after combining with AFP and liquid aptamer array.7.Analysis of the prognostic value of AFP for early liver cancer: Te demographic and clinical data of early liver cancer patients was extracted in the surveillance,epidemiology,and end results(SEER)database to evaluate the prognostic value of AFP for early liver cancer patients through big data,and develop a prediction model based on AFP.ROC curve and C-index are used to evaluate the discriminative ability of the model and the calibration curve was used to evaluate the calibration ability of the model.The decision curve analysis curve was used to evaluate the clinical utility of the model.Results:1.The result of small sample screening of liver serum aptamers with high diagnostic value: Using double-dye triple fluorescence analysis method to sequentially measure the serum fluorescence intensity of liver cirrhosis and liver cancer patients,the fluorescence intensity of after adding nucleic acid dyes and the fluorescence intensity of adding the liver cancer serum aptamer,16 serum aptamers with better diagnostic value were selected.2.The result of selection of candidate aptamers of liver cancer to construct the array based on the sequence and secondary structure of aptamers: Using RNA Structure 4.6 software to analyze the secondary structure of 16 serum aptamer with good diagnostic value,we combined the result of small sample screening of liver serum aptamers,and selected aptamers with complicated secondary structure including G-quadruplex and stem-loop structure.Finally,Ap-HCS-6-1-27,Ap-HCS-9-17,Ap-HCS-9-26,Ap-HCS-9-115 and Ap-HCS-9-144 were selected as 5 preferred aptamers were used to construct a liquid aptamer array.3.The result of expanding sample evaluation of the diagnostic value of candidate aptamers for liver cancer: According to the dual-dye triple fluorescence analysis method,the preferred five aptamers are used to evaluate the diagnostic value of liver cancer.When tow dyes were combined,the area under the receiver operating characteristics(AUROC)of discrimination liver cancer between cirrhosis,liver hepatitis and benign liver diseases(including cirrhosis and liver hepatitis)were above0.75.The five preferred aptamers have the potential to construct liquid-phase aptamer arrays.4.The result of construction of liver cancer liquid aptamer array and evaluation of the diagnostic value of liver cancer: According to correlation analysis and clustering results,a total of three liquid aptamer diagnostic arrays were constructed,namely AP-HCS-9-17,AP-HCS-9-115,AP-HCS-6-1-27;AP-HCS-9-17,AP-HCS-9-115,AP-HCS-9-26 and AP-HCS-9-17,AP-HCS-9-115,AP-HCS-9-144.The three aptamers of arrays of AUROCs of discrimination liver cancer between cirrhosis,liver hepatitis and benign liver diseases(including cirrhosis and liver hepatitis)were above 0.9.5.Comparison of diagnostic value of AFP and liquid aptamer arrays in liver cancer: Some clinical serological indicators had certain diagnostic value for distinguishing liver cancer from chronic hepatitis and cirrhosis respecetively,but they also had certain limitations.AFP's AUROC for the identification of liver cancer and chronic hepatitis was only 0.637,while the AUROCs for the identification of liver cancer and liver cirrhosis and benign liver disease(including cirrhosis and liver hepatitis)were 0.836 and 0.736,which were lower than the three liquid aptamer arrays.After combining the liquid aptamer array,the diagnostic value of the three liquid aptamers in AURCO has been improved to varying degrees,but the comprehensive discriminating ability is not obvious.6.The result of analysis of the prognostic value of AFP for early liver cancer: After adjusting age,gender,race,degree of differentiation,fibrosis score,and treatment method,AFP was still an independent risk factor of early liver cancer.The AUROCs and C-index of the early liver cancer prognostic model established based on AFP were above 0.70 in both training and validation group.However,the AUROC and C-index of the traditional TNM stage were lower than 0.6.The calibration curve shows a well consistency between the model's predicted probability and actual probability.Decision curve analysis curve indicated that the model had good clinical practicality.Conclusions:1.According to the results of the dual dye triple fluorescence analysis method, 16 aptamers were selected,and based on the combination of theoretical analysis,5 candidate aptamers were selected to construct liquid aptamer arrays,and the analysis of expanding sample showed the 5 aptamers had a good diagnostic value for primary liver cancer.2.Based on the analysis of correlation and clustering of 5 aptamers,3 liquid aptamer arrays which including 3 apatmers,respectively were constructed.The 3 models of liquid aptamer arrays were constructed by LASSO regression and stepwise backward analysis based on AIC and these three liquid aptamer arrays have a good effect on the diagnosis of primary liver cancer.3.Clinical serological examination and AFP had certain diagnostic value for liver cancer,but they were not as good as liquid aptamer arrays.After AFP combining with aptamer arrays,AUROC of aptamer arrays had improved to a certain extent,but the comprehensive discrimination ability was not obvious.4.The analysis based on SEER database data showed that AFP was an independent risk factor for early liver cancer patients,and liver cancer prognosis models based on AFP and clinical indicators had good prognosis prediction ability and clinical practical value. |
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