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Introduction

Recent advances in molecular oncology, genomics and bioinformatics have furthered the understanding of the biology of cancers and given researchers and clinicians the tools to detect and treat an individual's cancer based on the specific genomic profile of the tumour.

Obtaining information on a patient's tumour through a biopsy can be invasive, costly and difficult to repeat over time. Alternative ways are being developed that are less invasive, more accurately reflect the heterogeneity of the tumour and can easily be repeated, allowing real-time analysis of the genomic profile of the tumour.

Cell-free nucleic acids include cell-free DNA (plasma DNA), RNA and circulating microRNA (miRNA). These are nucleic acid fragments that are released into the bloodstream from both normal cells and tumour cells, most likely through necrosis and apoptosis, and can be analysed in a liquid biopsy. Of these, plasma DNA is the most established as having clinical relevance, although there is currently much research into the detection and use of free RNA, miRNA and exosomes. Free RNA and miRNA are present in blood either in ribonucleoprotein complexes or in extracellular vesicles such as exosomes. RNA packaged in exosomes is relatively stable, making it potentially suitable for analysis in a liquid biopsy. Exosomes are present in most body fluids and there is increasing interest in their use as cancer biomarkers and therapeutic targets.

The proportion of plasma DNA that is derived from the tumour is highly variable, often very low at <1% of total plasma DNA, and is known as circulating tumour DNA (ctDNA). There is a high degree of fragmentation of the ctDNA, with most between 160 and 180 base pairs long,1 reflecting fragments of DNA that in plasma are wound around a single nucleosome.

Methods for analysis

The extraction of plasma DNA from blood for analysis of tumour-specific alterations is relatively simple and cheap to achieve. It is important to minimize release of genomic DNA during sample processing; therefore, blood is collected most commonly in either EDTA or cell-free DNA blood collection tubes,2 centrifuged twice and plasma removed. Plasma DNA is extracted from the plasma using commercially available kits. Extraction of DNA from plasma, as opposed to serum, is preferred because of the lower concentrations of normal free DNA, with release of normal genomic DNA in the clotting of serum. EDTA is the preferred anticoagulant for plasma, as chelation by EDTA inactivates plasma DNAse enzymes. There are now several technologies that can be used to detect and characterize the extracted ctDNA. Two of the most common approaches include digital polymerase chain reaction (PCR) and massively parallel sequencing-based methods.

Digital PCR

Digital PCR is a highly sensitive and relatively inexpensive method used for the quantification of nucleic acids. It has many applications, including detection of mutations, quantification of copy number alterations and gene expression analysis. Methods include BEAMing, droplet digital PCR and array-based digital PCR.3 Digital PCR can detect mutations down to an allele fraction of 0.01% but can only detect a small number of mutations simultaneously and ...

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