The advent of liquid biopsy has sparked a revolution in cancer detection and monitoring. This non-invasive technique allows for the analysis of cell-free DNA (cfDNA) and other analytes found in bodily fluids, offering a promising alternative to traditional tissue biopsies. Liquid biopsy for cancer has gained significant attention due to its potential to detect malignancies at earlier stages, leading to improved patient outcomes and more effective treatment strategies.
DNA methylation, a key epigenetic modification, has emerged as a powerful biomarker in liquid biopsy applications. By examining methylation patterns in cell-free DNA, researchers can identify specific cancer signatures and distinguish between various tumour types. This approach has opened new avenues to explore early diagnosis, treatment monitoring, and personalised medicine. As the field continues to advance, liquid biopsy techniques that leverage methylation biomarkers are poised to transform cancer screening and management, offering hope for more accurate and timely interventions in oncology.
Understanding liquid biopsy
What is liquid biopsy?
Liquid biopsy represents a groundbreaking advancement in disease diagnostics, offering a less invasive alternative to traditional tissue biopsies, particularly for cancer. This innovative method detects and analyses fragments of cancer DNA or cells circulating in the blood, providing a dynamic snapshot of the disease. It’s a simple and non-invasive procedure that enables doctors to discover a range of information about a tumour through a blood sample.
The technique involves analysing various types of analytes in body fluids, including circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and cfDNA. These components provide valuable insights into tumour heterogeneity, treatment response, and personalised treatment options for cancer patients.
Liquid biopsies have the potential to revolutionise cancer care by enabling early detection, real-time monitoring of treatment response, and identification of molecular changes that may signal resistance to therapy. This approach is reshaping oncology by allowing more frequent, non-invasive patient cancer assessments and facilitating timely treatment plan adjustments.
Types of liquid biopsy samples
Liquid biopsies can be performed using various body fluids, offering flexibility and accessibility in sample collection. The most common types of liquid biopsy samples include:
- Blood: the primary source for liquid biopsies, requiring only 5 millilitres for analysis.
- Urine: a non-invasive alternative to blood samples.
- Saliva: another easily accessible bodily fluid for liquid biopsy.
- Cerebrospinal fluid: used for specific neurological cancers and conditions.
These samples can be analysed to detect:
- CTCs: cancer cells from the tumour travelling in the bloodstream.
- ctDNA: DNA fragments from tumour cells circulating in the blood.
- cfDNA: DNA released from both normal and cancerous cells.
Advantages over traditional biopsies
Liquid biopsies offer several advantages over traditional tissue biopsies:
- Non-invasive nature: requiring only a simple blood draw, liquid biopsies minimize patient risk and discomfort.
- Repeatability: they can be performed more frequently than tissue biopsies, allowing for real-time monitoring of disease progression and response to treatment.
- Early detection: liquid biopsies have the potential to detect cancer at earlier stages by identifying ctDNA or cells in the blood, possibly before the cancer is detectable by other means and before traditional symptoms arise.
- Comprehensive tumour profiling: they offer an improved chance of detecting genetic changes across the entire tumour, overcoming the limitations of tissue biopsies that may not capture all mutations.
- Real-time monitoring: liquid biopsies enable ongoing monitoring of tumour evolution and the detection of resistance mutations, offering a dynamic view of the disease over time.
- Accessibility: the technique is more suitable and more easily accessible for low- and middle-income countries due to lower procedural costs and easier repeatability.
- Sample quality: liquid biopsies provide a source of reliable tumour-derived components and materials, avoiding contamination from preservatives used in tissue biopsies.
- Rapid results: the analysis can be carried out quickly, providing genomic, epigenomic, proteomic, and metabolomic information.
While liquid biopsies offer numerous advantages, it’s important to note that they are not intended to replace tissue biopsies entirely. Tissue biopsies remain the gold standard for cancer diagnosis. Liquid biopsies complement traditional methods, allowing for more comprehensive and frequent monitoring of cancer progression and treatment effectiveness, particularly for early-stage or asymptomatic screening.
As research in this field continues to advance, liquid biopsies are poised to play an increasingly important role in cancer detection, monitoring, and personalised treatment strategies. However, further studies are needed to fully explore the potential of plasma-based testing and address questions about test accuracy across different tumour types and stages of disease.
