Liquid biopsy has emerged as a groundbreaking technique in the field of cancer diagnostics and personalised medicine. This non-invasive method allows for the detection and analysis of genetic biomarkers in bodily fluids, such as blood, offering a powerful tool for early cancer detection and monitoring. By examining circulating tumour DNA (ctDNA) and other cell-free DNA (cfDNA) present in the bloodstream, liquid biopsy provides valuable insights into tumour genetics without the need for invasive tissue sampling.
The use of liquid biopsy has an impact on various aspects of cancer management, from early diagnosis to treatment selection and disease monitoring. This technique relies on the analysis of genetic biomarkers, including single nucleotide variants (SNVs) and copy number variations (CNVs), to gain a comprehensive understanding of tumour biology. What’s more, liquid biopsy enables the detection of circulating tumour cells (CTCs) and the assessment of gene expression patterns, offering a holistic view of cancer progression. As research in this field continues to advance, liquid biopsy is poised to revolutionise cancer care, paving the way for more accurate diagnoses and tailored treatment strategies.
Genetic biomarkers in liquid biopsy
Liquid biopsy has emerged as a groundbreaking technique in cancer diagnostics, offering a non-invasive method to detect and analyse genetic biomarkers in bodily fluids. This approach has a significant impact on various aspects of cancer management, from early detection to treatment selection and disease monitoring. The analysis of genetic biomarkers in liquid biopsy provides valuable insights into tumour biology and enables a more comprehensive understanding of cancer progression.
Types of genetic biomarkers
Several types of genetic biomarkers can be identified through liquid biopsy. The most studied cancer non-invasive biomarkers include CTCs, ctDNA, and exosomes. These biomarkers are shed from tumours and their metastatic sites into the bloodstream and other peripheral fluids of cancer patients.
ctDNA is a crucial component of the total circulating cfDNA found in the bloodstream. It is released from tumour cells that have undergone apoptosis or necrosis. The concentration of ctDNA in the blood correlates with tumour burden, disease progression, and metastasis. This makes ctDNA a valuable biomarker for monitoring cancer development and treatment response.
Another important genetic biomarker is circulating tumour RNA (ctRNA). These RNA fragments originate from cancer cells and can provide additional information about gene expression patterns in tumours. The analysis of ctRNA can offer insights into tumour behaviour and potential therapeutic targets.
SNVs and CNVs explained
Single Nucleotide Variants (SNVs) and Copy Number Variations (CNVs) are two critical types of genetic alterations that can be detected through liquid biopsy. SNVs are changes in a single DNA base pair, representing the most frequently identified genetic variation. There are approximately 10,000,000 SNVs in the human genome. These variations can have an impact on gene function and may contribute to cancer development or progression.
CNVs refer to alterations in the number of copies of specific genes or genomic regions. In cancer, CNVs can lead to the amplification of oncogenes or the deletion of tumour suppressor genes, both of which can contribute to tumour growth and progression. Liquid biopsy allows for the detection and analysis of CNVs in circulating tumour DNA, providing valuable information about the genomic landscape of the cancer.
Advanced sequencing technologies, such as next-generation sequencing (NGS), have enabled researchers and clinicians to detect even very low levels of ctDNA in the blood. These techniques can identify different types of genetic alterations, including point mutations, translocations, and gene fusions, in multiple genes simultaneously. Currently, NGS technology is able to detect a minor allele frequency (MAF) of less than 1% in lung cancer, and this is constantly improving as technology is updated.
Importance in cancer detection
The use of genetic biomarkers in liquid biopsy has a significant impact on early cancer detection and monitoring. The concentration of these biomarkers in the bloodstream can contribute to earlier detection of cancer stages and more favourable prognosis predictions in patients. For instance, in prostate cancer, a study showed that more than 5 CTCs per 7.5 mL of blood and an approximate ctDNA concentration of 437 ng/mL of blood (compared to 99 ng/mL in healthy patients) could be considered unfavourable and potentially reduce overall survival.
Liquid biopsy offers several advantages over traditional tissue biopsies. It allows for real-time monitoring of tumour progression and therapeutic resistance, enabling clinicians to make timely adjustments to treatment plans. This is particularly valuable in cases where tumour heterogeneity may cause subclonal or emerging mutations to be overlooked in tissue biopsies, especially in metastatic cases.
The sensitivity and specificity of liquid biopsy tests can be further improved by combining them with conventional biomarkers for specific tumour types. For example, a test combining ctDNA with carcinoembryonic antigen (CEA) for colorectal cancer and pancreatic carcinoma, or ctDNA with prostate-specific antigen (PSA) for prostate cancer, could be a potentially useful tool for better understanding disease progression and early-stage diagnosis.
