The Diagnostic Value Of Aptamers Against Gastric Cancer Serum

Background and objective：Currently, the diagnosis of gastric cancer (GC) mainly depends on endoscopy.However, endoscopy is invasive and expensive, for which some patients rejectendoscopy. Serum tumor markers are useful for screening and diagnosis of cancers,but there is still a lack of valuable markers for the diagnosis of gastric cancer.Aptamers are single-stranded oligonucleotide ligands capable of binding to targetwith high specificity and affinity, which are selected from a synthesized library ofrandom single-stranded oligonucleotides by systematic evolution of ligands byexponential enrichment (SELEX) and considered valuable in clinical diagnosis. Inour previous study, we selected some aptamers against pooled serum of gastriccancer after10rounds of selection by SELEX. Although some of them werespecific to the pooled serum of gastric cancer, but not valuable in the diagnosis ofgastric cancer. Therefore, we will continue to select new aptamers by SELEX inwhich the last round of oligonucleotide sub-library were used as initial library andpooled serum of gastric serum as target, attempting to generate more specificaptamers and evaluate their value in the diagnosis of gastric cancer for developinga new method for gastric cancer screening.Methods：1. Serum specimen collection and pooled serum preparation: Serumspecimens were collected from the patients with GC, non-atrophic gastritis, pepticulcer and normal people for health checkup (normal control) in the First AffiliatedHospital of Nanchang University from2012to2013and refrigerated at-80oC.Pooled serum of gastric cancer was prepared by mixing equal serum aliquot of15cases of gastric cancer, and the pooled serum of normal people was preparedsamely.2. Re-selection and separation of aptamers against the pooled serum of gastriccancer: The10thround sub-library prepared in previous selection was used as the initial library. The enrichment and specificity of sub-library prepared after eachround of selection were analyzed by polyacrylamide gel electrophoresis, by whichthe end of selection was determined. The sub-libraries which specifically binds tothe pooled serum of gastric cancer were amplified by PCR for aptamers separationby cloning and sequencing.3. Characterization of the aptamers: The secondary structures of the aptamerswere analyzed with RNAStructure4.6and the aptamers which have less secondarystructures and low free energy were chosen for subsequent experiments. After theappropriate amounts of aptamers and serum used in analysis were determined bypolyacrylamide gel, aptamers were incubated with the16serum samples of gastriccancer and normal control, respectively, followed by polyacrylamide gelelectrophoresis and GelRed staining. The gray value of free band in each samplewas measured and its ratio to aptamer control was calculated. The aptamers withgray value ratio in gastric cancer group less than in normal control group and thedifference (nearly) statistically significant were considered specific to gastriccancer serum.4. The diagnostic value of aptamers in gastric cancer: The diagnostic value ofthe specific aptamers for gastric cancer was evaluated by fluorescence analysis.After determination of appropriate amounts of aptamer and serum used in reaction,each aptamer was incubated with15serum specimens of gastric cancer and normalcontrol, respectively, for preliminary selection of aptamers with diagnostic valuein gastric cancer. The aptamers with AUROC more than0.9were selected to beincubated with small samples of serum specimens (32cases of gastric cancer andnormal control, respectively) for verification the diagnostic value in gastric cancer.The aptamers with diagnostic value in gastric cancer were incubated with a largesample of serum specimens (more80cases of gastric cancer, benign gastricdiseases and normal control) for validation of their value in diagnosis of gastriccancer. Meanwhile, the natural fluorescence of each serum specimen wasmeasured with binding buffer instead of aptamers. The diagnostic value ofaptamers alone and combined with the natural serum fluorescence value wasevaluated with AUROC and diagnostic performance (sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihoodratio, negative likelihood ratio).Results：1. Re-selection and separation of aptamers against pooled serum of gastriccancer: during SELEX process, the sub-library band was gradually concentrated(enriched), and the specificity of sub-library binding to the pooled serum of gastriccancer showed a growing tendency in PAGE analysis. Selection was ended at18thround. The sub-libraries from14thto18throunds were cloned and160aptamerswere isolated.2. Characterization of the aptamers against pooled serum of gastric cancer:Secondary structure simulation analysis showed that the isolated aptamers havevarious secondary structures. There were21aptamers with less than2secondarystructures, in which there are11aptamers with lower gray ratio of free aptamerband in gastric cancer group than in normal group. The aptamers with (nearly)significant difference (P<0.1) between two groups were considered specificaptamers to gastric cancer serum.3. The diagnostic value of aptamers for gastric cancer: The11specificaptamers were incubated with15serum specimens of gastric cancer and normalcontrol, respectively, and the fluorescence values were detected. The resultsshowed3aptamers (AP-GCS-15-16, AP-GCS-16-17and AP-GCS-17-16) withAUROC more than0.9in diagnosis of gastric cancer. These3aptamers withdiagnostic value in preliminary analyses were farther verified in small-sample andshowed that aptamer AP-GCS-15-16, AP-GCS-17-16were also valuable in thediagnosis of gastric cancer with AUROC more than0.7. Their diagnostic value forgastric cancer were validated in a larger sample (106cases of gastric cancer,84cases of benign stomach diseases and94cases of normal control), and the resultsshowed that the fluorescence value of AP-GCS-15-16in gastric cancer group wassignificantly different from benign gastric disease group and normal control group(P <0.001), but no significant difference between benign gastric disease group andnormal group (P>0.05). The AUROC of AP-GCS-15-16in diagnosis of gastric cancer was at a "good" level (0.880), with sensitivity70.8%, specificity84.9%,and accuracy79.6%. The AUROC of AP-GCS-17-16was only0.656. In addition,natural serum fluorescence has limited diagnostic value in gastric cancer, with aAUROC0.632.4. Combined analysis of aptamers in the diagnosis of gastric cancer:Combined analysis could improve the diagnostic value of aptamers in gastriccancer. When AP-GCS-15-16was combined with the natural serum fluorescencevalue, AUROC increased from0.880to0.920, with sensitivity89.6%, specificity81.5%and accuracy84.5%. When AP-GCS-17-16was combined with the naturalserum fluorescence, the AUROC in diagnosis of gastric cancer increased from0.656to0.725. However, the combination of AP-GCS-15-16with AP-GCS-17-16could not enhance AUROC significantly.Conclusions:1. After re-selection by SELEX, a group of aptamers against gastric cancerserum was generated, and some of them are specific to gastric cancer serum.2. The aptamers with diagnostic value for gastric cancer were identified byfluorescence analysis, in which AP-GCS-15-16alone have good diagnostic valuefor gastric cancer, and its diagnostic value can improve by combining with naturalserum fluorescence.3. The natural fluorescence in gastric serum are higher than in benign gastricdiseases and normal control, but its diagnostic value for gastric cancer is limited,and combined analysis with aptamers can improve their diagnostic value.