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A guide to liquid biopsy and non-invasive diagnosis

Liquid biopsy offers an innovative solution for the diagnosis and monitoring of cancer and other diseases. However, traditional invasive biopsy procedures remain the primary method of diagnosis in the majority of cases. How can liquid biopsy deliver on its promise and move into widespread clinical use?

A brief history of liquid biopsy

In a review exploring the challenges and opportunities of circulating tumour cells (CTCs) in cancer in 2010, the term “liquid biopsy” was coined for the first time by Catherine Alix-Panabières and Klaus Pantel. However, the principles, techniques and candidate biomarkers that would go on to form the pillars of liquid biopsy research were established before this.

Circulating tumour cells (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.

Circulating tumour DNA (ctDNA)

Although the term liquid biopsy was initially only used to define CTCs, it was quickly expanded to include another circulating biomarker – ctDNA. The presence of cell-free nucleic acids in blood circulation had been discovered long before this. 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.

Establishing liquid biopsy

CTCs and ctDNA have now 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.

The scope of liquid biopsy has also expanded to include many other different types of circulating biomarkers that can be found not only in blood, but in all of the other liquids and biological matrices released from the human body.

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

The history and development of liquid biopsy

Liquid biopsy in cancer

The main application of liquid biopsy is cancer. Currently, the gold standard methods used to diagnose cancers are image-based, followed by surgical biopsies to remove tissue that will subsequently go under molecular testing and be pathologically examined.

Not only is this process often painful and distressing for patients, but there are also situations where surgical biopsies are difficult to access. For instance, in certain types of brain tumours or if a re-biopsy is needed following therapy, but the tumour has shrunk too significantly to remove a sample.

Early cancer detection

It is well established that when cancer is detected at an earlier stage, it is not only easier to treat, but also reduces patient suffering and increases survival.  However, there are several issues with the current gold-standard methods for the early detection of cancer, that limit this in clinical practice.

These include difficulties in accessing certain tumours, changes in tumour genetic composition arising from malignant neoplasms and intra-tumour heterogeneity. Liquid biopsy has shown that it is a technique that has the potential to address these problems, revolutionising the early detection and diagnosis of cancer.

Monitoring disease progression and therapeutic response

One of the unique aspects of liquid biopsy is the minimally invasive process involved when taking a blood sample. This considerably reduces patient discomfort when sampling and means that multiple samples can be taken from the same patient over a prolonged period of time. Liquid biopsy is therefore a particularly powerful tool for monitoring the progression of cancer and patient response to therapy.

The molecular and genetic profiling of circulating biomarkers using liquid biopsy delivers real-time monitoring of constantly changing tumours and their genotypes. These changes would otherwise not be detectable with conventional tissue biopsy. The molecular characterisation of tumours using liquid biopsy can therefore track disease progression, predict therapeutic outcomes and prevent disease relapses.

Cancer metastasis

The metastasis of a primary tumour to a secondary site is responsible for approximately 90% of all deaths caused by cancer. The process of metastasis and liquid biopsy are intrinsically linked. Tumour cells from the primary site are able to enter the bloodstream and travel as CTCs, before deposition at a distant site and the formation of secondary tumours. This process also means that the analysis of CTCs using liquid biopsy can reveal unique insights about metastasis and the types of primary tumours that are able to metastasise.

Measuring minimal residual disease

Minimal residual disease, also known as molecular residual disease, (MRD) is when cancer persists in a patient after treatment but is no longer detectable through current medical imaging procedures. The high sensitivity of liquid biopsy technologies has enabled the detection of MRD in patients, in whom the disease is not otherwise detectable through radiological imaging.

Relapse is thought to be caused by micro-metastases, where a small number of cancer cells have spread from the primary tumour to other parts of the body, but where there are too few to be picked up in imaging or using diagnostic tests. MRD detection is mainly done through ctDNA analysis, and can be helpful in the post-surgical setting, or to monitor for recurrence.

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

The application of liquid biopsy in cancer

Alternative liquid biopsy applications

Even though the majority of research focusses on cancer, it is not the only disease that stands to benefit from liquid biopsy. Recent reviews in liquid biopsy have covered the application of the technology across several other research areas.

For example, liquid biopsy targeting microbial cell-free DNA has emerged in infectious disease diagnosis, allowing non-invasive and rapid detection of a broad range of pathogens. In chronic liver disease, liquid biopsy is being explored as a cost-effective and safe alternative to liver biopsies, whilst still providing detailed molecular insights. Within Alzheimer’s disease, liquid biopsy has made great progress as a rapid and low-cost method for use in clinical diagnosis.

An application that has received much attention is non-invasive prenatal testing (NIPT). NIPT can be performed using liquid biopsy and cell-free foetal DNA. This gives an insight into pregnancy-related conditions and allows for non-invasive prenatal screening of pregnant individuals.

