The Application Value Of SHOX2 Gene Methylation By Droplet Digital PCR In Peripheral Blood Of Patients With Lung Cancer

Lung cancer is the most common malignant tumor.Beacause most patients have no obvious symptoms at the early stage and they are mostly found in the middle and late stage.So,they are easy to delay the optimal reatment period and five-year survival rate is low.It is very important to improve the diagnosis rate of lung cancer.Currently,the main methods for clinical diagnosis of lung cancer are histological cytology,imaging techniques and serum tumor markers.With the development of the research on tumor pathogenesis,the mechanism of DNA methylation in tumor development has attracted much attention,among which genomic hypomethylation and local hypermethylation of CpG islands could induce tumors by regulating the functional of tumor-related genes.Short stature homeobox 2(SHOX2)is a member of SHOX gene family and mainly encodes protein transcription factors.Abnormal expression of SHOX2 gene or epigenetic changes could cause certain diseases.Many studies have shown that SHOX2 gene methylation may be closely related to lung cancer.At present,the detection of SHOX2 gene methylation mainly relies on tissue biopsy,which is invasive,risky and heterogeneous in tumor tissue.However,liquid biopsy with blood or other body fluid specimens could compensate for the shortcomings of tissue biopsies in detecting SHOX2 gene methylation.Generally,the SHOX2 gene methylation is detected by methylation specific PCR,pyrosequencing and real time quantitative PCR.Although these methods have better sensitivity and specificity,there still have some shortcomings.For example,they are not easy to accurately quantify the amount of target nucleic acids from complex samples.In recent years,droplet digital PCR(ddPCR)could solve these problems.The ddPCR could absolutely quantitatively extremely small amounts of target nucleic acids from samples with complex or trace amounts of matrix with higher sensitivity and accuracy.However,SHOX2 gene methylation in peripheral blood was detected by ddPCR to analyze its value in diagnosis and efficacy evaluation of lung cancer has not been reported at home and abroad.Therefore,in this study,SHOX2 gene methylation was detected by ddPCR to analyze its application value in peripheral blood of patients with lung cancer,which may provide valuable and non-invasive auxiliary indicator for diagnosis and treatment of lung cancer.Methods1.Specimen collection and grouping:The specimens from lung cancer patients,patients with benign lung disease and healthy subjects from the First Affiliated Hospital of Zhengzhou University were collected during July 2017 to February 2019.(1)Peripheral blood samples from 100 patients with lung cancer before treatment were collected as lung cancer group,peripheral blood samples of 50 patients with benign lung disease and 50 healthy subjects were collected as benign lung disease group and normal group,respectively.(2)Tumor tissues from 40 patients with lung cancer were collected as and lung cancer tissue group.(3)Peripheral blood samples of 33 patients with non-small cell lung cancer who treated hemotherapy drugs(gemcitabine+cisplatin,paclitaxel+carboplatin,bevacizumab combined with TC regimen,pemetrexed+cisplatin,vinorelbine+cisplatin)were collected before and after one course of treatment,with reference to the curative effect of solid tumor evaluation criteria in solid tumors(RECIST)curative effect evaluation,divided into effective(CR and PR)and ineffective group(SD and PD).2.The ddPCR assay for SHOX2 gene methylation was optimizated and established:Primers and probes were designed for SHOX2 gene promoter;Select a temperature range of 50℃~60℃and amplify using the Thermal Cycler gradient program.The optimum annealing temperature of ddPCR was determined according to the degree of fluorescence amplitude separation;The methylated and non-methylated reference materials were amplified at optimal annealing temperature,and amplified products were electrophoresis and sequenced;ddPCR was applied to detect different levels of methylation standards(100%,20%,4%,0.8%,0.16%,0.032%,0.006%,0%)prepared by human methylation standards and human non-methylation standards,and then the sensitivity of method was analyzed.3.The ddPCR was applied to detect SHOX2 gene methylation in tumor tissues and peripheral blood samples.4.The levels of CEA、NSE、CA125 and CYFRA21-1 were determined by electrochemical luminescence immunoassay.5.SPSS19.0 software was applied to analyze data.The positive rate of SHOX2gene methylation was compared between two groups by chi-square test;The clinical value of SHOX2 gene methylation and tumor markers was analyzed by receiver operating characteristics(ROC)curve;The Pearson chi-square independence test was used to analyze the association between