As research in this field continues to advance, liquid biopsy is poised to revolutionise cancer care. The ability to detect and analyse genetic biomarkers through non-invasive means paves the way for more accurate diagnoses, tailored treatment strategies, and improved patient outcomes. The ongoing development of more sensitive and specific assays, coupled with advancements in bioinformatics and data analysis, will further enhance the utility of liquid biopsy in personalised medicine and early cancer detection.
Circulating Tumour DNA (ctDNA)
What is ctDNA?
ctDNA is a crucial component of liquid biopsy, representing a portion of cfDNA in the bloodstream that originates from tumour cells. This genetic material is released into circulation through various mechanisms, including apoptosis, necrosis, and active secretion by cancer cells. ctDNA carries the same genetic alterations as the tumour itself, making it a valuable biomarker for cancer detection and monitoring.
One of the distinguishing features of ctDNA is its size. Typically, ctDNA fragments are shorter than normal cfDNA, with lengths ranging from 70 to 200 base pairs. This characteristic can be used to differentiate ctDNA from other cfDNA sources in the blood. The concentration of ctDNA in the bloodstream can vary significantly, depending on factors such as tumour type, stage, and location.
A notable attribute of ctDNA is its short half-life, ranging from 16 minutes to 2.5 hours. This rapid turnover rate allows for real-time monitoring of tumour dynamics, providing a more current representation of the cancer’s genomic profile compared to traditional tissue biopsies. The ability to capture these dynamic changes makes ctDNA an attractive option for tracking tumour evolution and treatment response.
How ctDNA is analysed
The analysis of ctDNA involves sophisticated techniques to detect and quantify genetic alterations associated with cancer. Two main approaches are employed in ctDNA analysis: targeted and untargeted methods.
Targeted approaches focus on detecting specific known mutations or a small panel of genes. These methods include real-time PCR (RT-PCR), digital PCR (dPCR), and targeted next-generation sequencing (NGS). RT-PCR is widely used due to its relatively low cost and quick turnaround time, although its sensitivity is lower compared to other methods, with a detection limit of around 10% variant allele frequency (VAF).
Digital PCR offers improved sensitivity, capable of detecting mutations at VAFs as low as 0.1%. This technique partitions the DNA sample into thousands of individual reactions, allowing for the precise quantification of rare mutant alleles. Advanced dPCR methods, such as BEAMing (beads, emulsion, amplification, and magnetics), have further enhanced the sensitivity of ctDNA detection.
Untargeted approaches, primarily based on NGS, provide a broader view of the tumour genome. These methods can detect a wide range of genetic alterations, SNVs, CNVs, and gene rearrangements. NGS-based techniques, such as whole-genome sequencing, whole-exome sequencing, and targeted gene panels, offer high sensitivity, with some platforms capable of detecting mutations at VAFs below 1%.
One innovative NGS-based method is personalised cancer profiling by deep sequencing (CAPP-Seq). This technique combines molecular barcoding with targeted sequencing to improve sensitivity and reduce background noise. CAPP-Seq has shown promise in detecting ctDNA at very low concentrations, making it particularly useful for early cancer detection and minimal residual disease monitoring.
ctDNA as a biomarker
The potential of ctDNA as a biomarker spans various aspects of cancer management, from early detection to treatment monitoring and prognosis assessment. In early cancer detection, ctDNA analysis offers the possibility of identifying tumours before they become clinically apparent or detectable by conventional imaging techniques. This approach could be particularly valuable for cancers that currently lack effective screening methods, such as ovarian, pancreatic, and gastric cancers.
However, the use of ctDNA for early detection faces several challenges. The primary hurdle is the extremely low concentration of ctDNA in early-stage cancers, which can be as low as 0.01% of total cfDNA. This necessitates highly sensitive and specific detection methods to avoid false positives and negatives. Additionally, distinguishing cancer-specific mutations from benign somatic mutations, such as those arising from clonal haematopoiesis, remains a significant challenge.
Despite these obstacles, recent studies have shown promising results in using ctDNA for early cancer detection. For instance, a multi-analyte blood test called CancerSEEK, which combines ctDNA analysis with protein biomarkers, demonstrated the ability to detect eight common cancer types with a sensitivity ranging from 69% to 98% and a specificity of over 99%.
