Background
There is a high demand for noninvasive screening tools for gastrointestinal cancer (GIC) detection, and GIC-specific markers are required for such purposes. It is established that induction of the telomerase reverse transcriptase gene (TERT) coupled with telomerase activation is essential for cancer development/progression and aberrant TERT promoter methylation of specific 5'-C-phosphate-G-3' (CpGs) has been linked to TERT induction in oncogenesis. Here we analyzed TERT promoter methylation in fecal samples from GIC patients and healthy adults and determined its value as a stool biomarker for GIC detection. Materials and
Conclusion
This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses as an additional tool in noninvasive GIC screening. Implications for practice: Induction of telomerase reverse transcriptase (TERT) expression coupled with telomerase activation is essential for cancer development/progression, while aberrant TERT promoter methylation has been linked to TERT induction in oncogenesis. We identified two cancer-specific methylation sites (CpG1 and 2) in the TERT promoter in tumors from GIC patients. Methylated TERT promoter CpG sites 1 and 2 were detectable in patient stool, while only background levels were observed in healthy individuals. The sensitivity and specificity was comparable to other known stool methylation markers for GIC detection. This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses for noninvasive screening of GIC. 摘要 背景.对于用于胃肠癌（GIC）检测的无创筛查工具有很高的需求, 为达到该目的, 需要有GIC特异性标志物。可以确认端粒酶逆转录酶基因（TERT）诱导联合端粒酶激活是癌症的发展/进展的重要因素, 特定5'‐C‐磷酸‐G‐3'（CpG）的异常TERT启动子甲基化与肿瘤形成中的TERT诱导有关。在这里, 我们分析了取自GIC患者和健康成人的粪便样本中的TERT启动子甲基化, 并确定其作为一种用于GIC检测的粪便生物标志物的价值。 材料和方法.招募了69例GIC患者（34例结直肠癌和35例胃癌患者）和62例健康成年人, 并收集了其粪便样本。收集了12例健康个体的正常粘膜组织和34例患者的成对肿瘤和相邻的非癌组织。使用焦磷酸测序法测定了TERT启动子甲基化密度。 结果.我们在肿瘤组织TERT启动子的‐218（CpG位点1）和‐210（CpG位点2）处识别出了两个GIC特异性甲基化位点。在患者粪便中可检测到甲基化TERT启动子CpG位点1和2, 而在健康个体中仅观察到背景水平。90％特异性的粪便甲基化TERT启动子检测方法的总体灵敏度达到52.2％[95％置信区间（CI）：48.3‐56.0], 该检测方法与用于GIC检测的其它已知粪便甲基化标志物检测方法相当。粪便TERT启动子甲基化和潜血（OB）的联合检测显著提高了结直肠癌检测的灵敏度和特异性（单一甲基化的曲线下面积：0.798, 95％CI：0.707‐0.889 vs. 甲基化+OB：0.920, 95％CI：0.859‐0.981; p = 0.028）, 但没有显著提高胃癌检测的灵敏度和特异性。 结论.本项概念验证研究表明粪便TERT启动子甲基化分析可作为无创GIC筛查的一种额外工具。 对临床实践的提示：端粒酶逆转录酶基因（TERT）诱导联合端粒酶激活是癌症发展/进展的重要因素, 异常TERT启动子甲基化与肿瘤形成中的TERT诱导有关。我们在GIC患者的肿瘤TERT启动子中发现了两个癌症特异性甲基化位点（CpG1和2）。在患者粪便中可检测到甲基化TERT启动子CpG位点1和2, 而在健康个体中仅观察到背景水平。本检测方法的灵敏度和特异性与用于GIC检测的其它已知粪便甲基化标志物检测方法相当。本项概念验证研究表明粪便TERT启动子甲基化分析可用于无创GIC筛查。
Methods
Sixty-nine GIC patients (34 colorectal carcinoma and 35 gastric cancer) and 62 healthy adults were recruited and fecal samples were collected. Paired tumors and adjacent non-cancerous tissues from 34 patients and normal mucosa tissues from 12 healthy individuals were collected. TERT promoter methylation density was determined using pyrosequencing.
Results
We identified two GIC-specific methylation sites at -218 (CpG site 1) and -210 (CpG site 2) in the TERT promoter in tumor tissues. Methylated TERT promoter CpG sites 1 and 2 were also detectable in patient stool, while only background levels were observed in healthy individuals. The overall sensitivity reached 52.2% (95% confidence interval [CI]: 48.3-56.0) for fecal methylated TERT promoter assays at 90% specificity, which was comparable to other known stool methylation markers for GIC detection. The combined assays of fecal TERT promoter methylation and occult blood (OB) significantly improved sensitivity and specificity in colorectal cancer (area under curves for methylation alone: 0.798, 95% CI: 0.707-0.889 vs. methylation + OB: 0.920, 95% CI: 0.859-0.981; p = .028), but not in gastric cancer.