the status of SHOX2 gene methylation and clinicopathological features.Wilcoxon matched-pairs signed ranks sum test was applied to compare the groups before and after treatment.The test level was 0.05,the P<0.05 was considered statistically significant.Results1.Optimization and establishment of ddPCR method for detecting SHOX2 gene methylation:The intensity of fluorescence signal first increased and then decreased between 50℃~60℃.When the temperature was within the range of 56.3℃~58.3℃,the positive and negative microdroplets were significantly separated.In order to improve specificity of result,the optimum annealing temperature of SHOX2 gene methylation was detected by ddPCR at 58℃;As annealing temperature at 58℃,the level of SHOX2 gene methylation in methylated and unmethylated control was detected by ddPCR.The results of electrophoresis and sequencing were consistent with expected results.By detecting different methylation level standards,it was found that the positive of SHOX2 gene methylation could be detected in the standard samples with 100%,20%,4%,0.8%and 0.16%methylation levels,but not in the standard samples with 0.032%,0.006%and 0%methylation levels,which indicates that the sensitivity of ddPCR method for detecting SHOX2 gene methylation is0.16%.2.Comparative analysis of the status of SHOX2 gene methylation in peripheral blood and lung cancer tissue:The positive rate of SHOX2 gene methylation in lung cancer tissue group and matched peripheral blood group was 47.5%(19/40)and 40%(16/40),respectively.There was no significant difference in the positive rate of SHOX2 gene methylation between the two groups(χ~2=0.8,P=0.375).3.Determination of the cut-off value of SHOX2 gene methylation in peripheral blood of lung cancer patients by ddPCR and analysis of its clinical value:ROC curve was used to analyze the SHOX2 gene methylation levels in patients with lung cancer(stage III and IV)and normal group,combined with Youden index,it was found that the sensitivity and specificity of lung cancer diagnosis were best when 0.64 as the cut-off value.Therefore,0.64 was selected as cut-off value to distinguish the positive and negative of SHOX2 gene methylation.In lung cancer group and benign pulmonary disease group,the positive rate of SHOX2 gene methylation were 68.0%(68/100)and 8.0%(4/50),respectively,and difference was statistically significant(χ~2=48.077,P<0.0001).ROC results showed an AUC value of 0.838.4.Application value of SHOX2 gene methylation combined with serum tumor markers detection in lung cancer:ROC curve analysis showed that AUC values of CEA、NSE、CA125 and CYFRA21-1 were 0.735、0.663、0.631 and 0.862,respectively(all P<0.05).The AUC value of SHOX2 gene methylation combined with four indicators(CEA、NSE、CA125 and CYFRA21-1)was 0.929,which was higher than combined AUC value of 0.893(P=0.041).SHOX2 gene methylation was associated with CEA、NSE、CA125 and CYFRA21-1,with correlation coefficients of0.355、0.216、0.261 and 0.533,respectively(all P<0.05).Among them,SHOX2 gene methylation was significantly strongly correlated with CYFRA21-1(P<0.0001).5.Analysis of the correlation between SHOX2 gene methylation and clinicopathological characteristics of lung cancer.There was no association between the status of SHOX2 gene methylation in peripheral blood of patients with lung cancer and age,gender and smoking history(P=0.798,P=0.808,P=0.320).Pathological type(P=0.019)and TNM stage(P=0.009)were related to the status of SHOX2 gene methylation.The positive rate of SHOX2 gene methylation in small-cell lung cancer patients was 85.7%(18/21),squamous cell carcinoma 75.8%(25/33)and adenocarcinoma 54.3%(25/46).The positive rate of SHOX2 gene methylation in stage IV lung cancer patients was 86.4%(19/22),followed by stage III77.4%(24/31),stage II 65.0%(13/20),and stage I 44.4%(12/27).6.Comparative analysis of SHOX2 gene methylation in peripheral blood of patients with non-small cell lung cancer before and after one course of treatment:33patients with non-small cell lung cancer were found after one course of chemotherapy drug combination treatment,8 cases in effective group and 25 cases in ineffective group,with an effective rate of 24.2%(8/33).The level of SHOX2 gene methylation after treatment was lower than that before treatment(P=0.0006).Conclusions1.A ddPCR method for detecting the SHOX2 methylation was established.Peripheral blood could replace tumor tissues for the detection of SHOX2 gene methylation.2.The detection of SHOX2 gene methylation combined with four tumor markers(CEA、CA125、NSE and CYFRA21-1)can improve the application value of the combined detection of these four tumor markers in the diagnosis of lung cancer.3.SHOX2 gene methylation is involved in the occurrence and development of lung cancer and can be used as an auxiliary indicator for lung cancer diagnosis and efficacy evaluation.