In the context of treatment monitoring, ctDNA has shown great potential as a dynamic biomarker. Changes in ctDNA levels often correlate with treatment response, disease progression, and the development of drug resistance. This real-time monitoring capability allows for timely adjustments to treatment strategies, potentially improving patient outcomes.
Furthermore, ctDNA analysis can provide valuable prognostic information. Higher levels of ctDNA have been associated with worse prognoses in various cancer types, including breast, colorectal, and lung cancers. The ability to detect minimal residual disease through ctDNA analysis after curative treatment could help identify patients at high risk of recurrence who might benefit from additional therapy.
As research in this field continues to advance, ctDNA is poised to play an increasingly important role in personalised medicine, offering a non-invasive means of obtaining comprehensive genomic information throughout a patient’s cancer journey.
Single Nucleotide Variants (SNVs) in liquid biopsy
What are SNVs?
Single Nucleotide Variants (SNVs) are genetic alterations that involve a change in a single DNA base pair. These variations are the most frequently identified genetic changes in the human genome, with approximately 10,000,000 SNVs present. In the context of liquid biopsy, SNVs play a crucial role in detecting and monitoring cancer progression.
SNVs can be either germline or somatic. Germline variants occur in germ cells and can be passed on to future generations, while somatic variants arise in non-germ cells and are not inherited. In cancer, somatic SNVs are of particular interest as they can contribute to tumour development and progression.
The detection of SNVs in ctDNA has become a valuable tool in cancer diagnostics and personalised medicine. ctDNA, a component of cfDNA found in the bloodstream, carries genetic information from tumour cells. The presence and abundance of specific SNVs in ctDNA can provide insights into tumour burden, disease progression, and treatment response.
Detection methods for SNVs
Several methods have been developed to detect SNVs in liquid biopsy samples, each with its own strengths and limitations:
- Next-Generation Sequencing (NGS): This high-throughput method allows for the simultaneous sequencing of millions of DNA fragments. NGS technologies can detect SNVs with a minor allele frequency (MAF) of less than 1% in lung cancer. The use of unique molecular identifiers (UMIs) or molecular barcoding has further improved the accuracy and sensitivity of NGS-based liquid biopsy assays, especially for samples with low ctDNA concentrations.
- Digital PCR (dPCR): This technique offers improved sensitivity compared to traditional PCR methods. dPCR can detect mutations at variant allele frequencies (VAFs) as low as 0.1%. Advanced dPCR methods, such as BEAMing (beads, emulsion, amplification, and magnetics), have further enhanced the sensitivity of ctDNA detection.
- Targeted approaches: These methods focus on detecting specific known mutations or a small panel of genes. They can provide high coverage and depth of sequencing for selected targets, increasing the sensitivity for detecting low-frequency mutations.
- Untargeted approaches: These include whole-genome sequencing (WGS) or whole-exome sequencing (WES), which provide a comprehensive view of the entire genome or exome. While these methods can identify novel or unexpected mutations, they may have lower coverage and depth for specific regions.
- Ligase Chain Reaction (LCR): This method uses two sets of probes that hybridise to adjacent locations on the target DNA strand. LCR can achieve specific detection of SNVs, as a single base mismatch at the junction of two probes prevents ligation.
Clinical significance of SNVs
The detection of SNVs through liquid biopsy has significant clinical implications in cancer management:
- Early detection: SNVs in ctDNA can potentially identify tumours before they become clinically apparent or detectable by conventional imaging techniques. This approach could be particularly valuable for cancers that currently lack effective screening methods, such as ovarian, pancreatic, and gastric cancers.
- Treatment monitoring: Changes in SNV levels often correlate with treatment response, disease progression, and the development of drug resistance. This real-time monitoring capability allows for timely adjustments to treatment strategies, potentially improving patient outcomes.
- Prognostic information: Higher levels of certain SNVs in ctDNA have been associated with worse prognoses in various cancer types, including breast, colorectal, and lung cancers. The ability to detect minimal residual disease through SNV analysis after curative treatment could help identify patients at high risk of recurrence who might benefit from additional therapy.
- Personalised medicine: SNV detection from liquid biopsy enables the study of allelic expression and manifests the effects of SNVs on gene expression. This information can guide the selection of targeted therapies and contribute to more personalised treatment approaches.
- Clinical trials: SNVs detected in liquid biopsy can serve as inclusion criteria for clinical trials, helping to match patients with appropriate investigational therapies.