DNA methylation as a cancer biomarker
The role of DNA methylation in cancer
DNA methylation, a common epigenetic alteration, plays a crucial role in cancer development and progression. This process involves the addition of a methyl group to the fifth position of cytosine, forming 5‑methylcytosine (5mC). Typically occurring at CpG dinucleotides, DNA methylation is particularly prevalent in CpG islands, which are found in approximately 60% of human gene promoter regions.
In cancer, two distinct patterns of DNA methylation emerge: global hypomethylation and focal hypermethylation. Global hypomethylation contributes to genomic instability and the activation of silenced oncogenes, while focal hypermethylation often leads to the inactivation of tumour suppressor genes. These alterations in DNA methylation patterns are considered major contributors to neoplastic transformation.
Interestingly, aberrant DNA methylation is thought to occur at very early stages of cancer development, with specific genes becoming methylated at different tumour stages. This characteristic makes DNA methylation an attractive target for early cancer detection and monitoring.
Detecting methylation changes in liquid biopsies
Liquid biopsy has emerged as a promising approach for detecting cancer-specific DNA methylation patterns. This non-invasive technique allows for the analysis of cfDNA in various body fluids, including blood. By examining these methylation patterns, liquid biopsy testing can potentially identify cancer-specific methylation signals and aid in the early detection and classification of cancer molecules.
Several methods are available for methylation analysis in cancer cfDNA using next-generation sequencing (NGS) technology. While bisulfite sequencing is widely used for methylation analysis in genomic DNA, it presents challenges when applied to cfDNA from liquid biopsy samples due to DNA fragmentation, low input amounts, and DNA degradation. Newer techniques, such as duet multiomics solution evoC from biomodal, use enzymatic rather than chemical conversion, resulting in significant improvements for cfDNA analysis compared to traditional bisulfite sequencing.
Advantages of methylation biomarkers
DNA methylation biomarkers offer several advantages for cancer detection and monitoring:
- Stability: DNA methylation is an extremely stable epigenetic mark, making it an ideal candidate for biomarker development.
- Accessibility: methylation status can be readily obtained from various sources, including blood, urine sediments, and even highly processed tissues.
- Early detection: aberrant DNA methylation occurs at very early stages of cancer development, making it valuable for early diagnosis.
- Cancer type discrimination: some genes acquire tissue-specific DNA methylation, potentially allowing for discrimination between different cancer types in metastatic tumours or liquid biopsies.
- Prognostic and predictive value: DNA methylation biomarkers can be used to distinguish tumour subtypes, indicate treatment responsiveness, predict clinical outcomes, and determine therapeutic strategies.
5mC and 5hmC as distinct cancer biomarkers
While 5mC has been the primary focus of DNA methylation studies, our recent research into colorectal cancer (CRC) and the role of 5mC and 5hmC has highlighted the importance of 5hmC in both early and late stage diagnosis of the disease in addition to being a predictive indicator of disease progression.
Studies have shown that 5hmC and 5mC (total methylation) profiles exhibit low correlation, with only a small proportion of significant 5hmC loci overlapping with significant total methylation loci. This suggests that 5hmC and 5mC may provide distinct and complementary information as cancer biomarkers.
Key findings regarding 5hmC in colorectal cancer include:
- 5hmC offers additional discriminatory power in early stage disease progression.
- Methylation changes demonstrated by combining 5mC and 5hmC allow for the tracking of changes within tumour progression.
These observations demonstrate the potential of 5hmC as a novel and distinct biomarker for not only CRC cancer detection and monitoring but also the potential for other cancer types – shown below – complementing the information provided by 5mC profiles.
In conclusion, DNA methylation biomarkers, including both 5mC and 5hmC, offer promising avenues for cancer detection, monitoring, and personalised treatment strategies through liquid biopsy approaches. As research in this field continues to advance, these epigenetic markers are poised to play an increasingly important role in improving cancer diagnosis and management.
Methylation biomarkers across cancer types
DNA methylation patterns serve as valuable biomarkers for various cancer types, offering insights into early detection, diagnosis, and prognosis. This section explores the application of methylation biomarkers across different cancer types, highlighting their potential in liquid biopsy-based screening methods.
Lung cancer
Lung cancer remains a leading cause of cancer-related mortality worldwide. Early detection is crucial for improving patient outcomes, and liquid biopsy methods based on DNA methylation have shown promise in this regard. Studies have demonstrated that aberrant DNA methylation often occurs at an early stage of lung cancer development, making it a valuable target for early diagnosis.
