PO.CL01.18 · 临床研究
使用改良qPCR通过尿液DNA突变和甲基化检测膀胱癌
Detection of bladder cancer via urinary DNA mutation and methylation using modified qPCR
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：膀胱镜检查是诊断和监测膀胱癌（BC）的金标准，但其为侵入性且伴有疼痛。既往关于尿液活检的研究表明，尿液DNA突变和甲基化标志物是有前景的辅助诊断方法；然而，目前基于PCR检测基因突变或甲基化的方法准确性不足。这一缺陷凸显了对更精确的DNA标志物检测技术和膀胱癌辅助诊断方法的迫切需求。
方法：我们开发了MTPD-PCR，这是一种高特异性的基于PCR的检测方法，使用双脱氧核酸（ddNA）阻断PCR引物和焦磷酸解激活聚合，以防止常规PCR引物可能引入的人为错误扩增产物，实现对丰度低至0.01%突变的稳定检测。我们使用该方法整合膀胱癌DNA突变和甲基化生物标志物，用于尿液样本中膀胱癌的无创检测。我们在中国两家三级医院的双中心队列中开展了一项前瞻性研究，纳入302例经组织学确诊的膀胱癌患者和200例非膀胱癌对照。以膀胱镜检查联合组织病理学检查作为参考标准，计算诊断准确性参数（敏感性、特异性和准确性）。
结果：研究队列包括302例膀胱癌患者，其中114例（37.8%）为低级别，175例（58.0%）为高级别，13例（4.2%）未分级。对489份可分析样本的评估显示，总体敏感性为85.9%，特异性为95.8%（健康对照组为100%），诊断准确性为89.8%。阳性预测值和阴性预测值分别为97.0%和81.3%。对于不同的非膀胱癌对照，敏感性为85.95%，特异性范围为90%至100%。具有临床意义的是，该方法在91例早期肿瘤中达到70.3%的敏感性，并在191例T1/Tis/Ta期肿瘤的验证集中保持81.7%的敏感性，表明其在不同疾病分期中均有良好表现。
结论：该方法在489例患者的队列中表现良好，展现出高敏感性和准确性。这种无创分子检测方法可在早期检测膀胱癌并作为治疗参考，尤其适用于DNA含量低的病例，有望减轻膀胱镜检查的负担并避免不必要的经尿道膀胱肿瘤二次切除。
查看英文原文 English abstract
Background: Cystoscopy is the gold standard for diagnosing and monitoring bladder cancer (BC), but it is invasive and painful. Previous studies on urine biopsy have shown that urinary DNA mutation and methylation markers are promising auxiliary diagnostic methods; however, current PCR-based methods for detecting gene mutations or methylations are not accurate enough. This shortcoming highlights the urgent need for a more precise DNA marker detection technology and an auxiliary diagnostic method for bladder cancer.
Methods: We developed MTPD-PCR, a highly specific PCR-based detection method that uses dideoxyribonucleic acid (ddNA)-blocking PCR primers and pyrophosphorolysis-activated polymerization to prevent artificially erroneous amplification products that may be introduced by conventional PCR primers, achieving stable detection of mutations with an abundance of 0.01%. We used this method to integrate bladder cancer DNA mutation and methylation biomarkers for non-invasive detection of bladder cancer in urine samples. We conducted a prospective study in a two-center cohort from two tertiary hospitals in China, including 302 patients with histologically confirmed bladder cancer and 200 non-bladder cancer controls. Using cystoscopy combined with histopathological examination as the reference standard, diagnostic accuracy parameters (sensitivity, specificity, and accuracy) were calculated.
Results: The study cohort included 302 patients with bladder cancer, of which 114 (37.8%) were low-grade, 175 (58.0%) were high-grade, and 13 (4.2%) were ungraded. Evaluation of 489 analyzable samples showed an overall sensitivity of 85.9%, a specificity of 95.8% (100% in the healthy control group), and a diagnostic accuracy of 89.8%. The positive predictive value and negative predictive value were 97.0% and 81.3%, respectively. For different non-bladder cancer controls, the sensitivity was 85.95%, and the specificity ranged from 90% to 100%. Clinically significant, the method achieved a sensitivity of 70.3% in 91 early-stage tumors and maintained a sensitivity of 81.7% in a validation set of 191 T1/Tis/Ta stage tumors, indicating good performance across different disease stages.
Conclusion: This method demonstrated good performance in a cohort of 489 patients, exhibiting high sensitivity and accuracy. This non-invasive molecular detection method can detect bladder cancer at an early stage and serve as a treatment reference, especially suitable for cases with low DNA content, potentially reducing the burden of cystoscopy and avoiding unnecessary secondary transurethral resection of bladder tumors.
利益披露 Disclosure
D. Jin, None..
R. Tan, None..
R. Zhang, None..
H. Chen, None.