Despite the promising applications of SNV detection in liquid biopsy, challenges remain. These include the need for highly sensitive and specific detection methods to avoid false positives and negatives, especially in early-stage cancers where ctDNA concentrations can be extremely low. Additionally, distinguishing cancer-specific mutations from benign somatic mutations, such as those arising from clonal haematopoiesis, remains a significant challenge.
As research in this field continues to advance, the detection and analysis of SNVs in liquid biopsy are poised to play an increasingly important role in cancer diagnostics, treatment selection, and monitoring. The ongoing development of more sensitive and specific assays, coupled with advancements in bioinformatics and data analysis, will further enhance the utility of SNV detection in liquid biopsy for personalised cancer care.
Copy Number Variations (CNVs) in liquid biopsy
Understanding CNVs
CNVs are a significant component of genetic variation, affecting a larger portion of the genome than single nucleotide polymorphisms (SNPs). These structural alterations involve gains or losses of genomic DNA that can be either microscopic or submicroscopic. CNVs are now thought to cover at least 10% of the human genome. In the context of cancer, CNVs play a crucial role in defining tumour types and progression, making them tightly linked to diagnostic and prognostic processes.
CNVs can range from focal changes in localised genomic regions involving a small number of genes to large alterations such as the gain or loss of an entire chromosomal arm. Changes in chromosome number and chromosome arms are collectively referred to as aneuploidy. Both focal copy number changes and aneuploidy are common in cancer and carry important information for clinical research, which can be related to diagnosis, prognosis, and therapeutic response.
CNV detection in liquid biopsies
Traditionally, the characterisation of cancer genetic features, including CNVs, has been done on tissue samples obtained through surgical resections or biopsies. However, these methods are often invasive, harmful, and not easily repeatable. Liquid biopsy offers a non-invasive alternative for monitoring tumour features through the analysis of body fluids, particularly cfDNA from plasma.
The analysis of cfDNA presents significant challenges due to its low concentration, high fragmentation (approximately 169 bp fragments), and low tumour-derived cfDNA fraction (0.01–60%). Despite these obstacles, recent advancements in sequencing technologies and bioinformatics tools have made it possible to detect CNVs in liquid biopsy samples.
One promising approach is the use of low-coverage whole-genome sequencing (lcWGS) for CNV detection in ctDNA. This method has shown potential in identifying CNVs in ovarian cancer patients before and after chemotherapy, highlighting its possible application in cancer patient management. Another innovative technique involves using non-invasive prenatal testing (NIPT) platforms for cancer screening, as demonstrated in ovarian cancer patients.
Recent studies have also explored the use of Nanopore sequencing technology for obtaining CNV profiles from plasma cell-free DNA of cancer patients. This approach has shown high sensitivity, specificity, accuracy, and precision when compared to standard short-read sequencing methods. Importantly, it has been demonstrated that CNVs can be detected with as little as 5–10% of ctDNA fraction.
Importance of CNVs in cancer diagnosis
The role of CNVs as risk factors for cancer is currently underappreciated. However, the genomic instability and structural dynamism that characterise cancer cells make this form of genetic variation particularly intriguing to study in cancer. Both germline and somatic CNVs have been associated with a wide variety of human cancers.
CNVs can alter transcription of genes by changing dosage or by disrupting proximal or distant regulatory regions. In cancer, specific CNVs can define the type and progression of the tumour. For example, loss of chromosomal arms 1p and 19q are closely associated with oligodendrogliomas, a subtype of primary brain tumours, and with favourable prognosis in diffuse gliomas.
Focal copy number changes may serve as predictive biomarkers for certain therapeutics. A well-known example is ERBB2 amplification in breast cancer. Additionally, CNVs can be selected for as part of a mechanism of acquired resistance, such as MET amplification in non-small cell lung cancer (NSCLC).
The identification of CNVs through liquid biopsy has the potential to revolutionise cancer screening and monitoring. It offers a non-invasive means of obtaining comprehensive genomic information throughout a patient’s cancer journey, enabling real-time monitoring of tumour dynamics and treatment response. As research in this field continues to advance, the detection and analysis of CNVs in liquid biopsy are poised to play an increasingly important role in personalised medicine and early cancer detection.
Early cancer detection using liquid biopsy
Potential for early diagnosis
Liquid biopsy has emerged as a promising technique for early cancer detection, offering a non-invasive alternative to traditional tissue biopsies. This approach relies on the analysis of ctDNA, CTCs, and other tumour-derived components present in bodily fluids, particularly blood. The potential of liquid biopsy for early diagnosis lies in its ability to detect cancer-related genetic alterations before clinical symptoms appear or tumours become visible through conventional imaging techniques.
