Comparative study of the effect of plasma CLIP4 gene methylation and three serum tumor markers on the diagnosis of colorectal cancer
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摘要: 目的 异常DNA甲基化已成为结直肠癌(CRC)筛查的一类有前景的生物标志物，本研究旨在评估血浆CLIP4基因甲基化检测在CRC筛查中的可行性，并与目前临床上常用的血清肿瘤标记物癌胚抗原(CEA)、糖类抗原125(CA125)和糖类抗原19-9(CA19-9)相比较，评价其诊断价值。方法 研究纳入2020年12月—2021年12月于徐州医科大学附属医院就诊行结肠镜检查的研究对象共303例，其中包括CRC患者97例、结直肠息肉患者102例及接受体检的对照者104例。采用ABI7500实时荧光定量聚合酶链式反应谱仪检测研究对象血浆CLIP4基因甲基化的水平，罗氏Cobas 8000电化学紫外发光分析仪检测患者CEA、CA19-9、CA125的浓度。评估血浆CLIP4基因甲基化检测诊断CRC的可行性，同时比较血浆CLIP4基因甲基化检测与血清CEA、CA19-9、CA125联合检测对CRC的诊断价值，并进行统计学分析。结果 ① CEA、CA125、CA19-9、CLIP4基因甲基化在CRC中的阳性率分别为42.3%(41/97)、3.1%(3/97)、19.6%(19/97)、77.3%(75/97)，3种血清肿瘤标志物联合检测阳性率提升至66.0%(64/97)。3组研究对象中，除CA125外，CEA、CA19-9、CLIP4基因甲基化、CEA+CA125+CA19-9联合检测阳性率比较，差异均有统计学意义(P< 0.05)，其中，CRC组患者CEA、CA19-9、CLIP4基因甲基化、CEA+CA125+CA19-9联合检测的阳性率均明显高于健康对照组、结直肠息肉组，差异均有统计学意义(P< 0.05)。②在CRC组患者中，血浆CLIP4基因甲基化阳性率在不同年龄、肿瘤位置、浸润深度、淋巴结转移、临床分期中比较差异均无统计学意义(P>0.05)，在性别、远处转移中差异有统计学意义(P< 0.05)，男性患者血浆CLIP4基因甲基化阳性率明显高于女性患者，有远处转移CRC患者的血浆CLIP4基因甲基化阳性率明显高于无远处转移患者(P< 0.05)。③血浆CLIP4基因甲基化诊断CRC的曲线下面积为0.826(95%CI：76.9%~88.4%)，灵敏度为72.2%，特异度为88.9%，高于CEA、CA125、CA19-9三种肿瘤标志物联合诊断能力，该三种肿瘤标志物联合诊断CRC的曲线下面积为0.786(95%CI：72.8%~84.5%)，灵敏度为79.4%，特异度为67.0%。CLIP4、CEA、CA125、CA19-9四种指标联合诊断CRC的曲线下面积为0.871(95%CI：82.1%~92.1%)，灵敏度为74.2%，特异度为89.8%。血清肿瘤标志物CEA、CA125、CA19-9与血浆CLIP4基因甲基化联合检测诊断CRC的曲线下面积最大，并且有较高的灵敏度及特异度，能够更有效地筛查CRC。结论 ① 血浆CLIP4基因甲基化检测诊断CRC具有较高的灵敏度及特异度，可作为诊断CRC的新型标记物。②血浆CLIP4基因甲基化检测诊断CRC优于血清肿瘤标记物CEA、CA125、CA19-9，能够更有效地筛查CRC。③血浆CLIP4基因甲基化与血清肿瘤标记物(CEA+CA125+CA19-9)联合检测，可获得更高的灵敏度及特异度，能够更有效地用于临床CRC检测。Abstract: Objective Abnormal DNA methylation has become a promising biomarker for colorectal cancer screening. The purpose of this study is to evaluate the feasibility of plasma CLIP4 gene methylation detection in colorectal cancer screening, and to evaluate its diagnostic value compared with serum tumor markers CEA, CA19-9 and CA125 commonly used in clinic.Methods This study included 303 subjects who underwent colonoscopy in the Affiliated Hospital of Xuzhou Medical University from December 2020 to December 2021, including 97 patients with colorectal cancer, 102 patients with colorectal polyps and 104 controls. The methylation level of CLIP4 gene in plasma was detected by ABI7500 real-time fluorescence quantitative polymerase chain reaction spectrometer, and the concentrations of carcinoembryonic antigen(CEA), carbohydrate antigen 125(CA125) and carbohydrate antigen 19-9(CA19-9) in serum were detected by Roche Cobas 8000 electrochemical ultraviolet luminescence analyzer. To evaluate the feasibility