Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Erin  R. Bonner; Karim Saoud; Sulgi Lee; Eshini Panditharatna; Madhuri Kambhampati; Sabine Mueller; Javad Nazarian

doi:10.3791/59721

JoVE Journal
Cancer Research

This content is Free Access.

JoVE Journal Cancer Research

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

患者生物流体中肿瘤相关循环DNA的检测与监测

Full Text

9,297 Views

06:53 min

June 8, 2019

DOI: 10.3791/59721-v

Erin R. Bonner^1,2, Karim Saoud¹, Sulgi Lee^1,2, Eshini Panditharatna³, Madhuri Kambhampati¹, Sabine Mueller^4,5, Javad Nazarian^1,2,5

¹Center for Genetic Medicine,Children's National Health System, ²Institute for Biomedical Sciences,The George Washington University School of Medicine and Health Sciences, ³Dana-Farber Cancer Institute, ⁴Department of Neurology,University of California San Francisco, ⁵DIPG Centre of Expertise Zurich,Universitats-Kinderspital Zurich

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This protocol enables the detection of tumor-associated mutations in circulating DNA from patient biofluids, providing a minimally invasive alternative to surgical biopsies. It allows for the monitoring of tumor mutation load over time, which is particularly beneficial for tumors in difficult-to-access locations.

Key Study Components

Area of Science

Oncology
Genomics
Molecular Biology

Background

Detection of tumor mutations is crucial for diagnosis and treatment.
Traditional biopsies can be invasive and challenging for certain tumors.
Monitoring tumor mutation load can inform treatment decisions.
Digital PCR offers higher sensitivity compared to conventional methods.

Purpose of Study

To present a protocol for detecting tumor somatic mutations in biofluids.
To facilitate a non-invasive method for monitoring tumor response.
To provide a reliable alternative to surgical tissue biopsies.

Methods Used

Droplet digital PCR (dPCR) for quantifying mutation allelic frequency.
Preparation of reagents and samples for dPCR analysis.
Thermal cycling and quantification of PCR products.
Data analysis using specialized software to identify mutant and wild type alleles.

Main Results

Successful detection of mutations in plasma and cerebrospinal fluid samples.
Mutation allelic frequencies of 1.6% and 39.32% observed.
Clear separation of mutant and wild type clusters in data analysis.
Negative controls showed no contamination, confirming assay reliability.

Conclusions

The protocol provides a sensitive method for tumor mutation detection.
It allows for longitudinal monitoring of tumor dynamics.
False negatives can occur, emphasizing the need for comprehensive analysis.

Frequently Asked Questions

What is droplet digital PCR?

Droplet digital PCR (dPCR) is a highly sensitive technique used to quantify nucleic acids by partitioning a sample into thousands of droplets.

How does this protocol improve tumor monitoring?

It allows for non-invasive monitoring of tumor mutations over time, reducing the need for repeated biopsies.

What are the advantages of using biofluids?

Biofluids provide a less invasive means of obtaining genetic information compared to surgical tissue biopsies.

Can this method be used for all types of tumors?

While it is particularly useful for tumors in hard-to-reach locations, its applicability may vary based on tumor type and mutation presence.

What precautions should be taken during the procedure?

It is crucial to maintain a clean workspace and properly handle reagents to avoid contamination.

What does a negative result indicate?

A negative result does not necessarily mean the absence of mutation; false negatives can occur, and further testing may be required.

在这里,我们提出一个协议,以检测肿瘤体突变在循环DNA存在于患者生物流体(生物流体)。我们的滴数字聚合酶链反应(dPCR)方法能够定量肿瘤突变等位基因频率(MAF),促进对肿瘤反应的诊断和时间监测的微创补充。

该协议能够检测肿瘤相关突变，目前补充侵入性外科手术。另一个优势是能够使用此协议监测肿瘤突变负荷加班。该技术使肿瘤基因分型无需实际手术组织活检，这特别有助于肿瘤在具有挑战性的位置，如脑干与有限的组织访问。

