Monitoring and Assessment of Circulating Tumor DNA in Cancers Using Ultrarapid Sensitivity as an Innovative Practice

ABSTRACT

Background

Liquid biopsy with circulating tumor DNA (ctDNA) has rapidly emerged as a new paradigm for assessing tumor burden, genetic heterogeneity, and therapeutic response in a real‐time, noninvasive manner. However, ctDNA is often visually low (sometimes < 0.1% of the total circulating cell‐free DNA), creating a significant challenge for reliable detection (especially for early‐stage disease and minimal residual disease).

Discussion

New technologies for structural variant (SV)‐based ctDNA assays, nanomaterial‐based electrochemical sensors, magnetic nano‐electrode platforms, and fragment‐enriched library preparation have improved sensitivity to attomolar concentrations and less in some populations. In some cancers, ctDNA may provide early evidence of recurrence (i.e., > 1 year) before being clinically evident using traditional metrics. These technologies allow for unprecedented opportunities and sensitivity for early detection, monitoring of treatment response, and early detection of molecular recurrence. Nevertheless, a barrier remains for widespread clinical application owing to pre‐analytical technique variability, analytical platform variability, cost, and the necessity of large‐scale, prospective trials.

Conclusion

This study will analyze new innovative technology‐based ultrasensitive ctDNA assay detection and future research and clinical applications for breast, colorectal, lung, lymphoid, and gastroesophageal cancers, and studies assessing ctDNA for monitoring treatment. Prospects for ctDNA detection utilizing multiplexed CRISPR‐Cas ctDNA assays, microfluidic point‐of‐care (POC) devices, and AI‐based error suppression methods may be the next horizon for ctDNA liquid biopsy technology.

1. Introduction

Circulating tumor DNA (ctDNA), a subset of cell‐free DNA derived from tumor tissue, is emerging as an essential biomarker for the real‐time and noninvasive assessment of cancer burden, an indication of the molecular response, and for the early evaluation of recurrence. The analysis of ctDNA is increasingly becoming a noninvasive alternative to tissue biopsy and a potential standard in precision oncology. However, existing technologies are limited in their ability to identify ctDNA at very low variant allele frequencies, particularly in early‐stage cancers or minimal residual disease. Overcoming these limitations applies to the development and implementation of ultrasensitive detection methods, which is the focus of this study.

Over the last decade, the idea of a “liquid biopsy”—collecting tumor‐based biomarker(s) from blood instead of tumor tissue—has fascinated and stimulated our engagement in the field of precision oncology [1, 2]. ctDNA is one of the most promising and readily available analytes because it represents the dynamic genomic landscape of cancer [3]. Acquiring ctDNA, which is a molecular sample from nearby body fluid, is less invasive, has a lower sampling bias, and has a lower procedural risk in comparison to repeated biopsies of tumor tissue [4, 5]. The initial uses were highly focused on detecting single nucleotide variants (SNVs) predicated on a mostly unseen technology such as droplet digital PCR (ddPCR) and targeted sequencing panels (QIAseq Ultra Panels), where a limit of detection (LOD) of ~0.1% VAF was perceived to be significant [6, 7]. However, with the increasing specificity of therapeutic regimens and the intent to cure in patients with earlier‐stage disease, there is a greater need for detection of ctDNA at < 0.01% VAF, which has become a priority and ultimately a pressing need [8, 9, 10]. This has led to the development of technology with ultrahigh sensitivity that has the ability to detect a low number of mutant molecules in a large pool of wild‐type molecules and potentially tens of thousands of molecules. This study discusses breakthroughs in the field of assay development, nanotechnology, and bioinformatics, which have transformed ctDNA detection from sensitivities ranging from low ng/mL to attomolar sensitivity. This study will evaluate innovation from data sets across multiple cancers and consider the remaining barriers to readership‐targeted use in clinical practice.