The MRE-Seq (Methylation-sensitive Restriction Enzyme digestion followed by Sequencing) protocol has emerged as a highly sensitive and effective liquid biopsy method for detecting cancer-specific DNA methylation patterns in lung cancer. This technique achieved an area under the receiver operating characteristic curve (AUC) of 0.956 with a sensitivity of 66.3% for lung cancer at a specificity of 99.2%. Sensitivities for stages I-IV ranged from 44.4% to 78.9%, again at a specificity of 99.2%.
Colorectal cancer
CRC is one of the most commonly occurring cancers and the second leading cause of cancer-related deaths in the United States. Methylation biomarkers have been integrated into clinical practice for CRC screening. For instance, Cologuard®, an approved multi-target stool-based DNA test, includes two DNA methylation biomarkers (BMP3 and NDRG4) along with seven distinct KRAS variants and haemoglobin. In a clinical trial, Cologuard® demonstrated a sensitivity of 92.3% for detecting any stage cancer and 93.3% for stages I-III.
Epi proColon®, an FDA-approved CRC detection test that uses patient blood, relies solely on the detection of methylated SEPT9 (mSEPT9). A meta-analysis of studies utilising mSEPT9 for CRC showed a pooled sensitivity of 0.69 and specificity of 0.92. Combining mSEPT9 with other markers has improved overall CRC detection.
Other promising methylation biomarkers for CRC include:
- mSDC2: A pooled sensitivity of 0.81 and specificity of 0.95 have been reported for mSDC2 detection in stool and blood samples.
- mALX4: An exploratory study found plasma-derived mALX4 and mSEPT9 positivity in 60% of polyps and CRCs, compared to 18% in healthy control plasma.
- LINE-1 hypomethylation: This marker has been associated with larger tumours, advanced stage, and metastasis in CRC.
Recent advances in methylation-based methods have shown robust improvements in CRC detection. For example, the ColonES Assay, using a 239-marker panel, demonstrated sensitivities of 79% for advanced adenoma and 86.6% for CRC detection.
Breast cancer
Breast cancer methylation biomarkers have been extensively studied, with several specific sites showing promise for early detection and classification. A study analysing 10 specific breast cancer methylation sites identified nine genes involved: C9orf125, RARB, ESR1, RUNX3, PCDHGB7, DBC1, PDGFRB, TIMP3, and APC.
Four methylation sites were found to effectively distinguish breast cancer from other cancer types:
- Two sites in the DBC1 gene (cg03625109 and cg24818566)
- One site in the C9orf125 gene (cg13683194)
- One site in the PDGFRB gene (cg16429070)
Furthermore, six methylation sites in other genes could be used to distinguish between different cancer species. Notably, two methylation sites (cg21646032 and cg23601468) could significantly distinguish Basal-like types from other types of breast cancer.
Genitourinary cancers
For urological cancers, urine often serves as the preferred liquid biopsy source due to its non-invasive nature and high content of exfoliated tumour cells and cell-free tumour DNA. DNA hypermethylation events represent versatile biomarkers for these cancers, as they are common and can be easily assessed using well-established techniques.
- Bladder cancer: studies have analysed 114 different DNA methylation biomarkers in urine samples from bladder cancer patients. Two biomarkers, ZNF154 and POU4F2, each reached a median sensitivity of >80%, although with large variability across studies.
- Prostate cancer: GSTP1 was the most extensively studied biomarker (19 studies). The highest median sensitivity was reported for HOXD3 (76%), while most other biomarkers had sensitivities of <50%.
- Renal cancer: TCF21 was the only biomarker tested in more than one study, achieving 100% specificity in both studies, but with sensitivity varying from 28% to 79%.
- Upper urinary tract cancer: VIM was investigated in two studies, achieving sensitivities of 82% and 73%, and specificities of 100% and 61%, respectively.
In conclusion, methylation biomarkers show great promise across various cancer types for early detection, diagnosis, and monitoring through liquid biopsy. As research continues to advance, these epigenetic markers are poised to play an increasingly important role in improving cancer diagnosis and management, potentially revolutionising cancer screening and personalised medicine approaches.
Multi-cancer detection using methylation signatures
Pan-cancer methylation panels
The development of comprehensive methylation panels has opened new avenues for multi-cancer detection using liquid biopsy. Researchers have identified specific CpG sites that are consistently hypermethylated in tumours across various cancer types. One study developed a panel targeting more than 9,223 CpG sites by analysing 32 cancer types. This approach has shown promising results in detecting multiple cancer types simultaneously.
