Understanding The Key Biomarkers of cfDNA and ctDNA in Cancer Biology and Diagnostics
What is Cell Free DNA (cfDNA)?
Fragmented DNA can be found in the cell free fraction of whole blood and other bodily fluids. Known as cell-free DNA (cfDNA), these nucleic acids vary in size (~40-1000 base pairs (bp)) but average approximately 166 bp.1 CfDNA, released into the blood plasma by normal cellular processes and dead and dying cells, has a relatively short half-life of about 5-150 minutes.2 In healthy individuals, levels of plasma DNA is typically lower than 10 ng/mL but can drastically increase in specific disease states such as myocardial infarction, stroke, and diabetes.3,4 Given the transient and dynamic nature of cfDNA in addition to being elevated in specific disease states, cfDNA can be a biomarker to detect, stage, and assess a variety of different health conditions and disease states.
CfDNA can be harvested from a variety of different biofluids, including plasma or serum, cerebrospinal fluid, urine, saliva, and pleural effusions. 5 Known as liquid biopsy, sampling these various biofluids is more economical and less invasive than radiological exams and biopsies.2,6,7 As such, utilization of cfDNA as a diagnostic marker has proven feasible compared to standard routes to gain biological samples in multiple clinical studies and applications. The use of cfDNA has been proven to be suitable for prenatal diagnostic purposes in expecting mothers.8–10 Further, it has been demonstrated that cfDNA can be used for the early detection of graft rejection in human organ transplants.11 During the COVID-19 pandemic, cfDNA was utilized to assess the degree of tissue damage in COVID-19 patients.12 Finally, methylation patterns in cfDNA is currently used in population wide colorectal cancer screening.13
Circulating Tumor DNA, A Subtype of cfDNA
Given the ability of cfDNA to represent a biological “snapshot” of underlying processes, significant changes in either cell populations or cellular function would be expected to be reflected in both detectable cfDNA content and abundance. In the specific context of cancer, rapid clonal expansion and selective pressure will drive disease progression and at the same time increase circulating tumor DNA (ctDNA).14 Considered to be a subset of cfDNA, ctDNA is typically shorter than nonmutant cfDNA molecules.7 Detection of ctDNA has gained significant popularity over the past few years in cancer diagnostics given the unrivaled specificity of the biomarkers utilized. These tumor specific biomarkers are detection of DNA mutations, most commonly single base-pair substitutions, in precancerous or cancerous cells.
As a result of this unrivaled tumor biomarker specificity, the interest in ctDNA as a diagnostic, prognostic and research topic has exploded. Though this is still an emerging field where the true utility of ctDNA is still being understood, what has been proven thus far has supported the hypothesis that ctDNA is the proverbial smoke before the fire. In the context of early diagnosis, studies have demonstrated that the detection of ctDNA is a powerful asset in the earlier detection of cancer prior to metastatic disease. Indeed, mutations have been detected in saliva and plasma up to 2 years before an actual cancer diagnosis.15 In cancer patients who are being actively staged and treated, it has been found that the detection of ctDNA has been shown to correlate with both tumor size and stage.16,17 Furthermore, ctDNA has been shown to have higher prognostic power than other commonly used tumor markers.7 In scenarios where the primary tumor source is unknown, utilization of specific methylation and nucleosome occupancy patterns can encode tissue or cell specific information.18
Diagnostic and staging of cancer aside, utilization of ctDNA can also help to better inform individual treatment plans based on the presence or absence of specific mutations by obtaining a global view of the malignancy. Traditional biopsy methods may underestimate or otherwise introduce bias to the selection and efficacy of personalized medicines through localized sampling. Indeed, multiregional sequencing studies have demonstrated heterogeneity in mutation profiles of different tumor regions of the same patient.19,20 Thus, ctDNA analysis from a liquid biopsy released from multiple tumor regions may better reflect overall tumor heterogeneity and prevent the bias introduced by sampling a single region via biopsy.
Finally, ctDNA can provide longitudinal monitoring both during and after treatment. Multiple studies have correlated ctDNA dynamics with treatment response.7 Monitoring of ctDNA provided the earliest measurable response to treatment, exhibited the greatest dynamic range, and was the earliest indication of relapse when compared to other tumor markers utilized.21 Furthermore, monitoring ctDNA can provide insight into clonal evolution and the development of resistance to current therapeutic protocols. Multiplexing of tumor mutation profiles would enable the detection of relative changes and could provide insight into the molecular evolution of the tumor.22,23
Comprehensive cfDNA and ctDNA Testing Through Precise Extraction and Isolation
Given the benefits of using ctDNA as a diagnostic and prognostic indicator, this approach has gained signficant traction. However, ctDNA testing and discrimination from otherwise normal cfDNA has been a technical challenge. The presence of target mutated sequences, especially in the context of early stages of disease or low tumor burden after treatment, are present in extremely low copy numbers in comparison to the overall cfDNA content.6 Indeed, less than 1% of the overall cfDNA content is ctDNA in early stages of disease.24,25 However, parallel improvements in the detection and resolution capabilities in nucleic acid amplification technologies have enabled the detection of these extremely low copy number events. Novel digital PCR-based methods in combination with next generation sequencing have enabled the more intense investigation of ctDNA due the enhanced ability to detect rare sequences of interest as well as multiplexing of several genes of interest.21,26
Although improvements in the detection methodology of gene mutations have vastly improved, the preparation of these samples is equally as important. Isolating DNA is as a critical piece of the workflow for cfDNA detection and ctDNA discrimination from normal cfDNA. Sample purification tools like MagMAX kits and the KingFisher instruments are critical for the continued success of leveraging cfDNA for both basic understanding of pathogenic mechanisms in cancer as well as furthering diagnostics for the earlier detection of cancer. The Applied BiosystemsTM MagMAXTM Cell-Free DNA Isolation Kit is designed for enrichment of circulating cfDNA and optimized for use with biological samples such as serum and plasma. This technology utilizes magnetic bead technology to reproducibly recover high-quality DNA suitable for a broad range of applications, including real-time PCR, digital PCR, and next-generation sequencing. Likewise, KingFisher instruments are extremely versatile and automated purification systems that enable efficient, reproducible and reliable results for both low and high throughput workflows. Finally, Thermo Fisher also hosts a rich source of information in the latest developments of biomarker research to assist with all aspects of method development. Together, enhanced high throughput sample preparations in combination with more sensitive amplification methodologies are continuing to fuel both basic cancer research and diagnostic development to enable earlier detection and more complete treatment strategies for cancer.
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