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

项目摘要 循环肿瘤DNA（ctDNA）分析使晚期乳腺癌患者能够进行非侵入性肿瘤基因分型。 转移性癌症现在越来越有兴趣在早期患者中复制这种成功 癌的ctDNA分析可以在接受抗肿瘤治疗的患者中进行基于血液的微小残留病检测。 治疗和早期发现症状前个体的癌症。然而，ctDNA水平为10 - 100 在局部癌症中比在转移性癌症中低1倍，限制了当前测定的灵敏度。这是 进一步受到分析前变异性的混淆，如血液采集和处理以及DNA的差异 萃取例如，不适当或延迟的血液处理可导致外周细胞溶解和稀释。 血液样品中的ctDNA分数。在ctDNA水平已经相当低的早期癌症患者中， 会导致假阴性结果分析前影响因素对宫颈癌患者ctDNA检测结果的影响 局限性癌症还没有被很好地理解。 为了解决这一差距，我们建议对180例早期和局部晚期乳腺癌患者进行研究， 研究分析前变异的三个方面：1）DNA提取方法（目的1），2）血液采集 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测定法来研究这些因素：1）质量评估测定法， 测量总的无细胞DNA浓度和片段大小和2）靶向数字测序（TARDIS）， 一种同时测量血浆DNA中多达30种患者特异性创始者突变的测定法， ctDNA水平。通过利用每个患者的多个突变，改善错误抑制并最小化 输入DNA材料的损失，TARDIS能够灵敏地检测和精确定量患者的ctDNA 与局部癌症相比，检测限提高了10-100倍。 使用ctDNA分析的微小残留病检测和早期检测为 个体化癌症治疗和改善结果。我们的研究将澄清分析前的因素， 对于未来在多种亚型的局部癌症中进行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.