of plasma CLIP4 gene methylation detection in the diagnosis of colorectal cancer, and to compare the diagnostic value of plasma CLIP4 gene methylation detection with serum CEA, CA19-9 and CA125 in the diagnosis of colorectal cancer.Results ① The positive rates of CEA, CA125, CA19-9 and CLIP4 gene methylation in colorectal cancer were 42.3%(41/97), 3.1%(3/97), 19.6%(19/97) and 77.3%(75/97), respectively. The positive rate of combined detection of three serum tumor markers increased to 66.0%(64/97). Among the three groups, except CA125, there were significant differences in the positive rates of CEA, CA19-9, CLIP4 gene methylation and the combination of three tumor markers(P< 0.05).②There was no significant difference in the positive rate of CLIP4 methylation in different age, location, depth of invasion, lymph node metastasis and clinical stage(P>0.05), but there was significant difference in gender and distant metastasis(P< 0.05). The positive rate of CLIP4 methylation in male patients was significantly higher than that in female patients, patients with distant metastasis were significantly higher than those without distant metastasis.③The area under the curve of plasma CLIP4 gene in the diagnosis of CRC was 0.826(0.769-0.884), the sensitivity was 72.2%, and the specificity was 88.9%, which was higher than the combined diagnosis ability of CEA, CA125 and CA19-9. The area under the curve of the combined diagnosis of CRC was 0.786(0.728-0.845), the sensitivity was 79.4%, and the specificity was 67.0%. The area under the curve of CLIP4, CEA, CA125 and CA19-9 in the combined diagnosis of CRC was 0.871(0.821-0.921), the sensitivity was 74.2%, and the specificity was 89.8%(P< 0.001). The combination of serum tumor markers CEA, CA125, CA19-9 and plasma CLIP4 methylation has the best detection method, which can obtain high sensitivity and specificity, and can screen colorectal cancer more effectively.Conclusion ① The detection of CLIP4 gene methylation in plasma has high sensitivity and specificity in the diagnosis of colorectal cancer. It can be used as a new marker for the diagnosis of colorectal cancer. ②The detection of CLIP4 gene methylation in plasma is superior to serum tumor markers CEA, CA125 and CA19-9 in the diagnosis of colorectal cancer, and has high sensitivity and specificity, which can screen colorectal cancer more effectively. ③The combined detection of plasma CLIP4 gene methylation and serum tumor marker(CEA + CA125 + CA19-9) can obtain higher sensitivity and specificity, which can be more effectively used in clinical colorectal cancer detection.
