A Review of Circulating Tumor DNA (ctDNA) and the Liquid Biopsy in Cancer Diagnosis, Screening, and Monitoring Treatment Response

Introduction

The gold standard in cancer diagnosis is tissue biopsy, which allows for the typing and grading of the tumor cells and the identification of target expression for targeted therapies [1,2]. There is a need for less invasive early cancer diagnosis and techniques that can evaluate prognosis and treatment response by detecting cancer recurrence [3,4]. The tissue biopsy is an invasive procedure that is not suitable for early detection or continuous monitoring of cancer progression and recurrence [5].

The concept of liquid biopsy is based on the knowledge that blood or secretions from the body contain tumor cells, nucleic acids, cellular components, and metabolites from tumors (Table 1) [6]. The liquid biopsy may contain circulating tumor cells (CTCs), tumor-derived extracellular vesicles (EVs), tumor-educated platelets (TEPs), circulating free RNA (cfRNA), and circulating tumor DNA (ctDNA) [4]. CTCs are tumor cells that have been shed from the primary tumor and EVs include nano-particles with a lipid bilayer membrane that have central roles in tumor invasion and metastasis [4]. However, ctDNA consists of small fragments of DNA released by tumor cells into the blood and tissue fluids [7].

The detection of ctDNA in liquid biopsy material shows the most promise due to the advances in DNA technologies that have made detection and sample screening possible. In 1977, Leon and colleagues identified that plasma levels of free DNA increased in cancer patients [8]. In 2008, Diehl and colleagues studied ctDNA of 18 patients with colorectal cancer and identified mutations in several genes, including KRAS and PIK3CA [9]. They also showed that the rate of ctDNA mutations changed following cancer treatment and correlated with both carcinoembryonic antigen (CEA) concentrations and tumor volume [9]. In 2014, the use of ctDNA to identify EGFR mutations in non-small cell lung cancer (NSCLC) was authorized by the European Medicines Agency (EMA) [10]. In 2021, the International Association for the Study of Lung Cancer (IASLC) issued a consensus statement on using liquid biopsy in advanced NSCLC [11]. In 2022, the European Society for Medical Oncology (ESMO) Precision Medicine Working Group issued a report on the recommendations for circulating tumor DNA assays for patients with cancer [12].

Cell-free DNA (cfDNA) normally circulates in all people due to the effects of cell proliferation, apoptosis, and other normal cellular physiological functions [13]. Liquid biopsy analysis of ctDNA involves detecting a tumor-specific characteristic such as somatic mutation, viral sequence, or methylation profile, distinguishing it from cfDNA of non-tumor origin [13]. Currently, several molecular technologies are used to detect ctDNA in liquid biopsy, which mainly includes polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques (Table 2) [12]. The PCR-based approaches include digital droplet PCR (ddPCR) and BEAMing (beads, emulsion, amplification, magnetics) to identify single or few well-characterized mutations suitable for targeted therapy [12]. Also, because cfDNA is released following cell death, cfDNA can indicate cancer treatment response [13]. The role of ctDNA has been investigated for cancer diagnosis and prognosis. However, a further important clinical application is to evaluate cancer treatment response and minimal residual disease (MRD) across various tumor types [13]. This article aims to review ctDNA and liquid biopsy in the diagnosis, early detection, and monitoring of treatment response in cancer.

Methods Used for ctDNA Analysis

POLYMERASE CHAIN REACTION (PCR)-BASED METHODS:

Targeted ctDNA analysis includes PCR methods, which can rapidly detect mutations with high sensitivity and a rapid turnaround, including quantitative PCR (qPCR), digital (d)PCR, and BEAMing (beads, emulsion, amplification, and magnetics) [14]. There are also commonly mutated genes in specific cancers, including the BRAF gene in melanoma, KRAS in lung and colorectal cancer (CRC), PIK3CA in breast cancer, and androgen receptor (AR) in prostate cancer, which are tumor targets that can be monitored for each assay [14].

NEXT-GENERATION SEQUENCING (NGS) METHODS:

NGS methods have recently developed to detect a broad range of genomic alterations and include whole-exome sequencing (WES) and whole-genome sequencing (WGS), targeted methods including tagged-amplicon deep sequencing (TAm-Seq), CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), and targeted error correction sequencing (TEC-Seq) [15]. Because heterogeneous cancers have high genomic instability, these advanced methods have shown some advantages, but sequencing artifacts can occur [13]. The development of more sensitive NGS methodologies has begun to overcome the limitations of low levels of ctDNA in early-stage cancers or low-shedding tumors [14]. In 2024, Martin-Alonso and colleagues proposed using priming agents to transiently reduce cfDNA clearance in vivo to overcome the low levels of ctDNA in the circulation, which may be a future approach to improving the detection of ctDNA [16]. Currently, sequencing methods still face challenges associated with cost and longer analysis times [15]. There are also non-mutation-based methods to undertake ctDNA monitoring and detection of viral DNA for human papillomavirus (HPV) in cases of oropharyngeal and cervical carcinoma and hepatitis B virus (HBV) in cases of hepatocellular carcinoma (HCC) [13].