在没有重复活检的情况下，纵向分子数据无法补充MRI。数字 PCR 监测有助于进行分子性诊断补充，从而实现更明智的治疗决策过程。类似的方法可用于检测特定系统中基因的表达，使用传统的 Fluo-4 和淬火器链接探针，但灵敏度高于定量 PCR。

在开始液滴数字 PCR 程序之前，使用 10% 漂白剂和 70% 乙醇清洁工作台空间和设备，并轻轻涡旋并短暂离离除液滴稳定油以外的所有试剂。确认压缩氮气瓶连接到液滴生成仪器，且气瓶罐设置为每平方英寸 90 磅。38微升数字PCR反应混合物直接进入8井PCR管带的每一个井的底部，用干净的移液器尖端去除任何气泡。

在管条的适当孔中加入12微升预放大DNA样品的三分之一。轻轻移液10次，将反应混合物与DNA样品混合，将每井的全音量移液到液滴生成仪器芯片的相应 A 通道中。将新的 PCR 管条插入液滴生成仪器，并将液滴生成仪器芯片 ID 扫描到仪器计算机上的软件中。

然后单击"开始运行"以开始滴滴样本。液滴化完成后，拆下 PCR 管条并涂抹管条盖。将管带转移到热循环器中，将管带与每井包含 80 微升水的管带进行平衡。

然后在适当的热循环条件下运行热循环器。用高速盖更换带盖。热循环完成后，将管条传输到定量仪器，单击仪器软件上的"设置运行"，然后将管条放入定量仪器中。

将金属屏蔽放在新的定量仪器芯片上，将芯片 ID 扫描到仪器中，然后将芯片插入机器中。合上仪器的盖子。在计算机软件中，输入数字 PCR 运行和八个通道中每个通道的名称，然后选择"快速模式"并单击"开始"开始量化。

要分析原始光谱数据，请启动分析软件并打开 fcs 文件。在分析视图中，选择"完好无损"。在示例视图下，单击八个样本旁边的框。

该软件将分别沿 x 轴和 y 轴绘制突变类型和野生等位线的信号。使用计算的矩阵函数根据制造商指导器对完整液滴应用光谱补偿，并在轴选项下调整轴设置。将 x 轴设置为最小零，最大值为 30，000，y 轴设置为负 5，000，最大值为 10，000。

识别液滴聚类后，调整轴以减少图形中的空间。选择与阳性对照肿瘤组织基因组DNA对应的样本，设置阴性突变体和野生型门，确保聚类清晰易识别。然后右键单击并选择"将所有设置应用于选定示例"，将正控的门设置应用于所有样本。

在图形视图下，单击多个样本以查看所有选定样本的绘图，然后将图像导出为 tif 文件。然后在"工作区"下，选择"导出分析"，然后将分析的数据文件另存为 csv 文件。在这里，显示了成功检测前扩增血浆和脑脊液细胞自由DNA的代表性结果，从两个儿童与弥漫中线胶质瘤。

在此实验中，可以分别沿 x 轴和 y 轴观察到突变类型和野生类型聚类之间的清晰分离。强大的野生类型聚类表明，细胞自由DNA提取是成功的，因为模板DNA存在。对于这些患者，突变组显示血浆和脑脊液样本的突变频率为1.6%和39.32%，根据肿瘤突变状态，通过活检肿瘤组织的基因组分析确认。

另一方面，负对照显示零突变和零野生型液滴，表明PCR反应混合物没有污染。阳性对照肿瘤组织基因组DNA显示所选特定肿瘤样本的预期全体频率检测到的突变。血浆中缺乏突变检测可能没有必要意味着患者是野生类型的兴趣突变，因为假阴性确实发生。

例如，在这个患者中，虽然没有检测到感兴趣的突变，但肿瘤组织的基因组分析证实了突变状态。PCR 是一种非常敏感的技术，需要频繁对工作区和设备进行净化，以避免获取误报数据。

Explore More Videos

癌症研究第148期液体活检数字液滴PCR 循环细胞游出DNA 血浆血清脑脊液