When cells become apoptotic, their DNA gets fragmented to a distinct size of 160–180 bp and released into the bloodstream. These cell-free DNAs remain for some time as circulating fragments in the blood and – similarly to other blood analytes – can be assessed through a simple blood draw. As apoptosis is a normal cellular process that all cells in the human body eventually undergo, isolating and analyzing these cell-free DNA fragments from a blood sample can provide a comprehensive picture of the mutational proﬁle of cells across the human body.
However, the analysis of cell-free DNA requires sensitive and comprehensive high-throughput methods to capture the full complexity and diversity of cell-free DNA molecules. Such analysis tools have only become broadly available and affordable with the advent of Next-Generation Sequencing (NGS) methods in the last 5–8 years.
Tumor cells in a cancer patient undergo exactly the same mechanism of releasing their DNA to the bloodstream when they become apoptotic. Thus, the cell-free DNA obtained from a blood draw of a cancer patient contains not only normal cell-free DNA molecules, but also tumor-speciﬁc molecules which resemble the complete tumor genome (1) and provide a view on the tumor-speciﬁc mutational proﬁle. The analysis of cell-free DNA from a cancer patient to get speciﬁc information about the tumor is referred to as a “Liquid Biopsy” and is an especially promising technique for advanced tumor cases where invasive approaches to obtain tissue biopsy are life-threatening to the patient or simply not possible.
Since this potential has been embraced by the scientiﬁc and clinical community, a lot of research effort has been put into better understanding the molecular mechanisms and properties of cell-free DNA and has generated a lot of exciting ﬁndings around cell-free DNA in the last couple of years – even going towards methylation proﬁling of cell-free DNA to investigate the tissue origin of circulating DNA. Methylation proﬁling of ccfDNA can further hint at the origin of tumor-speciﬁc aberrations (2) and can show tumor-speciﬁc methylation patterns, e.g. at transcription start sites (3).
Cell-free DNA remains a challenging analyte
Despite the strong potential of cell-free DNA as a source for biomarker discoveries, it remains a challenging analyte. ccfDNA is highly variable in the plasma and varies not only from person to person, but also depending on the disease status (4). Amounts of cell-free DNA in plasma are usually limited with 1–100 ng/ ml plasma and additionally the signal-to-noise ratio between circulating tumor DNA fragments and normal circulating DNA is low. Cell-free DNA fragments are also fairly small with a mean size of 160–180 bp and require speciﬁc extraction and NGS library size selection protocols.
Another aspect about ccfDNA analysis is the accurate quantiﬁcation of tumor-derived vs. normal DNA fragments. The amount of circulating tumor DNA in the blood can be directly linked to tumor burden, cancer therapy success and overall prognosis (3). Thus, accurate quantiﬁcation of ccfDNA is essential to accurately monitor a cancer patient with a liquid biopsy.
Considering these points, dedicated solutions are needed for the analysis of cell-free DNA to maximize yields, capture the full sample complexity and obtain highly-sensitive and reliable results.
Learn more about the dedicated solutions QIAGEN offers in the ﬁeld of Liquid Biopsy at http://www.qiagen.com/liquidbiopsy.
Are you specifically looking for a solution to go from plasma to NGS-ready library? Then the QIAseq cfDNA Library Kit is the right solution for you.
Interested in knowing more about ccfDNA? Check out the recordings for our ccfDNA webinar series!
Part 1: Less is more – improved methods for fast and efﬁcient ccfDNA puriﬁcation using spin-column-based methods (Learn more)
Part 2: Standardize and streamline ccfDNA-based liquid biopsy workﬂows through automation using the QIAsymphony SP (Learn more)
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