METHYLOMICS METHODS:

DNA methylation and fragmentome analysis are used for ctDNA analysis to overcome the limitations of genomic ctDNA studies. DNA methylation analysis has been done using bisulfite conversion and analytical methods that include whole genome bisulfite sequencing (WGBS) and targeted bisulfite sequencing for longitudinal monitoring of cancer patients [13]. Recently, bisulfite-free methods have been developed to overcome the limitations of DNA degradation caused by bisulfite conversion, including chromatin immunoprecipitation sequencing (ChIP-Seq) and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) [14,17].

FRAGMENTOMICS METHODS:

Fragmentomics of cfDNA is a new field in liquid biopsy diagnosis, which includes fragmentation patterns, fragment sizes, and end characteristics [14,18]. Cancer patients show more diverse fragmentation patterns that can be used to distinguish cancer from non-cancer-derived cfDNA [9]. Novel bioinformatics methods have resulted in more detailed fragmentomic data [19]. In 2019, Cristiano and colleagues developed a new method for genome-wide analysis of cfDNA fragmentation patterns using a low-coverage WGS method called DELFI (DNA evaluation of fragments for early interception) [20]. This machine learning model incorporates genome-wide fragmentation profiles and can be combined with mutation-based cfDNA analyses, which results in a sensitivity of cancer detection of 91% [20].

MULTIMODAL METHODS:

Multimodal combinations of analyses are used, including analysis of copy number alterations (CNA) and epigenetic, genomic, and fragmentomic analyses of cfDNA samples [13]. In 2021, Parikh and colleagues showed that the integration of epigenomic signatures increased the sensitivity for detection of recurrence by 25–36% when compared with genomic alterations alone [21]. Currently, the range of biofluids for liquid biopsy analysis and detection of ctDNA in response monitoring has gone beyond serum and plasma to include urine, saliva, and cerebrospinal fluid (CSF) (Table 1) [13].

Limitations of ctDNA Analysis in Clinical Practice

Currently, the main clinical role of ctDNA is as a tool for monitoring treatment response in patients with a history of cancer, including lung, colorectal, and breast cancer [13]. However, several limitations to this liquid biopsy approach have prevented its widespread clinical use. There is still a lack of liquid biopsy sample collection and analysis standardization, and clinical and laboratory guidelines for optimal time points for taking samples [22]. Also, it is still unclear what the optimal sampling times after cancer treatment best predict clinical relapse [22]. These limitations may also affect the results of ongoing clinical trials. Also, ctDNA analysis of liquid biopsies may be limited by low levels of DNA due to fragment degradation on sampling and storage [22]. Importantly, there will be potential confounding DNA data from patient comorbidities, particularly chronic diseases, inflammatory diseases, and diseases that involve cell proliferation [22]. These limitations highlight the importance of establishing standardized protocols for ctDNA analysis in patients with various cancer types [13,22].

However, clinical trials have begun to evaluate ctDNA monitoring for response to cancer treatment in clinical settings. Table 3 summarizes the use of ctDNA to diagnose and monitor treatment response in common solid cancers, including non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC) in some reported clinical trials [23–37]. Therefore, ctDNA analysis from liquid biopsies has the potential for cancer diagnosis, selection of targeted therapies, treatment modification, and response to treatment.

Future Directions: Development of a Routine ctDNA Diagnostic for Cancer

Hopes for the application of liquid biopsy technologies as less invasive methods for cancer diagnosis and early detection (screening) have driven recent research. However, most liquid biopsy tests introduced into the clinic have only been able to identify one or two features of tumor DNA, which limits the specificity of the test. In early 2025, a study led by a research team at Oxford University identified a new blood test, developed using machine learning, to detect multiple types of cancer at an early stage [38]. This innovative test, named TriOx, analyses multiple features of DNA in the peripheral blood across six types of cancer (colorectal, oesophageal, pancreatic, renal, ovarian, and breast) and can distinguish between individuals with and without cancer [38]. The research team developed a new methodology for ctDNA detection using deep (80x) whole-genome TET-Assisted Pyridine Borane Sequencing (TAPS), which is a less destructive method than bisulfite sequencing [36]. TAPS, combined with machine learning, permits the simultaneous analysis of genomic and methylation modification data to analyze and combine multiple features from the ctDNA circulating in the blood to improve the detection rate for small fractions of DNA [38]. The research team tested diagnostic accuracy across multiple cancer types in patients with symptoms and matched tumor biopsies, which showed 94.9% sensitivity and 88.8% specificity [38]. Further in silico validation showed strong discrimination at ctDNA fractions, which were as low as 0.7% [38]. An important part of this study showed that tumor burden and ctDNA shedding could be tracked from early or pre-malignant lesions following treatment and without requiring matched tumor biopsies [38]. TriOx is still in the development phase, but this study has shown that blood-based early cancer detection is possible [38].

Conclusions

Because there are still many gaps in understanding the fundamental origins of and dynamics of tumor-derived molecules and DNA, further research must address these gaps before standardizing ctDNA analysis and interpretation can lead to improved clinical utility in different types of cancer as part of a non-invasive method for diagnosis, prognosis, and treatment response in a personalized medicine approach in oncology. Recent research, as shown by TriOx, has highlighted the possibility that blood tests for ctDNA may revolutionize cancer screening, monitoring, and diagnosis to make early cancer detection as routine as other diagnostic blood tests, such as blood glucose testing.