Department of Molecular Pathology, Nara Medical University, 840, Shijyo-cho, Kashihara, Japan.
BMC Cancer. 2013 Jun 4;13:275. doi: 10.1186/1471-2407-13-275.
Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation.
In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers.
Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity.
Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.
早期检测和风险评估对于治疗尿路上皮癌（UC）至关重要，UC 的特点是复发率高，需要频繁且有创的监测。我们旨在基于 DNA 甲基化建立 UC 的诊断标志物。
在这项多中心研究中，我们准备了三个独立的样本集。首先，使用 Illumina Golden Gate 甲基化分析测量训练集（91 名 UC 患者的肿瘤样本、这些患者的相应正常组织以及 12 名年龄匹配的无膀胱癌患者的正常组织）中 CpG 位点的 DNA 甲基化水平，以鉴定差异甲基化位点。然后，使用另一个组织样本队列（组织验证集）通过焦磷酸测序定量 DNA 甲基化来验证这些甲基化位点。最后，在独立的尿液样本（尿液验证集）中分析这些标志物的甲基化。进行 ROC 分析以评估这 12 个选定标志物的诊断准确性。
与正常组织相比，在肿瘤组织中，1303 个 CpG 位点中有 158 个呈高甲基化，356 个呈低甲基化。在面板分析中，12 个位点在肿瘤和正常样本之间表现出明显的改变，具有 94.3%的敏感性和 97.8%的特异性。同样，相应的正常组织也可以与正常组织区分开来，具有 76.0%的敏感性和 100%的特异性。此外，这些在尿液样本中确定的标记物对 UC 的诊断准确性也很高，具有 100%的敏感性和 100%的特异性。
基于这些初步发现，基于特定位点差异 DNA 甲基化的诊断标志物可用于 UC 的非侵入性和可靠检测以及表观遗传学领域缺陷。
