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Cell-free DNA (cfDNA) refers to DNA fragments present in extracellular fluids in the human body. In physiological or pathological states – for example, during pregnancy or in tumours – the levels and molecular signature of cfDNA undergo transformations, which rank it among the markers for diagnosis and treatment. cfDNA tests are now diagnostic by definition, as cfDNA enrichment methods and newer detection techniques such as sequencing and mass spectrometry have enabled. Liquid biopsy with cfDNA detection is an accepted technology in clinical oncology and prenatal diagnosis, and has great promise in the diagnosis and treatment of autoimmune disorders.
What is Cell Free DNA?
Cell-free DNA (cfDNA) are DNA fragments that exist in body fluids in the form of single-stranded, double-stranded, or circular DNA, typically ranging from 20 to 200 base pairs in length, with some fragments reaching up to 1,000 base pairs. Exogenous cfDNA mainly come from bacteria, fungi, viruses and other microorganisms that get into the body via processes such as endocytosis and cell-to-cell exchange. In contrast, natural cfDNA is expunged into the extracellular world from the human body via apoptosis and necrosis and carries genes from the nuclear and mitochondrial genomes. cfDNA was first identified in human peripheral blood in 1948, and has since been found to be elevated in individuals with autoimmune diseases and cancers. With cfDNA detection systems now available, cfDNA-based liquid biopsy has become routinely used in clinical non-invasive prenatal diagnosis and oncology.
The applications of cfDNA in cancer. (Yan, Y.Y.; et al, 2021)
Sources of Cell Free DNA
- Normal Cellular Turnover: Under normal physiological conditions, cfDNA is released into circulation through processes like apoptosis and necrosis. In apoptosis, cells undergo controlled death, releasing DNA fragments into the bloodstream.
- Cancer Cells: Tumors, particularly those undergoing rapid growth or necrosis, shed cfDNA into the bloodstream. This tumor-derived cfDNA (ctDNA) can carry genetic mutations, making it a valuable marker for early cancer detection and monitoring therapeutic responses.
- Fetal Cells: In pregnant women, a portion of cfDNA in the maternal bloodstream is derived from the fetus. This fetal cfDNA has revolutionized non-invasive prenatal testing (NIPT), enabling the detection of chromosomal abnormalities like Down syndrome.
- Organ Transplants: cfDNA from the transplanted organ can be detected in the blood, offering a non-invasive method for monitoring organ rejection.
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Cell Free DNA Methylation
Methylation of DNA is a key epigenetic modification that regulates gene expression without altering the underlying genetic code. In the context of cfDNA, methylation patterns can serve as biomarkers for various diseases, including cancer. Unlike genetic mutations, which represent permanent changes in the DNA sequence, methylation modifications are dynamic and can change over time in response to disease progression or treatment.
cfDNA Methylation Analysis
The analysis of cfDNA methylation patterns has proven to be highly effective in cancer diagnostics. Tumors often exhibit abnormal DNA methylation patterns that are detectable in cfDNA, even in the early stages of cancer. These methylation markers are tissue-specific, allowing for the identification of the origin of the tumor and providing insight into its aggressiveness. In addition to cancer, aberrant methylation patterns in cfDNA have been implicated in other diseases such as neurological disorders, cardiovascular diseases, and autoimmune conditions. The ability to detect these changes non-invasively provides significant advantages over traditional biopsy-based methods.
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Cell Free DNA Test
The ability to analyze cfDNA offers numerous diagnostic advantages. Traditional biopsies require invasive tissue samples, which can be painful, expensive, and time-consuming. In contrast, cfDNA testing is non-invasive, with a simple blood draw being sufficient to obtain genetic material for analysis. This process is often referred to as "liquid biopsy" and is increasingly being used in clinical settings for cancer detection, prenatal screening, and monitoring genetic diseases. cfDNA testing involves the extraction of DNA from plasma or serum samples, followed by the analysis of genetic markers, mutations, or epigenetic modifications. For example, mutations in the KRAS gene or other oncogenes can be detected in cfDNA, providing early indications of cancer. Additionally, cfDNA testing can be used for monitoring minimal residual disease (MRD) in cancers, offering insights into treatment efficacy and recurrence.
