cfDNA Quantitation for Research and Clinical Applications

Jan 31, 2019 | Blog

Liquid biopsy gains traction as a powerful minimally invasive sampling method. Circulating cell-free DNA (cfDNA) is often analyzed from liquid biopsy samples for molecular diagnostics. cfDNA is small DNA fragments found free-floating (therefore cell-free) in blood or other bodily fluids. The origin of the cfDNA is apoptosis (which generates smaller fragments such as 100 to 500 bp, but average at 170 bp) and necrosis (around 1000 bp).

cfDNA becomes Important for Cancer Diagnostics

Most of the cfDNA authors found low concentration of cfDNA from 0 to a few ng/ml in healthy individuals while pregnant women or people with different diseases or medical treatments have elevated level of cfDNA.

cfDNA has been extensively studied for the capacity to be used as a clinical biomarker in cancer diagnostics. Not only the quantitation has been used for study of tumor progression in various types of cancers, the gene mutations and epigenetic changes in cfDNA at different stages of cancers have been widely used for clinical diagnostic tests using different mutation detection technologies including next-generation sequencing (NGS).

Early cfDNA Discovery

Here is the timeline for early cfDNA discovery:

  •  1948: Mandel and Metais discovered cfDNA present in blood plasma
  • 1965: Bendich et al. hypothesized that cfDNA from cancer could be involved in metastasis
  • 1966: Tan et al. detected high levels of cfDNA in the blood of systemic lupus erythematosus patients  
  • 1977: Leon et al. demonstrated that for the level of cfDNA was significantly higher in at least half of cancer patients than in normal control subjects.

cfDNA studies grow exponentially in recent years and research and clinical studies are evaluating cfDNA as a predictive biomarker for different applications.

cfDNA Clinical Trials

The clinical trials using cfDNA started in 2009 and the number of trials has been increasing over the years. More than 20 clinical trials were conducted in 2014 and the number reached about 80 in 2016. Three major types of clinical trials have been conducted:

  • Concordance trials assess if plasma genotyping assays using cfDNA can accurately detect mutations in tumor tissue.
  • Observational trials determine if the cfDNA can be used as a predictive biomarker.
  • Interventional trials focus on treatment outcome comparison between decisions made from plasma genotyping and from the standard of care.

cfDNA Quantitation is Challenging

Different methods have been used for quantitation of cfDNA., but accurate quantitation of cfDNA is challenging because of its lower amount in plasma. Direct measurement has been hampered since complex plasma matrix causes analytical inaccuracy with commonly used methods. These methods such as spectrometry and dye analysis can only be used after cfDNA extraction to achieve reliable measurement. Some other methods use quantitative real-time PCR with different house-keeping or abundantly expressed genes to quantify cfDNA after extraction and require method development and optimization.

The above traditional methods failed to directly and accurately measure cfDNA concentration because some of the low level of cfDNA can get lost in the extraction process, in addition to the bias of different measurements following the extraction.

Our QuantiDNA® Alu-assay applied in our Radiation toxicity measurement, uses a few microliters of plasma for determination of cfDNA without DNA extraction or PCR using SuperbDNA technology. This method is especially useful when only quantitation of cfDNA is required and multiple samples are processed at the same time in a time-course study. The method provides great value for direct quantitation of cfDNA to follow cfDNA timely changes in a disease study process while DNA extraction followed by measurement is better used for cfDNA quantitation for genetic mutation detection or epigenetics characterization applications.

cfDNA Quantitation Applications

cfDNA quantitation can be used, but not limited, in the following applications:

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