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An overview of alternative biomarkers in liquid biopsy

The most common analytes in liquid biopsies are CTCs and ctDNA. However, there is so much more that can be found circulating around the human body that shouldn’t be ignored.

Extracellular vesicles (EVs)

EVs have gained attention as they are mediators of intercellular communication, and transporters of genetic material and signalling molecules between cells. As EVs reflect the contents of the cell of origin, studies have been conducted that demonstrate that they have good potential to be a biomarker source for cancer diagnostics. EVs are classified based on their size and the biological mechanism by which they are generated (see Figure 1).

Figure 1: EV classification.(a)Exosomes are generated through the endocytic pathway and released via exocytosis, are spherical in shape and have size range of 30–200 nm in diameter. (b) Microvesicles are released through budding from plasma membrane, are irregular in shape and range in size between 100–1000 nm in diameter. (c) Apoptotic bodies are released through blebbing by cells undergoing apoptosis and are > 1000 nm in size. Taken from: The exosome journey: from biogenesis to uptake and intracellular signalling (Gurung et al., 2021)

The use of EVs as targets for liquid biopsy has been extensively explored – with a particular interest shown towards exosomes. Exosomes are released into blood circulation and other body fluids, meaning that they can be sampled through liquid biopsy.

Owing to their complex molecular cargoes, exosomes are following in the footsteps of CTCs and ctDNA as exciting and novel liquid biopsy biomarkers. It has even been suggested that exosomes may be superior to CTCs and ctDNA for early diagnosis, disease monitoring and prognosis prediction in cancer.

Alternative nucleic acids

Although circulating DNA has been leading the way in liquid biopsy, there are other types of nucleic acids that can be non-invasively detected and shouldn’t be ignored. Analysis of these alternative nucleic acids can also provide complementary diagnostic information that can’t be found by analysing DNA alone.

DNA methylation

Although not technically a different biomarker, analysing the methylation status of ctDNA allows liquid biopsy to move past genomics and gain insight into epigenetic modifications. As analysing methylation status uses ctDNA as a template, much of the liquid biopsy sample preparation remains the same as genomic analysis. Following ctDNA extraction, many different technologies are available for the detection of ctDNA methylation status.

Cell-free RNA (cfRNA)

Unlike ctDNA, cfRNA is released from both cancerous and non-cancerous cells. It can be derived from the non-malignant cells in the tumour microenvironment, such as stromal cells and immune cells. This means that cfRNA can be a valuable source of diagnostic information, revealing insights about the tumour that would be missed by analysing ctDNA alone. Changes in RNA expression are also more reflective of the dynamic processes going on within cells – and when these processes go wrong – whereas DNA provides only a static readout of cellular genomics.

Micro-RNA (miRNA)

miRNAs are one of the most important components of circulating cell-free nucleic acids which can be found in bodily fluids. Circulating miRNAs are potentially a very useful biomarker as they are relatively stable and are found in multiple bodily fluids, including serum, plasma and saliva.

MiRNAs can be found circulating freely, also known as circulating cell-free microRNAs (cfmiRNAs) or found in exosomes, also known as exosomal miRNAs (exomiRs).

Circular RNA (circRNA)

circRNAs are a novel class of single-stranded RNAs with a closed loop structure, formed by a back-splicing process in pre-mRNA splicing. circRNAs have diverse functions as miRNA decoys, protein regulators and translation templates, impacting many different biological functions. Aberrant expression of circRNAs is therefore implicated in human disease.

The high stability, abundance and spatiotemporal expression of circRNAs make them ideal candidate biomarkers for liquid biopsy. However, challenges need to be addressed before circRNA biomarkers can be translated clinically.

Despite substantial advances, the methods to discover and profile circRNAs are far from optimal. Future studies should focus on applying different analytical techniques, such as RNA sequencing and circRNA microarray, to test the analytical performance and move towards clinical validation.

Proteins and proteomics

As with other circulating biomarkers, detectable proteins in liquid biopsies fall into two categories. The first category consists of cell-free proteins and the second category encompasses proteins that are present in circulating cells or subcellular structures such as EVs.

The diagnostic use of protein biomarkers in blood pre-dates the development of liquid biopsy as it is known today.  Traditional techniques (ELISA and immunohistochemistry) are used to analyse proteins present in blood, which suffer from a low throughput compared to other techniques used in liquid biopsy.

High-plex proteomics

Recent years have seen the advancement of high-plex proteomic technologies that can facilitate the integration of proteomics into liquid biopsy. High-plex proteomic techniques include: mass spectrometry, antibody/antigen arrays, aptamer-based arrays, proximity extension assays and reverse phase protein arrays.

Single-cell proteomics

Single-cell proteomics is forging a path across all fields of cancer biomarker discovery, including liquid biopsy. Circulating cells are prime targets for single-cell proteomics as they are already dissociated from tissue and therefore compatible with analysis.

Combining existing CTC technologies and assays with single-cell proteomics has the potential to completely redefine circulating cellular analysis within liquid biopsy.

Report: Liquid Biopsy – A New Era in Disease Detection and Surveillance

A guide to liquid biopsy and non-invasive diagnosis


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Cancer / Clinical Trials / ctDNA / Liquid Biopsy