Obtaining an effective diagnosis is the crucial first step in the treatment of cancer and many other diseases. Unfortunately, invasive biopsy procedures are currently often the gold-standard used to obtain the material required for molecular diagnostic testing.

Liquid biopsy offers another proposition – the ability to perform molecular diagnostic testing using a simple blood test; a minimally invasive alternative to traditional biopsies. Over the past decade, this central proposition has led to the progression of liquid biopsy and the development of many novel technologies. Liquid biopsy has been applied to a number of different areas and has spawned a thriving research community.

Here, we take a look at the history and development of this rapidly progressing field.

Circulating tumour cells (CTCs)

The history of liquid biopsy starts with CTCs. It has long been understood that cancer cells can be found circulating in the bloodstream. The dissemination of cancer cells into blood circulation is involved in the process of metastasis and explains how primary tumours are able to form secondary sites in distant organs and tissues.

In the early-mid 2000s, the diagnostic relevance of CTCs was demonstrated when higher levels were found in cancer patients compared to healthy controls. As interest into CTCs grew, subsequent studies integrated an element of CTC research into established cancer biology. For example, analysis of EGFR mutations in non-small cell lung cancer patients was performed using DNA extracted from CTCs.

Establishing liquid biopsy

In a review exploring the challenges and opportunities of CTCs in cancer in 2010, the term “liquid biopsy” was coined for the first time by Catherine Alix-Panabières and Klaus Pantel. Although liquid biopsy was initially only used to define CTCs, the term was quickly expanded to include another circulating biomarker – circulating tumour DNA (ctDNA).

Despite this late inclusion, the presence of cell-free nucleic acids in blood circulation had been discovered long before. Research into common cancer mutations, such as those in the RAS gene, had also been explored using ctDNA prior to its uptake in the field of liquid biopsy.

With CTCs and ctDNA established as the two key components, research in the field of liquid biopsy expanded rapidly. CTCs and ctDNA have been highlighted as prognostic biomarkers in the majority of solid tumour types, including breast, prostate, lung and colorectal.

Ongoing studies aim to demonstrate the clinical utility of CTCs and ctDNA in areas such as early cancer detection, improved cancer staging, detection of relapse, real-time monitoring of therapeutic response and the detection of novel therapeutic targets and disease mechanisms.

This has led to the development of many technologies for the detection, enrichment and characterisation of CTCs and ctDNA, as well as multiple assays that can be performed on the targets (see Figure 1). Both of these liquid biomarkers are also currently being pursued in hundreds of active clinical trials.

The application of liquid biopsy in cancer

Figure 1: Technologies for CTC and ctDNA enrichment, detection, and characterisation. Analysis that can be performed on ctDNA: chromosomal aberrations, epigenetic marks, translocation analysis, fragmentomics, detection of deletions and point mutations, amplification. CTCs can be enriched by their biological or physical properties. Detection of CTCs can be performed with molecular biology, immunocytology and functional assays. CTCs can be characterised at the molecular level and their functional properties can be investigated by using xenograft models and *in vitro* CTC lines. CNA – copy-number alteration; DEP – dielectrophoresis; NGS – next-generation sequencing; WGA – whole-genome analysis. Taken from Liquid biopsy: from discovery to clinical application (Alix-Panabières & Pantel, 2021).

More biomarkers, more liquids

As the liquid biopsy field progressed, it was discovered that a single blood sample can yield much more molecular information than just CTCs and ctDNA. Alongside ctDNA, the plasma fraction of blood also contains extracellular vesicles (EVs), proteins, cell-free RNA and tumour-educated platelets (TEPs).

In the cellular fraction of blood, CTCs can be found as single cells or in clusters with other CTCs or immune cells. Other cell types present include immune cells, circulating endothelial cells (CECs) and cancer-associated (CA) fibroblasts.

An overview of alternative biomarkers in liquid biopsy

The term liquid biopsy has become synonymous with blood and the circulating biomarkers that can be extracted from blood. However, there are many other liquids that can be extracted, or released naturally, from the human body. Each of these is an important source of circulating cells, DNA, or other biomarkers.

Liquids such as saliva and urine can offer a less invasive diagnostic approach than even a minimally invasive blood test. Cerebrospinal fluid (CSF) can be enriched in molecular information regarding primary and metastatic brain tumours. Saliva and sputum can present a more targeted approach for the analysis of head, neck and lung cancers just as urine can for bladder, renal and prostate cancers. Finally, bone marrow can act as a source of biomarkers from multiple cancers such as breast, prostate, lung and colorectal.

An overview of alternative fluids in liquid biopsy

A guide to liquid biopsy and non-invasive diagnosis

Report: Liquid Biopsy – A Non-Invasive Approach to Health and Disease