Liquid Biopsy: A Timely Technology Waiting to be Taken Seriously

Biological components of LB

LB targets can be broadly categorised into two groups based on their biological nature. The first includes cell-free molecules such as proteins and nucleic acids (cell-free [cf] DNA and circulating tumour [ct] DNA), and to a much lesser extent, lipids, carbohydrates, metal ions, and small metabolites. The second consists of cellular components such as circulating tumour cells (CTCs), circulating cancer-associated fibroblasts (CAFs),² and myeloid derived suppressor cells (MDSCs),³ or subcellular components, including extracellular vesicles and circulating mitochondria.¹ Detection methods vary depending on the specific target analyte.

LB biomarkers that have progressed to clinical practice address CTCs, ctDNA and cfDNA. These biomarkers and their analytic technologies are summarised below.

Circulating tumour cells

Perhaps the most promising LB approach is to isolate CTCs directly from the peripheral blood and use them as surrogate cancer tissue. Besides being clinically useful via enumeration, CTCs also offer a full spectrum of analytes (proteins, DNA and RNA) to comprehensively access clinically relevant information.⁴ Isolation of CTCs can be performed by immunoaffinity for specific cell surface biomarkers (like epithelial cell adhesion molecule, EpCAM), by various enrichment techniques or by combining immunomagnetic enrichment and size-based microfluidics.

Detection of CTC typically involves three main stages: enrichment, detection, and analysis. Originating from primary solid tumours or metastatic sites, CTCs enter the peripheral circulation following matrix degradation, angiogenesis, invasion into blood vessels and extravasation. CTCs can also be carried beyond the primary cancer site via drainage into the lymphatic system. Only a small subset of CTCs can survive and migrate, as most CTCs are quickly eliminated by the immune system or destroyed by shear stresses in the peripheral blood. CTCs have a very short half-life, ranging from 1 to 2.4 hours, and are rare, with typically less than 50 detected in 7.5 ml of blood.

Recent technical advances have resolved this limitation owing to the rarity of CTCs somewhat, and enabled molecular characterization even at the single-cell level.⁵ Some commercially available technologies allow capabilities for accessing not only the CTCs but also hold the potential to examine the other components of LB like ctDNA, exosomal RNA, and tumour marker proteins from a single blood draw. The surface or intracellular proteins of CTCs can be analysed by microfluidics or enzyme-linked immunospot assay (ELISPOT).⁶ Finally, CTCs can be analysed for their function via in vitro culture to study their proliferation, transformation, and invasion capabilities. While highly specific, this approach is among the slowest progressing CTC analytic avenues due to the inherently low viability and heterogeneity of CTCs, as well as low initial seeding cell counts.

Limitations of CTC analysis and ways to address them

The detection of CTCs can be unreliable due to their rarity and low frequency, even in metastatic cancer. Also, their high heterogeneity leads to variable surface biomarker expression, making isolation for guiding treatment challenging. These factors have contributed to the slow uptake of CTC tests in routine clinical practice. Use of surface-antigen-agnostic CTC isolation methods could be a way to address these limitations. Since the conventional 8–10 ml LB collection tubes cannot represent the whole circulating blood volume, litre-scale LB is being pursued to substantially increase the chance of catching rare CTCs in sufficient numbers.⁷

ctDNA and cfDNA

Extracellular DNA fragments, such as cfDNA, are released into the bloodstream via secretion or through cell death processes, including necrosis and apoptosis. These single- or double-stranded fragments stabilise in circulation by binding to cell membranes and extracellular proteins, protecting them from nuclease-mediated degradation and rapid clearance. The cfDNA fragments originate from all dead cells floating in the blood, a very small fraction of which is contributed by tumour cells. ctDNA, on the other hand, is released by cancer cells, reflecting the tumour genomes from various sites, including primary tumours, CTCs, as well as metastases. Thus, unlike a single-site tissue biopsy, ctDNA captures the spatial molecular heterogeneity of cancer and reflects key genetic alterations found in tumour tissues, such as chromosomal rearrangements, point mutations, copy number variations, epigenetic modifications, insertions, and deletions.⁸

LB ctDNA tests can address detecting genetic mutations as well as epigenetic changes. LB can detect the following 2 most common cancer-specific genetic alterations:

Single-nucleotide variants (SNVs) are the single DNA base pair changes that are the most frequently identified genetic variation impacting gene function and contributing to cancer development.

Copy number variations (CNVs) are alterations in the number of copies of specific genes or genomic regions that can lead to the amplification of oncogenes or deletion of tumour suppressor genes, promoting tumour growth and progression.

ctDNA levels range from 0.01% to 10% in cancer patients. ctDNA is typically shorter than cfDNA (134 to 144 base pairs) with a half-life of approximately 2 hours, and is a valuable tool for real-time tumour monitoring and assessing treatment response. Relative to cfDNA, the concentration of ctDNA in plasma is lower, but because it is uncontaminated with non-tumour cell DNA, it is more representative of cancer tissue. A tumour volume of 10 cubic cm was required for ideal sensitivity to ctDNA tests, which is far larger than an early-stage/asymptomatic tumour, and the ctDNA test may be ineffective for a tumour <1 cm in size.⁹ This presents major sensitivity caveats in using ctDNA for detection in asymptomatic individuals, where the tumours would be much smaller. Consequently, the current literature is not supportive of using ctDNA for the detection of small cancers in asymptomatic individuals. The limited ability of both quantitative PCR and digital PCR to detect multiple mutations has led to the growing use of Next-Generation Sequencing (NGS) techniques for more comprehensive analysis, such as Cancer Personalised Profiling by deep Sequencing (CAPP-Seq), but resorting to NGS or CAPP-Seq adds substantially to the cost of LB. Three primary ctDNA detection technologies have translated into clinical practice. These are discussed below.