One of the key advantages of liquid biopsy is its ability to provide a comprehensive view of tumour genetics through the analysis of cfDNA. This approach allows for the detection of various genetic biomarkers, including SNVs and CNVs, which can be indicative of early-stage cancer. The analysis of these biomarkers can contribute to earlier detection of cancer stages and more favourable prognosis predictions in patients.
Recent advancements in sequencing technologies have significantly improved the sensitivity of liquid biopsy tests. For instance, next-generation sequencing (NGS) techniques can now detect mutations at very low concentrations, with some platforms capable of identifying alterations at variant allele frequencies below 1%. This enhanced sensitivity is crucial for early cancer detection, as the amount of ctDNA in early-stage cancers can be extremely low.
Current limitations
Despite its potential, liquid biopsy for early cancer detection faces several challenges. One of the primary obstacles is the low concentration of ctDNA in early-stage cancers. In fact, ctDNA is found in only 75% of patients with metastatic disease, and its concentration in plasma correlates with both tumour stage and size. This makes detecting ctDNA in early-stage cancers particularly challenging, as the amount of tumour-derived genetic material in the bloodstream may be insufficient for reliable detection.
Another limitation is the potential for false-positive results. The high sensitivity required for early detection can sometimes lead to the identification of benign mutations or age-related clonal haematopoiesis, which can be mistaken for cancer-related alterations. This issue highlights the need for improved specificity in liquid biopsy tests to avoid unnecessary follow-up procedures and patient anxiety.
The cost of liquid biopsy tests, particularly those involving extensive sequencing, remains a significant barrier to their widespread adoption as a screening tool. Currently, the economic burden associated with these tests may limit their use in population-wide screening programs.
Furthermore, there is still a lack of standardisation in liquid biopsy techniques, including sample collection, cfDNA isolation, and data analysis. This variability can lead to inconsistencies in results and make it difficult to compare findings across different studies and clinical settings.
Future prospects
Despite these challenges, the future of liquid biopsy in early cancer detection looks promising. Ongoing research primarily aims to improve the sensitivity and specificity of liquid biopsy tests, as well as develop multi-analyte approaches that combine ctDNA analysis with other biomarkers, such as proteins or metabolites. These multi-modal strategies may enhance the accuracy of early cancer detection and provide more comprehensive information about tumour characteristics.
Advancements in technology are also paving the way for more sensitive and cost-effective liquid biopsy tests. For example, the development of novel sequencing techniques and bioinformatics tools may allow for the detection of even lower concentrations of ctDNA in the future.
The potential for liquid biopsy to detect multiple cancer types simultaneously is an area of active research. Multi-cancer early detection (MCED) tests are being developed and evaluated for their ability to screen for various cancer types using a single blood draw. While these tests show promise, further clinical validation is needed to determine their effectiveness in real-world screening scenarios.
As research in this field continues to advance, liquid biopsy may play an increasingly important role in cancer screening and early detection strategies. Its non-invasive nature and potential for repeated sampling make it an attractive option for longitudinal monitoring of high-risk individuals. However, it is important to note that liquid biopsy is unlikely to replace traditional tissue biopsies entirely, at least in the near future. Instead, it is more likely to complement existing diagnostic methods, providing additional information to guide clinical decision-making and personalised treatment strategies.
In conclusion, while liquid biopsy shows great promise for early cancer detection, significant work remains to be done to overcome current limitations and fully realise its potential. As technology continues to improve and our understanding of cancer biology deepens, liquid biopsy may become an invaluable tool in the fight against cancer, enabling earlier interventions and ultimately improving patient outcomes.
Conclusion
The utilisation of genetic biomarkers in liquid biopsy has caused a revolution in cancer diagnostics and personalised medicine. This non-invasive technique allows for the detection and analysis of ctDNA, offering valuable insights into tumour genetics without the need for invasive tissue sampling. The analysis of single nucleotide variants and copy number variations through liquid biopsy provides a comprehensive view of tumour biology, enabling earlier detection of cancer stages and more favourable prognosis predictions in patients.
As research in this field continues to advance, liquid biopsy is poised to play an increasingly important role in cancer care. The ongoing development of more sensitive and specific assays, coupled with improvements in bioinformatics and data analysis, will further enhance the usefulness of liquid biopsy in personalised medicine and early cancer detection. This groundbreaking approach has an impact on various aspects of cancer management, from early diagnosis to treatment selection and disease monitoring, paving the way for more accurate diagnoses and tailored treatment strategies.
