PO.BCS01.14 · 生物信息与计算
基于尿液的NGS检测方法在膀胱癌微小残留病灶检测中的优化
Optimization of a urine based NGS assay for detecting minimal residual disease in bladder cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：目前的临床工具难以在接受根治性膀胱切除术的患者中，准确区分新辅助化疗后的微小残留肿瘤与良性膀胱组织。识别完全缓解者或可使部分患者避免根治性膀胱切除。为满足这一需求，我们开发了一种基于NGS的尿液检测方法，以支持对化疗反应的无创检测。本研究着重优化关键的分析前因素，包括保存剂的选择和DNA提取方法。
方法：为克服即时-80 ℃冷冻在流程上的限制，我们评估了多种尿液保存剂维持DNA完整性的能力。比较了两种DNA提取策略：基于Sepharose的提取法和一种常用的商业试剂盒。对每种条件下获得的DNA进行质量控制检测，并进行浅通量全基因组测序，随后开展片段组学分析、片段末端基序图谱分析以及拷贝数变异（CNA）估计。
结果：Sepharose提取法结合专门设计的盐缓冲液可获得最高的DNA回收率。商业产品未能促进片段稳定性。片段组学分析显示，采用Sepharose提取法处理时，癌症患者的异常片段比例（FAF）高于健康对照。两组之间还观察到不同的片段末端基序模式。CNA分析表明，缓冲液保存的样本产生的估计肿瘤比例高于即时冷冻处理的样本，提示肿瘤信号保留得到改善。出乎意料的是，Sepharose提取法还提高了信噪比。
结论：该检测方法在检测膀胱癌微小残留病灶以及支持化疗后完全缓解的无创评估方面显示出强大潜力。与即时冷冻相比，盐缓冲液能有效增强尿液DNA的保存并改善肿瘤来源信号的检测。我们正在继续收集更多尿液样本，以进一步优化并临床验证该检测方法，使其可用于临床。
查看英文原文 English abstract
Background: Current clinical tools struggle to accurately distinguish minimal residual tumor from benign bladder tissue following neoadjuvant chemotherapy in radicl cystectomy patients. Identification of complete responders might permit avoidance of radical cystectomy. To address this need, we developed an NGS-based urine assay to support non-invasive detection of chemotherapy response. This study focuses on optimizing key pre-analytical factors, including preservative selection and DNA extraction methods.
Methods: To overcome the logistical constraints of immediate -80 °C freezing, multiple urine preservatives were evaluated for their ability to maintain DNA integrity. Two DNA extraction strategies were compared: Sepharose-based extraction and a popular commercial kit. DNA from each condition was subjected quality control testing and to shallow pass whole-genome sequencing, followed by fragmentomics analysis, fragment end-motif profiling, and copy-number alteration (CNA) estimation.
Results: Sepharose extraction combined with a specially designed salt buffer yielded the highest DNA recovery. Commercial products did not promote fragment stability. Fragmentomics analyses showed that cancer patients exhibited higher fraction of aberrant fragments (FAF) than healthy controls when processed with Sepharose extraction. Distinct fragment end-motif patterns were also observed between the two groups. CNA analysis demonstrated that buffer-preserved samples produced higher estimated tumor fractions than samples processed with immediate freezing, indicating improved tumor signal retention. Unexpectedly, Sepharose extraction yielded improved signal-to-noise ratio as well.
Conclusions: The assay shows strong potential for detecting minimal residual bladder cancer and supporting non-invasive evaluation of complete response after chemotherapy. Salt buffer effectively enhances urinary DNA preservation and improves tumor-derived signal detection compared with immediate freezing. We are continuing to collect more urine samples to further refine and clinically validate the assay for clinical use.
利益披露 Disclosure
Y. Zheng, None.