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Key words:
- colorectal cancer /
- DNA methylation /
- CLIP4 /
- serum tumor markers
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表 1 3组患者CLIP4基因甲基化及肿瘤标记物检测水平比较
M(P25，P75) 组别 例数 CEA/(ng·mL-1) CA125/(U·mL-1) CA19-9/(U·mL-1) CLIP4基因甲基化(ct值) 健康对照组 104 2.21(1.30，3.15) 7.40(5.95，8.89) 8.50(6.63，11.46) 50.00(50.00，50.00) 结直肠息肉组 102 2.63(1.89，3.69) 7.80(6.28，8.99) 11.09(7.96，15.19)1) 50.00(50.00，50.00) CRC组 97 4.22(2.82，9.20)1)2) 9.08(7.61，11.63)1)2) 13.32(8.35，31.40)1)2) 37.59(35.07，50.00)1)2) χ2 53.958 24.214 26.825 119.446 P < 0.001 < 0.001 < 0.001 < 0.001 与健康对照组比较，1)P < 0.05；与结直肠息肉组比较，2)P < 0.05。 
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表 2 3组患者肿瘤标记物及CLIP4基因甲基化阳性率比较
例(%) 组别 例数 CEA CA125 CA19-9 CLIP4基因甲基化 CEA+CA125+ CA19-9联合检测 健康对照组 104 6(5.8) 2(1.9) 3(2.9) 17(16.3) 8(7.7) 结直肠息肉组 102 8(7.8) 0 2(2.0) 20(19.6) 12(11.8) CRC组 97 41(42.3)1)2) 3(3.1) 19(19.6)1)2) 75(77.3)1)2) 64(66.0)1)2) χ2 56.001 2.957 26.687 99.958 104.639 P < 0.001 0.207 < 0.001 < 0.001 < 0.001 与健康对照组比较，1)P < 0.05；与结直肠息肉组比较，2)P < 0.05。
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表 3 血浆CLIP4基因甲基化阳性率与CRC患者的临床特征关系分析
例(%) 临床特征 例数 CLIP4基因甲基化检测 χ2 P 阴性 阳性 年龄/岁 0.573 0.449 < 60 39(40.2) 15(38.5) 24(61.5) ≥60 58(59.8) 18(31.0) 40(69.0) 性别 6.893 0.009 男 56(57.7) 13(23.2) 43(76.8) 女 41(42.3) 20(48.8) 21(51.2) 位置 0.011 0.918 结肠 33(34.0) 11(33.3) 22(66.7) 直肠 64(66.0) 22(34.4) 42(65.6) 浸润深度 0.831 0.362 T1+T2 24(24.7) 10(41.7) 14(58.3) T3+T4 73(75.3) 23(31.5) 50(68.5) 淋巴结转移 0.336 0.562 有 51(52.6) 16(31.4) 35(68.6) 无 46(47.4) 17(37.0) 29(63.0) 远处转移 4.802 0.028 有 11(11.3) 0 11(100.0) 无 86(88.7) 33(38.4) 53(61.6) 临床分期 0.016 0.901 Ⅰ+Ⅱ 42(43.3) 14(33.3) 28(66.7) Ⅲ+Ⅳ 55(56.7) 19(34.5) 36(65.5)
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表 4 各指标对CRC诊断价值分析
检测指标 AUC(95%CI) P 阈值 灵敏度/% 特异度/% CEA 0.752(0.689~0.814) < 0.001 2.765 76.3 64.1 CA125 0.675(0.607~0.743) < 0.001 8.630 61.9 70.9 CA19-9 0.647(0.573~0.721) < 0.001 16.895 44.3 88.3 CLIP4基因甲基化 0.826(0.769~0.884) < 0.001 44.613 72.2 88.9 CEA+CA125+CA19-9 0.786(0.728~0.845) < 0.001 0.227 79.4 67.0 CEA+CA125+CA19-9+CLIP4基因甲基化 0.871(0.821~0.921) < 0.001 0.413 74.2 89.8
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表 5 各指标诊断CRC的曲线下面积比较
检测指标对比 Z P CEA vs. CLIP4基因甲基化 2.120 0.034 CA125 vs. CLIP4基因甲基化 3.558 ＜0.001 CA19-9 vs. CLIP4基因甲基化 4.222 ＜0.001 CEA vs. 4种指标联合 4.648 ＜0.001 CA125 vs. 4种指标联合 5.133 ＜0.001 CA19-9 vs. 4种指标联合 5.820 ＜0.001 CLIP4基因甲基化vs. 4种指标联合 2.404 0.016 3种肿瘤标志物联合vs. 4种指标联合 3.994 ＜0.001
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