The realtime quantitative PCR (qPCR) assay to examine ctDNA has historically been used widely for endometrial cancer, non-small cell lung cancer (NSCLC), colorectal cancer, etc., and has received approval from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the detection, activation or identification of resistance to epidermal growth factor receptor (EGFR) targeted therapies in NSCLC. However, these tests may increase the risk of false positives when qPCR is used in isolation and need to be supplemented by tissue biopsy and other clinical assessment methods.

The NGS approach is being increasingly brought into clinical practice since it was approved by the US FDA as Guardant360® CDx (Guardant Health, Inc.) assay for analysing EGFR mutations in NSCLC patients undergoing treatment with tyrosine kinase inhibitor osimertinib. It is the preferred LB technique for metastatic NSCLC according to the current European Society for Medical Oncology guidelines.¹⁰ However, NGS has a potential for mislocalization of mutations, requires extensive data analysis, is substantially expensive and needs a turnaround time of 7–14 days.

Microdroplet digital PCR or Microdroplet ddPCR technique divides mixed ctDNA nucleic acid molecules and PCR solution into small droplets. While qPCR quantifies ctDNA through amplification and, hence, results in a relative quantification of the target, ddPCR partitions the sample into thousands of droplets, performing PCR in each droplet, and provides an absolute quantification of the target DNA, making it more precise and less susceptible to PCR efficiency variations. However, compared with NGS, ddPCR has a narrower reference range and covers fewer variants. In addition, ddPCR requires specialized operators and substantial data analysis, which increases complexity and operational costs.

Clinical role of ctDNA assays

The most impactful role that ctDNA tests can have is in screening and management of patients with cancer, not only as a biomarker for early diagnosis but also as a prognostic tool for designing treatment and monitoring treatment response and disease progression. Monitoring ctDNA levels at baseline, during neoadjuvant and adjuvant therapy and after radical therapy can help oncologists in assessing drug response and adjusting treatment regimens. This dynamic longitudinal monitoring ability will ultimately improve prognostic evaluation and permit informed clinical decision-making.

Substantial savings in follow-up time and offering better clinical decision-making ability can result if the ctDNA test can identify pseudoprogression, a space-occupying lesion and oedema following treatment, which typically resolves spontaneously in 4–8 weeks.¹¹ Detecting ctDNA also provides identification of minimal residual disease (MRD) at the molecular level. It has been suggested that a key treatment endpoint for colorectal cancer should be complete clearance of ctDNA.¹²

Limitations of ctDNA assays

Two important limitations of the ctDNA-based LB include low ctDNA levels to begin with and low mutation detection capability due to lower abundance in ctDNA than in localized tumour tissues. Another limitation is the non-uniformity of the assays across the testing laboratories, resulting in discrepancies. Hence, standardized testing protocols and interpretative guidelines are necessary.

Epigenetic modifications

Epigenetic modifications, mainly alterations in methylation patterns of ctDNA, have emerged as important biomarkers for cancer diagnosis and prognosis. PCR-based methods such as methylation-specific PCR (MSPCR) and droplet digital MSPCR (ddMSPCR) are the assays of choice; other technologies are still being established (target bisulfite sequencing, whole-genome bisulfite sequencing).

LB biomarkers and technologies that are still largely in the experimental phase include:

1. Examination of cfRNA and ctRNA derived from cancer cells, represented by circular RNA (circRNA), microRNA (miRNA), and long non-coding RNA (lncRNA). Technologies to analyse these are the qRT-PCR and NGS.

2. Extracellular vesicles (EVs), including nanoscale exosomes, microvesicles, and apoptotic bodies. Methods for EV analysis include ultracentrifugation, ultrafiltration, precipitation, immunoaffinity capture, and lipid-based isolation.

3. Metabolomic markers like low molecular-weight metabolites (<1500 Da), which are studied using nuclear magnetic resonance (NMR) and mass spectroscopy (MS), along with computational data analysis.

4. Proteomics, which entails analysing the distribution, structure, interactions and alterations of proteins that dynamically vary in keeping with cancer stage. Analytic methods include traditional ELISA and CLIA, and higher throughput techniques like antibody/antigen arrays, proximity extension assays (PEAs), and reverse-phase protein arrays (RPPAs).

Future potential and challenges for LB

Ongoing research and various clinical trials are focused on improving the detection capabilities of LB, enhancing the sensitivity and specificity of the assays and expanding applications to an increasing number of cancers. Integrating LB with other diagnostic technologies, such as imaging, promises to improve cancer detection and monitoring. While these advances take place, it will be critical to develop in parallel an international consensus for a robust regulatory framework to ensure the cross-platform reproducibility, reliability, accuracy, and safety of LB. This will require collaboration between medical oncologists, pathologists, academic research laboratories and small biomedical setups as well as corporates.