cfDNA and Cancer Diagnostics

By Priyanka Varma

BURLINGAME, Calif.

Mar. 15th, 2022

“cfDNA” or cell free DNA is double stranded and highly fragmented and is released from the cells on their death via necrosis or apoptosis [1, 2]. They are highly stable in body fluids as they are transported via microvesicles or protein-cf-Nucleic acid complexes, making them ideal biomarker candidates for liquid biopsy [1]. They can then be utilized for assessing DNA methylation with array-based technologies (Methylation specific Polymerase Chain Reaction (PCR) and pyrosequencing. cfDNA is mostly tested in blood, but other bodily fluids like urine, saliva, Cerebrospinal fluid, seminal plasma, tears etc. can also be used for its detection [1]. cfDNA is not specific to cancer detection and has been earlier recognised as a marker for prenatal genetic testing, inflammation, diabetes, sepsis, myocardial infarction etc [3]. 

The concentration of cfDNA ranges from 0 to 1000 ng/ml in cancer versus 0 to 100 ng/ml in healthy subjects [3]. The size of cfDNA ranges from 40-200 base pairs or more (in 1000s), depending on the type of cfDNA, whether it is genomic, mitochondrial, or microbial [1, 3]. The integrity of cfDNA has clinical implications as cfDNA from cancer patients have higher cfDNA integrity compared to healthy volunteers. The cfDNA integrity is ascertained by the ratio of long to short product amplified from one genetic locus [3]. The concentration of cfDNA is also determined by its very short estimated half life (several minutes to 1-2 hours) which is related to its clearance and rapid turnover [2, 3]. 

If blood is the body fluid of choice for collecting cfDNA, then the collection of blood or sampling procedure can affect the type of cell death, by releasing necrotic DNA from cells, resulting in elevated concentration of cfDNA [3]. Ruptured blood cells have been recognized to be the primary contaminant in cfDNA collection.  While the yield of cfDNA is higher in serum versus plasma, in theory, the contamination of cfDNA is lesser in plasma [2].

While the advantages of cfDNA are varied in cancer diagnostics, the limitations are low sensitivity and specificity, given the low concentration of cfDNA, high degradation, as well as high admixture of normal DNA in cfDNA. PCR-based methods involve detection of mutation and can be either qualitative or quantitative in nature [2]. Whole exome/ genome sequencing assists in identifying all the aberrations in the DNA. The limitations can be overcome by PCR amplification and hybridization, or by using methods like CAPP-Seq to improve the sensitivity of mutation detection [1].

‍

Bibliography:

1. Szilágyi M, Pös O, Márton É, Buglyó G, Soltész B, Keserű J, Penyige A, Szemes T, Nagy B. Circulating Cell-Free Nucleic Acids: Main Characteristics and Clinical Application. Int J Mol Sci. 2020 Sep 17;21(18):6827. doi: 10.3390/ijms21186827. PMID: 32957662; PMCID: PMC7555669.
2. Stanislav Volik; Miguel Alcaide; Ryan D. Morin; Colin Collins Mol Cancer Res (2016) 14 (10): 898–908.https://doi.org/10.1158/1541-7786.MCR-16-0044
3. Anatoli Kustanovich, Ruth Schwartz, Tamar Peretz & Albert Grinshpun (2019) Life and death of circulating cell-free DNA, Cancer Biology & Therapy, 20:8, 1057-1067, DOI: 10.1080/15384047.2019.1598759

‍