Profilome SAS, Paris Biotech 24 rue du Faubourg St Jacques, Paris 75014, France.
BMC Cancer. 2013 Dec 1;13:566. doi: 10.1186/1471-2407-13-566.
DNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.
We used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.
We obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.
We showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.
DNA 甲基化是一种众所周知的表观遗传机制，参与表观遗传基因调控。已有报道称，CRC 中存在多个基因过度甲基化，尽管在常规临床实践中，没有一种基因标志物被证明具有足够的敏感性或特异性。在这里，我们鉴定了新的表观遗传标志物，并评估了它们联合使用的诊断准确性。
我们使用来自多个流出物的甲基化阵列来描述 CRC 样本和对照的甲基化图谱，这些样本和对照是通过结肠镜检查和病理发现确定的，并选择了两个差异甲基化的候选表观遗传基因（NPY、PENK）。基于文献报道，WIF 在包括 CRC 在内的多种癌症中因启动子超甲基化而沉默，我们将其添加到该基因面板中。我们通过定量多重甲基化特异性 PCR（QM-MSP）测量了 15 对癌组织和相邻非癌直肠组织中这两个基因的甲基化程度，随后在两个不同的 266 份血清系列中进行了临床验证，这些血清分为 32 例 CRC、26 例息肉、47 例其他癌症和 161 例结肠镜检查正常。我们使用累积甲基化指数（CMI）作为变量阈值，通过接收者操作特征曲线（ROC）评估结果。
我们在组织中获得了 100%的 CRC 检测敏感性和特异性。在 CRC 血清样本中，我们获得了例如 87%/80%、78%/90%和 59%/95%的敏感性/特异性值，以及 97%/47%、95%/61%和 92%/70%的阴性预测值/阳性预测值。在来自其他癌症的血清样本中，我们获得了例如 89%/25%、43%/80%和 28%/91%的敏感性/特异性值。
我们证明了 NPY、PENK 和 WIF1 作为 CRC 诊断的联合表观遗传标志物的潜力，无论是在组织还是血清中，并测试了它们作为其他癌症血清生物标志物的用途。我们优化了一种 QM-MSP 来同时定量它们的甲基化水平。我们的检测方法可以成为一种有效的血液检测方法，用于存在 CRC 风险但难以评估的患者（例如，症状轻微且没有 CRC 家族史），因此他们不一定会选择进一步检查。如果该标志物组合得到验证，也可以成为一种具有成本效益的筛查工具，用于检测无症状癌症患者进行结肠镜检查。
