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

影响早期和局部晚期乳腺癌 ctDNA 分析的分析前因素

基本信息

批准号：
10304711
负责人：
Muhammed Murtaza
金额：
$ 37.11万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-18 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10304711
关键词：
Address Advanced Malignant Neoplasm Affect Aliquot Biological Assay Blood Blood Cells Blood specimen Breast Cancer Patient Cancer Patient Cell Extracts Cells Centrifugation Clinical Research Clinical Sensitivity Collection Cytolysis DNA DNA analysis Detection Detection of Minimal Residual Disease Disseminated Malignant Neoplasm Early Diagnosis Edetic Acid Enrollment Estrogen receptor positive Future Genotype Gold Hour Individual Localized Malignant Neoplasm Malignant Neoplasms Measures Metastatic breast cancer Methods Molecular Mutation No Evidence of Disease Noise Nonmetastatic Observational Study Oncology Operative Surgical Procedures Outcome Patients Peripheral Plasma Process Prospective cohort Protocols documentation RNA Residual Tumors Sampling Screening for cancer Shipping Ships Speed Temperature Testing Tube Variant Venipunctures advanced breast cancer base cancer therapy cell free DNA cohort detection limit digital founder mutation frontier genotyped patients healthy volunteer improved improved outcome interest magnetic beads malignant breast neoplasm prospective success tumor tumor DNA

项目摘要

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.

项目概要循环肿瘤 DNA (ctDNA) 分析使晚期晚期患者能够进行无创肿瘤基因分型转移性癌症。现在人们越来越有兴趣在早期患者中复制这一成功癌症。 ctDNA 分析可以对接受治疗的患者进行基于血液的微小残留病检测对有症状的个体进行癌症治疗和早期检测。然而，ctDNA 水平为 10s-100s 局部癌症中的细胞因子比转移性癌症中的细胞因子低几倍，限制了当前检测的灵敏度。这是分析前的变异性进一步造成混淆，例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能导致外周细胞裂解并稀释血液样本中的 ctDNA 分数。在 ctDNA 水平已经相当低的早期癌症患者中，这可能会导致假阴性结果。分析前因素对 ctDNA 检测的影响局部癌症尚不清楚。为了弥补这一差距，我们提议对 180 名早期和局部晚期乳腺癌患者进行一项研究，以期研究分析前变异的三个方面：1) DNA 提取方法（目标 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游离 DNA 浓度和片段大小，2) 靶向数字测序 (TARDIS)，一种可同时测量血浆 DNA 中多达 30 个患者特异性起始突变的检测方法，以进行定量 ctDNA 水平。通过利用每个患者的多个突变，改善错误抑制并最大限度地减少错误由于输入 DNA 材料丢失，TARDIS 能够对患者体内的 ctDNA 进行灵敏检测和精确定量与现有检测方法相比，检测限提高了 10-100 倍。使用 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个体化癌症治疗并改善结果。我们的研究将澄清可能的分析前因素对于未来针对多种亚型局部癌症的临床研究的成功至关重要。