2. Enabling Technologies for Ultra‐Sensitive Detection

2.1. ctDNA‐Based Assays for Somatic Mutations

Assays relying on the older SNV‐targeting metric can be confounded by the potential of added error rates from sequencing errors and/or artifacts inherent in PCR amplification [11]. Next‐generation sequencing (NGS) assays that seek to conduct structural‐variant (SV) analyses can mitigate many of the challenges described above and identify karyotype‐specific rearrangements (translocations, insertions, or deletions) with breakpoint sequences that are unique to the tumor, thus eliminating the aforementioned sequencing concerns [12, 13]. SV‐based assays can employ either multiplexed PCR panels or hybrid‐capture probes that are personalized to individual breakpoints and achieve parts‐per‐million sensitivity, with specificity that is unique to the tumor based on the fact that normal cells do not have combinations of rearrangements in the DNA [14]. An SV‐based ctDNA assay in patients with early‐stage breast cancer detected ctDNA in 96% (91/95) of participants at baseline with a median variant allele frequency of 0.15% (range: 0.0011%–38.7%); of these, 10% (9/91) had a variant allele frequency of < 0.01% [15]. In addition, phased variant approaches, such as PhasED‐Seq, improve the sensitivity of detecting circulating tumor DNA (ctDNA) by targeting phased variants, which are multiple single‐nucleotide variants (SNVs) on the same DNA fragment [16].

2.2. Electrochemical Biosensors Based on Nanomaterials

In conjunction with genomic platforms, bioelectronic sensors utilize the high surface area and conductive properties of nanomaterials to transduce DNA‐binding events to recordable electrical signals [17, 18, 19]. Magnetic nanoparticles coated with gold and conjugated with complementary DNA probes can capture and enrich target ctDNA fragments in proximity to the surface of the electrode with attomolar limits of detection within 20 min [20, 21]. Furthermore, graphene or molybdenum disulfide (MoS₂) can facilitate label‐free sensing methods, whereby ctDNA hybridization is detected through parallel decreases in impedance or current‐voltage characteristics [22, 23, 24]. These technologies can also be configured for rapid assays with minimal processing and are sufficiently small to leverage point‐of‐care or portable devices.

2.3. Magnetic Nano‐Electrode Systems

Magnetic nano‐electrode systems harness the synergies of nucleic acid amplification via PCR and magnetic nano‐technology using superparamagnetic Fe₃O₄–Au core–shell particles for both PCR substrates and electrochemical modifications [25, 26]. In short, the ctDNA amplification products from PCR are held on the nanoparticle for electrochemical probe readout with high sensitivity (three attomolar signal‐to‐noise ratio), within 7 min of PCR [27, 28]. These hybrid metabolic systems combine the sensitivity of nucleic acid amplification with speed, and easily formed electrochemical adaptations.

2.4. Enrichment of Fragments and Specialized Library Preparation

In addition to the previously described fork assay systems, the pre‐sequencing workflow yields exceptionally optimized and improved ctDNA [29]. The enrichment of short fragments takes advantage of a distinct property of tumor‐derived circulating cell‐free DNA (cfDNA), which is fragmented to lengths of 90‐150 base pairs (bp), whereas DNA derived from nontumor cells tends to be longer [29, 30]. The differences in cfDNA length indicate that nontumor cell‐derived cfDNA was artificially expanded. The use of a library preparation method of either bead‐based or enzymatic size selection of cfDNA, specifically for the enrichment and capture of short fragments, can yield an increase in fractional abundance by several folds within ctDNA sequencing libraries [31, 32]. The enrichment of short fragments can also increase the detection yield of low‐frequency variants in combination with error‐corrected next‐generation sequencing [31, 33]. To detect minimal residual disease, shorter fragments can reduce the required depth of sequencing, making it more efficient and cost‐effective.

2.5. Clinical Utility Across Cancer Types

2.5.1. Early Detection and Prediction of the Prognosis

In breast cancer, structural variant‐informative ctDNA assays may allow for the assessment of residual disease for several months to years after resection and adjuvant therapy, whereas clinical relapse may occur much sooner than the assessment grants the opportunity for salvage strategies to be implemented [15, 34, 35]. Notably, ctDNA findings, which assign a clinically actionable risk associated with detectable ctDNA after the completion of treatment, are associated with an observably higher rate of clinical recurrence. In addition to assigning risk probability, the findings can be used to determine the adjust the approach to actionable monitoring upon detection after treatment. Longitudinal ctDNA monitoring during adjuvant chemotherapy and long after for identifying molecular relapse has been revealed to be significantly faster and more reliable than carcinoembryonic antigen (CEA) and imaging assessment in the context of colorectal cancer, improving the precision of treatment intensification and de‐escalation [36, 37, 38].