The PanSEER test, a blood-based cancer screening method, relies on a large panel of ctDNA-methylation biomarkers for the early detection of five major cancer types: colorectal, aesophageal, liver, lung, and stomach. Remarkably, this test has demonstrated the ability to detect cancer up to four years before conventional diagnostic programmes. This highlights the potential of methylation-based liquid biopsy tests for early cancer detection across multiple cancer types.
Tissue-of-origin prediction
One of the significant advantages of using methylation signatures in liquid biopsy is the ability to predict the tissue of origin (TOO) for detected cancers. DNA methylation patterns can be tissue-specific and maintained during neoplastic transformation, creating epiclone signatures that allow for tumour origin identification. This feature is particularly valuable in cases where the primary tumour site is unknown.
A study developed a highly accurate TOO classifier derived from methylation profiles and applied it to a pilot cohort of cancer of unknown primary (CUP) patients. The classifier, named CUPiD, was built using an ensemble of 100 individual gradient-boosted tree sub-classifiers. When tested on an independent cohort of 170 cfDNA samples, including 143 cancer patient samples from 13 different tumour types, CUPiD correctly predicted the tumour type for 121 out of 143 patients, achieving an overall sensitivity of 84.6%.
In the CUP cohort, CUPiD yielded a tumour prediction in 32 out of 41 cases (78.0%). For the 33 patients with clinically resolved or suspected diagnoses, 26 had a CUPiD tumour type prediction, and 23 out of 26 (88.5%) of these predictions aligned with the confirmed primary tumour type or one of the suspected diagnoses. These results demonstrate the potential of methylation-based TOO prediction in guiding treatment decisions for patients with unknown primary tumours.
Commercial multi-cancer detection tests
Several commercial multi-cancer detection tests based on methylation signatures have been developed and are being evaluated for clinical use. One such test is a cell-free DNA targeted methylation sequencing assay with machine learning classifiers for multi-cancer detection and cancer signal origin prediction. This multi-cancer early detection (MCED) test has shown promising results in detecting clinically significant cancers and providing prognostic information beyond clinical stage and method of clinical diagnosis.
Another example is the BC-mqmsPCR assay, developed for the early detection of breast cancer. This assay is based on selected methylation markers identified through genome-wide DNA methylation profiling of peripheral blood mononuclear cells (PBMCs). The BC-mqmsPCR demonstrated high performance in diagnosing early-stage breast cancer and minimal breast tumours in a multicentre cohort. Notably, it showed the ability to diagnose tumours ≤1.5 cm with AUCs up to 0.945 and 0.936, and sensitivities up to 93.2% and 90.0% in the training and validation sets, respectively.
These commercial tests highlight the potential of methylation-based liquid biopsy assays to complement existing screening paradigms. They offer the advantage of detecting multiple cancer types simultaneously while providing information on the tissue of origin. The ability to detect clinically significant cancers at early stages without contributing to overdiagnosis makes these tests particularly promising for improving cancer screening and management.
In conclusion, the use of methylation signatures in liquid biopsy for multi-cancer detection represents a significant advancement in cancer diagnostics. Pan-cancer methylation panels, tissue-of-origin prediction capabilities, and commercial multi-cancer detection tests are paving the way for more comprehensive and efficient cancer screening strategies. As research in this field continues to progress, these approaches have the potential to revolutionise early cancer detection and improve patient outcomes across multiple cancer types.
From discovery to clinical application
The field of liquid biopsy has made significant strides, with methylation biomarkers emerging as powerful tools to detect and monitor cancer. This approach has a profound impact on early diagnosis, treatment monitoring, and personalised medicine. The use of both 5mC and 5hmC as distinct methylation biomarkers adds depth to our understanding of cancer biology, offering complementary insights that enhance the accuracy of liquid biopsy tests.
Looking ahead, liquid biopsy is poised to revolutionise cancer screening and management. The development of multi-cancer detection tests and tissue-of-origin prediction capabilities opens up new possibilities to improve patient outcomes. As research in this area continues to advance, we can expect liquid biopsy techniques that leverage methylation biomarkers to play a crucial role in shaping the future of oncology, offering hope for more timely and precise interventions in cancer care.