cfDNA Test Samples
The process of collecting and processing cfDNA for analysis begins with a blood draw. Plasma or serum is then separated from the whole blood sample by centrifugation. This is a critical step, as it ensures that only the extracellular DNA, and not genomic DNA from the cells, is collected for further analysis. The quality of cfDNA extraction can influence the accuracy of downstream analyses, making the choice of extraction method crucial for obtaining reliable results. Several methods are available for extracting cfDNA, ranging from silica-based column kits to magnetic bead-based extraction systems. The choice of method depends on the intended downstream analysis, as well as the specific requirements of the study. For example, if a high-quality cfDNA sample is required for methylation analysis, more stringent extraction methods may be employed.
cfDNA Extraction
The extraction of cfDNA from blood samples is a key step in the analysis process. High-quality cfDNA extraction ensures that the material is suitable for a variety of downstream applications, including next-generation sequencing (NGS), quantitative PCR, and methylation analysis. Different methods are used for cfDNA extraction, with the most common being:
- Silica-based columns: These provide a quick and efficient method for isolating cfDNA from plasma. They rely on the selective binding of DNA to silica membranes in the presence of certain buffers.
- Magnetic bead-based methods: These are often used for more efficient extraction and for dealing with low cfDNA concentrations, which is common in early-stage cancers or during disease monitoring.
- Automated extraction systems: For high-throughput studies, automated systems can process a large number of samples quickly and efficiently, ensuring consistency and reproducibility.
cfDNA Analysis
Once cfDNA has been extracted, it undergoes various types of analysis to identify specific genetic or epigenetic markers. Two of the most common analytical techniques used for cfDNA include:
- Next-Generation Sequencing (NGS): NGS allows for the comprehensive analysis of cfDNA, identifying mutations, structural variants, and methylation patterns across the genome. This method can provide valuable insights into cancer genomics, such as the detection of somatic mutations that drive tumor growth.
- Digital PCR and Quantitative PCR (qPCR): These techniques allow for the precise quantification of specific cfDNA markers, including mutations or epigenetic alterations. These methods are particularly useful for monitoring treatment response and detecting minimal residual disease.
With advances in technology, the accuracy and sensitivity of cfDNA analysis have significantly improved, making it a powerful tool for disease monitoring and personalized treatment approaches.
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cfDNA vs ctDNA
Although cfDNA and circulating tumor DNA (ctDNA) are both found in the blood, there are important differences between the two. cfDNA refers to the total DNA present in the plasma, including both normal and tumor-derived fragments. On the other hand, ctDNA specifically refers to DNA fragments that are shed by tumor cells into the bloodstream.
ctDNA analysis is a subset of cfDNA testing and is primarily used for cancer diagnostics. ctDNA has been shown to harbor tumor-specific mutations, allowing for the detection of cancers at early stages, monitoring of treatment responses, and identification of minimal residual disease. cfDNA, in contrast, may contain fragments from both healthy and diseased cells, making it more suitable for broader applications such as prenatal testing, genetic screening, and overall disease monitoring.
Applications of Cell Free DNA Testing
The applications of cfDNA testing are vast and rapidly expanding across multiple fields of medicine. Some of the most prominent applications include:
Cancer Detection and Monitoring
CfDNA, particularly ctDNA, is an important biomarker for detecting cancer in its early stages. Liquid biopsies are being increasingly used to detect genetic mutations associated with cancers such as lung, colorectal, and breast cancer. These tests can help monitor tumor evolution and treatment efficacy without the need for invasive tissue biopsies.
Prenatal Testing
Prenatal testing using cfDNA has revolutionized non-invasive screening for fetal genetic disorders such as Down syndrome, trisomy 18, and trisomy 13. This approach allows for early detection of chromosomal abnormalities, offering an alternative to traditional amniocentesis, which carries a risk of miscarriage.
Transplantation Monitoring
In organ transplantation, cfDNA can be used to monitor graft rejection. The presence of donor-derived cfDNA in the recipient's bloodstream can be an early indicator of transplant rejection, allowing for timely intervention.
Genetic Screening
cfDNA is a valuable resource for detecting genetic disorders in newborns, as well as for broader population screening. The ability to analyze cfDNA from a blood sample makes it an excellent tool for non-invasive genetic screening.
The advancements in cfDNA research and technology have led to transformative changes in diagnostic practices.
* Only for research. Not suitable for any diagnostic or therapeutic use.
