Pre-analytical factors affecting ctDNA analysis in early and locally advanced breast cancer

项目总结 循环肿瘤DNA(CtDNA)分析使晚期癌症患者的非侵入性肿瘤基因分型成为可能 转移性癌症。现在，越来越多的人有兴趣在早期糖尿病患者中复制这种成功 癌症。CtDNA分析可以在接受治疗的患者中实现基于血液的微小残留病检测 对有症状的个体进行癌症的治疗和早期发现。然而，ctDNA水平是10s-100s 局限性癌症的折叠率低于转移性癌症，限制了目前检测的灵敏度。这是 进一步混淆了分析前的可变性，如血液采集和处理以及DNA的差异 拔牙。例如，不适当或延迟的血液处理会导致外周细胞溶解和稀释。 血液样本中的ctDNA组分。在ctDNA水平已经相当低的早期癌症患者中，这 会导致假阴性结果。分析前因素对慢性支气管炎患者ctDNA检测的影响 人们对局部癌症还没有很好的了解。 为了解决这一差距，我们建议对180名早期和局部晚期乳腺癌患者进行研究，以 调查分析前变异的三个方面：1)DNA提取方法(目标1)，2)采血 试管和处理方案(目标2)和3)血浆和提取的DNA的长期储存(目标3)。我们 将使用我们最近开发的两种分析方法来研究这些因素：1)质量评估分析， 测量总的无细胞DNA浓度和片段大小以及2)靶向数字测序(TARDIS)， 一种同时测量多达30个患者特定的创始人血浆DNA突变的方法，以量化 CtDNA水平。通过利用每个患者的多个突变，改进错误抑制并最大限度地减少 失去输入DNA材料，TARDIS能够对患者的ctDNA进行灵敏的检测和精确的定量 与目前的检测方法相比，检测下限提高了10-100倍。 利用ctDNA分析进行最小残留疾病检测和早期检测具有巨大的前景 使癌症治疗个体化，并改善结果。我们的研究将澄清分析前的因素， 对于未来多亚型局部癌症的临床研究的成功至关重要。

PROJECT SUMMARY Circulating tumor DNA (ctDNA) analysis has enabled noninvasive tumor genotyping for patients with advanced metastatic cancers. There is now growing interest in replicating this success in patients with early stage cancers. ctDNA analysis could enable blood-based minimal residual disease detection in patients receiving treatment and early detection of cancers in pre-symptomatic individuals. However, ctDNA levels are 10s-100s fold lower in localized cancers than in metastatic cancers, limiting the sensitivity of current assays. This is further confounded by pre-analytical variability such as differences in blood collection and processing and DNA extraction. For example, inappropriate or delayed blood processing can cause peripheral cell lysis and dilute ctDNA fraction in blood samples. In early stage cancer patients where ctDNA levels are already quite low, this can cause false-negative results. The effects of pre-analytical factors on ctDNA detection in patients with localized cancers are not well understood. To address this gap, we propose a study of 180 patients with early and locally advanced breast cancer to investigate three aspects of pre-analytical variation: 1) DNA extraction methods (Aim 1), 2) blood collection tubes and processing protocols (Aim 2) and 3) long-term storage of plasma and extracted DNA (Aim 3). We will investigate these factors using two assays we have recently developed: 1) a quality assessment assay that measures total cell-free DNA concentration and fragment size and 2) TARgeted DIgital Sequencing (TARDIS), an assay that simultaneously measures up to 30 patient-specific founder mutations in plasma DNA to quantify ctDNA levels. By leveraging multiple mutations for each patient, improving error suppression and minimizing loss of input DNA material, TARDIS enables sensitive detection and precise quantification of ctDNA in patients with localized cancers and improves limit of detection by 10-100 fold over current assays. Minimal residual disease detection and early detection using ctDNA analysis hold tremendous promise to individualize cancer treatment and to improve outcomes. Our study will clarify pre-analytical factors that could be critical to the success of future clinical studies across localized cancers of multiple subtypes.