粪便多基因甲基化联合检测在结直肠癌及癌前病变诊断中的价值

Values of combined detection of polygene methylation in stool for the diagnosis of colorectal cancer and precancerous lesions

目的探讨粪便中SDC2、PPP2R5C及ADHFE1基因甲基化状态及其在结直肠癌和癌前病变筛查中的价值。方法招募青岛大学附属青岛市中心医院2020年8月至2021年3月结直肠癌患者64例、腺瘤患者72例、增生性息肉患者33例和健康体检者59名,收集研究对象清晨粪便标本,提取基因组DNA并进行亚硫酸盐修饰处理,采用甲基化特异性聚合酶链反应(MSP)检测SDC2、PPP2R5C及ADHFE1基因的甲基化状态;同时进行粪便隐血试验(FOBT)。以病理结果为金标准,通过受试者工作特征(ROC)曲线及曲线下面积(AUC)比较3个基因甲基化联合检测与FOBT预测结直肠癌和癌前病变的效果。采用R-Studio软件构建粪便基因甲基化联合检测与其他临床特征预测结直肠癌列线图,并行校准验证。结果粪便SDC2、PPP2R5C和ADHFE1基因甲基化联合检测在结直肠癌+腺瘤[74.3%(101/136)比47.1%(64/136),χ^(2)=23.20,P=0.001]、结直肠癌[90.6%(58/64)比70.3%(45/64),χ^(2)=8.91,P=0.003]、腺瘤[59.7%(43/72)比26.4%(19/72),χ^(2)=14.43,P=0.002]中的阳性率均高于FOBT,在增生性息肉[21.2%(7/33)比6.1%(2/33),χ^(2)=0.12,P=0.125]、健康对照[10.2%(6/59)比8.5%(5/59),χ^(2)=4.01,P=1.000]中阳性率差异均无统计学意义。基因甲基化联合检测预测结直肠癌+腺瘤的效能优于FOBT[AUC:0.85(95%CI 0.80~0.91)比0.71(95%CI 0.64~0.78),P<0.05],尤其对腺瘤的预测更优于FOBT[AUC:0.82(95%CI 0.74~0.89)比0.64(95%CI 0.57~0.69),P<0.001]。ADHFE1基因甲基化状态预测结直肠癌的灵敏度和特异度均较高(90.6%、96.6%)。在>50岁结直肠癌患者中,基因甲基化联合检测阳性率高于FOBT[90.2%(55/61)比68.9%(42/61),P<0.05]。依据基因甲基化联合检测和各临床特征构建的预测结直肠癌列线图校准曲线显示,预测和观察到的结直肠癌诊断效能之间具有高度一致性。结论粪便中SDC2、PPP2R5C及ADHFE1基因甲基化水平在结直肠癌和腺瘤患者中升高,基因甲基化联合检测有望成为结直肠癌及癌前病变筛查的非侵入性检测方法。

Objective To investigate the methylation status of SDC2,PPP2R5C and ADHFE1 genes in stool and their values in the screening of colorectal cancer and precancerous lesions.Methods From August 2020 to March 2021,64 patients with colorectal cancer,72 patients with adenoma,33 patients with hyperplastic polyps and 59 healthy people were recruited from Qingdao Central Hospital Affiliated to Qingdao University,and the morning stool samples were collected from the research subjects.The genomic DNA was extracted and modified with sulfite.The methylation status of SDC2,PPP2R5C and ADHFE1 genes were detected by methylation specific polymerase chain reaction(MSP),and the fecal occult blood test(FOBT)was performed.Taking the pathological results as the gold standard,receiver operating characteristic(ROC)curve and area under the curve(AUC)were used to compare the effect of combined detection of methylation of three genes and FOBT in predicting colorectal cancer and precancerous lesions.R-Studio software was used to construct a nomogram for the prediction of colorectal cancer with combined detection of gene methylation in stool and other clinical features,and the calibration and validation were performed.Results The positive rates of combined detection of methylation of SDC2,PPP2R5C and ADHFE1 genes in stool were higher than those of FOBT in colorectal cancer+adenoma[74.3%(101/136)vs.47.1%(64/136),χ^(2)=23.20,P=0.001],colorectal cancer[90.6%(58/64)vs.70.3%(45/64),χ^(2)=8.91,P=0.003]and adenoma[59.7%(43/72)vs.26.4%(19/72),χ^(2)=14.43,P=0.002].There was no significant difference in the positive rates in hyperplastic polyps[21.2%(7/33)vs.6.1%(2/33),χ^(2)=0.12,P=0.125]and healthy controls[10.2%(6/59)vs.8.5%(5/59),χ^(2)=4.01,P=1.000].The combined detection of gene methylation was better than FOBT in the prediction of colorectal cancer+adenoma[AUC:0.85(95%CI 0.80-0.91)vs.0.71(95%CI 0.64-0.78),P<0.05],especially in the prediction of adenoma[AUC:0.82(95%CI 0.74-0.89)vs 0.64(95%CI 0.57-0.69),P<0.001].The sensitivity and specificity of ADHFE1 gene methylation status in predicting colorectal cancer were high(90.6%and 96.6%).In colorectal cancer patients over 50 years old,the positive rate of combined detection of gene methylation was higher than that of FOBT[90.2%(55/61)vs.68.9%(42/61),P<0.05].The nomogram calibration curve for predicting colorectal cancer constructed based on the combined detection of gene methylation and each clinical feature showed a high degree of concordance between the predicted and observed diagnostic performance of colorectal cancer.Conclusions The methylation levels of SDC2,PPP2R5C AND ADHFE1 genes in stool are increased in patients with colorectal cancer or adenoma.The combined detection of gene methylation is expected to be a non-invasive method for the screening of colorectal cancer and precancerous lesions.