What Is cfDNA?

The advancement of liquid biopsies in the clinic have made these processes invaluable in the search for disease biomarkers in a minimally invasive manner. While a wide variety of biomolecules can be isolated from bodily fluids, free-floating nucleic acids are particularly useful when it comes to the detection of clinically relevant biomarkers in blood. In this article, we will define what circulating free DNA are and how these biomarkers are analyzed in the clinic.

Circulating free DNA (cfDNA) are degraded DNA molecules that are released into the bloodstream by cells. These fragments, which can vary in length between 50 and 300 base pairs, are generally found at low concentrations in the blood of a healthy individual. However, elevated levels of cfDNA in blood are commonly observed with the progression of cancers and other health conditions. The various types of cfDNA isolated from human blood for diagnostic and screening purposes include circulating tumor DNA, mitochondrial DNA and fetal DNA.

The bulk of cfDNA research is founded upon DNA originating from cancer cells. Circulating tumor DNA (ctDNA) are nucleic biomarkers used to detect or monitor the advancement of cancer over time. These cancer biomarkers are released by a tumor or circulating cancer cells as they undergo apoptosis or necrosis. As a tumor grows and matures, the concentration of fragmented ctDNA increases in circulation.

Cell-free mitochondrial DNA (cf mtDNA) are another type of cfDNA that are released by damaged or stressed cells. The circulation of these stress-derived fragments throughout the body can activate an inflammatory response by our immune system. While cf mtDNA has not been as readily studied as ctDNA, it is understood cancer patients have a measurable difference of circulating mtDNA in their blood when compared to healthy individuals. This finding exhibits the potential value of cf mtDNA for precise liquid biopsies.

Cell-free fetal DNA (cffDNA) are circulating fragments of fetal DNA that can be found in maternal blood during pregnancy. Detectable as early as seven weeks after gestation, cffDNA fragments are released into the maternal blood circulation by the death of placental cells. cffDNA fragments make up to 13% of cfDNA in maternal blood, which gives clinicians the ability to perform prenatal screening for genetic conditions in a non-invasive manner.

The advancement of liquid biopsies in oncology and prenatal medicine has demonstrated the importance of cfDNA as a valuable target in the 21^st century. When it comes to cancer, the discovery of cfDNA with tumor-specific changes opened the door for researchers and clinicians to noninvasively screen the blood of cancer patients for ctDNA. Liquid biopsies provide an attractive alternative to surgical biopsies since tumor-derived cfDNA can be collected repeatedly over time, which enables clinicians to monitor the progression of a patient’s cancer. The implications of this discovery continue to impact the resolution of clinical observations during diagnosis or prognosis today.¹

While cancer research is responsible for progressing the technological advances of cfDNA research, the discovery of cffDNA further established the importance of cfDNA as an essential diagnostic marker. With the advancement of next generation sequencing, cffDNA detection techniques have improved our ability to screen for genetic abnormalities in the fetal DNA.²

The testing of cfDNA involves the collection and processing of circulating DNA derived from a patient’s bodily fluid. Since cfDNA generally comes from dead or damaged cells, cfDNA testing methods need to be optimized for their target(s) of interest while minimizing the impact of cfDNA contamination.

To purify cfDNA from a sample, the collection of blood is carried out manually through venipuncture or with automated sampling systems designed for high throughput applications.³ Once cfDNA has been successfully isolated, genomic analysis can be performed. Sequence-specific detection methods such as quantitative PCR are one of the ways researchers can quantify cfDNA of a single sequence of interest. If deeper analysis is required to detect mutations amongst a fraction of cfDNA, next generation sequencing techniques can be used to detect such changes in DNA sequence.

Non-invasive prenatal testing (NIPT) is a relatively new type of cfDNA testing that presents no risks to expecting mothers interested in prenatal screening and diagnosis. NIPT is a blood test designed to analyze fetal DNA derived from maternal blood for chromosomal abnormalities such as Down Syndrome or trisomy 18. NIPT can also be used to screen for the sex of the developing fetus. Even though NIPT generally reports the likelihood of a chromosomal abnormality as a probability, it is a convenient method for screening genetic conditions when compared to amniocentesis.

Beyond these applications in prenatal care, cfDNA can also be exploited by oncologists to guide treatment or monitoring programs for tumors detected relatively early on. For example, the concentration of cfDNA in a patient’s bloodstream can be measured over time to assess tumor growth or regression in response to targeted treatments against the tumor(s). This approach can be similarly applied for patients who experience a stroke, sepsis, or myocardial infarction.⁴

Another useful application for cfDNA testing involves the monitoring of solid organ transplants. The levels of cfDNA derived from a donor’s organ can be measured in the transplant patient’s blood to monitor the likelihood of organ rejection or acceptance.