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

Clinical liquid biopsy applications

To date, several liquid biopsy tests have been approved by the FDA:

  • FoundationOne® CDx is a tissue-based, comprehensive genomic profiling service for solid tumours to help guide informed, personalised treatment decisions.
  • Guardant360® is a clinically validated liquid biopsy test that allows a comprehensive molecular analysis by analysing ctDNA.
  • The cobas® EGFR Mutation Test v2 is a real-time PCR assay targeting 42 mutations in the EGFR gene, including the T790M resistance mutation using cfDNA.
  • The therascreen PIK3CA RGQ PCR Kit is a real-time qualitative diagnostic PCR test for the detection of 11 mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene.
  • Epi proColon® 2.0 CE is a real-time PCR detection assay of methylated Septin 9 DNA in bisulphite converted DNA from human plasma samples, to aid in the detection of CRC.
  • The CELLSEARCH® Circulating Tumor Cell Kit detects circulating tumour cells (CTC) of epithelial origin in whole blood, which are associated with decreased survival in metastatic breast, colorectal and prostate cancer.

The EMA has also approved the use of liquid biopsies in non-small cell lung cancer (NSCLC) when material from tissue biopsies is not available in sufficient quantity.

To routinely implement liquid biopsies into the cancer diagnostic pathway, clinical trials must address the following challenges:

  • Demonstrate reproducibility and sensitivity
  • Demonstrate the effectiveness of the technology in distinguishing positive results from negative results
  • Showcase the utility of the biomarker to answer an actionable clinical question

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

Alternative circulating biomarkers

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. They contain multiple analytes of interest, including DNA, RNA, and proteins.

The contents of EVs and particles have already been associated with tumour progression, immune regulation and metastasis. Mounting evidence supports the notion that EV proteins can be used for early cancer detection, predict prognosis and guide therapy selection.

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).

Circulating proteins

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.

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

An overview of alternative biomarkers in liquid biopsy

Alternative fluids in liquid biopsy

The “liquid” in liquid biopsy most commonly refers to blood. However, the human body contains many other fluids and biological matrices that can be collected easily and non-invasively, whilst providing an important source of molecular information.

Saliva

Saliva is one of the easiest to collect biofluids and is already used routinely for diagnostic purposes. It is a promising source of cancer biomarkers and has already been demonstrated to contain multiple different tumour biomarkers, particularly for head and neck cancers.

The standout feature of saliva-based liquid biopsy is the ease of sampling, which can be performed without specialist training or expensive equipment. This makes saliva a good candidate in the application of liquid biopsy in disease monitoring, as multiple repeat samples can easily be taken. The qualities of saliva also open up the possibility of point-of-care testing, with individuals collecting their own samples to be sent for analysis.

Sputum

Sputum is the combination of saliva, phlegm, and mucus derived from the upper respiratory tract. Although sputum has many similarities with saliva, it has not seen as much development within liquid biopsy. Sampling of sputum can be performed using spontaneous production or through induction by inhalation of warm saline aerosol.

Urine

Urine is also a liquid that is straightforward to collect, with large volumes available compared to other types of liquid biopsy. As with saliva, the ease of collecting urine makes it suitable for disease monitoring using repeat samples. Compared to other fluids, urine contains smaller numbers of circulating cells, but it is a comprehensive source of other biomarkers such as circulating nucleic acids, proteins and extracellular vesicles.

Sweat

Sweat is secreted from the sweat glands onto the surface of the skin and its primary role is the thermoregulation of body temperature. In doing so, sweat also removes waste products from the body and is therefore a viable source of molecular biomarkers. Candidate biomarkers present in sweat include electrolytes, metabolites, hormones, proteins, nucleic acids, carbohydrates and urea.

Cerebrospinal fluid (CSF)

The gold standard for diagnosing and monitoring central nervous system (CNS) tumours is via imaging techniques. However, imaging doesn’t provide molecular information that can be crucial to therapeutic decision making. To compound matters, surgical sampling of malignancies of the central nervous system can be challenging. The tumours can be in hard-to-reach areas, and this may limit our ability to correctly characterise specimens.

It has recently been demonstrated that the detection of circulating tumour biomarkers in CSF can be used to characterise and monitor CNS malignancies. ctDNA is highly present in CSF (in larger amounts than plasma). Its analysis can inform therapeutic decision making and help track tumour evolution.

An overview of alternative fluids in liquid biopsy

Further reading:

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

The history and development of liquid biopsy

The application of liquid biopsy in cancer

An overview of alternative biomarkers in liquid biopsy

An overview of alternative fluids in liquid biopsy


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Cancer / Clinical Trials / ctDNA / Liquid Biopsy