2.6. Treatment Response and Minimal Residual Disease

Evidence has demonstrated that the concentrations of ctDNA over time are well correlated with tumor burden [39]. For instance, a decline in ctDNA levels predicted the radiographic response to therapy more accurately than follow‐up imaging in patients with NSCLC treated with anticancer drugs [40, 41]. Resistance mutations have also been observed in the plasma weeks before clinical or radiographic evidence of disease progression [42]. To improve the prognosis of the risk of relapse and guide immunochemotherapy for aggressive B‐cell lymphoma, ctDNA‐based MRD assays are more sensitive and informative than standard PET or CT imaging, even though the disease is frequently subclinical and not seen on imaging [43, 44, 45].

2.7. Noninvasive Genotyping and Resistance Monitoring

In advanced disease, tissue biopsies are sometimes unreasonably impractical, or even ethically contraindicated, and can only temporally assess run‐off tumor heterogeneity. Ultrasensitive ctDNA workflows enable the assessment of actionable driver mutations, copy number alterations, and resistance variants using a simple blood sample [46, 47]. For instance, in EGFR‐mutant NSCLC, monitoring of the T790M resistance mutation has the potential to switch to third‐generation inhibitors without repeated tissue sampling [48, 49].

2.8. Emerging Applications in Methylation and Epigenetic Profiling

In addition to measuring sequence variants in tumor specimens, ctDNA methylation changes can measure an orthogonal layer of tumor‐specific information [50, 51]. Tumor‐agnostic hypermethylated gene promoter panels can detect and quantify tumor development in patients with early‐stage gastroesophageal cancer by analyzing cell‐free DNA in the plasma, achieving greater concordance with tumor tissues and quantifying tumor development [52, 53]. The combination of mutations and methylation in cell‐free DNA (cfDNA) may lead to pan‐cancer screening initiatives [54, 55].

2.9. Clinical Applications of ctDNA Across Cancer Types

Extensive clinical studies have validated the clinical utility of ctDNA in various settings. For example, mature DYNAMIC outcome data support a ctDNA‐guided approach to adjuvant chemotherapy (ACT) in patients with stage II colon cancer, with potential for further risk stratification for ctDNA‐positive patients with ctDNA burden and EOT results [56]. A separate study demonstrated that ctDNA presence after neoadjuvant chemotherapy is associated with relapse in early stage breast cancer, thus supporting interventional trials that would test the clinical utility of ctDNA monitoring [57]. In another study, sustained ctDNA clearance by ACT was an indicator of favorable DFS and OS relative to transient clearance (24‐month DFS: 89.0% vs. 3.3%; 24‐month OS: 100.0% vs. 82.3%) [58]. This study provides evidence for the utility of ctDNA post‐resection monitoring for risk stratification of recurrence and mortality, which could guide adjuvant therapy [58]. More broadly, the clinical utility of ctDNAs is not limited to the studies described above. For example, an interesting study indicated that the plasma EGFR T790M ctDNA status is associated with clinical outcomes in acquired EGFR‐TKI resistance in patients with advanced NSCLC [59]. These examples demonstrate the potential of ctDNA to inform multiple therapeutic decisions, monitor minimal residual disease (MRD), and ultimately allow intervention sooner relative to routine diagnostic studies. Table 1 summarizes studies that have evaluated ultrasensitive ctDNA detection across different cancer types to provide a clearer picture of clinical evidence.

Table 1.

Ultrasensitive ctDNA detection in representative clinical studies.

Methodology	Cancer type	Key findings	Reference
Phased variant enrichment and detection sequencing (PhasED‐seq)	B cell lymphoma	PhasED‐seq improves ctDNA detection and have worse outcomes	[16]
SV‐based dPCR ctDNA assay	Early‐stage breast cancer	Provides ultrasensitive ctDNA detection, treatment monitoring, and prognostications	[15]
Droplet digital PCR (ddPCR)	Colorectal liver metastases (CRLM)	Reduce time‐to‐intervention, contributing to clinical decision‐making in indeterminate CT findings	[60]
Digital PCR	Early‐stage triple‐negative breast cancer (TNBC)	Identify patients at higher risk of relapse and those with MRD not visible on imaging	[61]
Electrochemical biosensor	Gastric cancer	Contribute to early screening for gastric cancer	[62]
CRISPR Cas9n‐driven DNA walker + COFs‐AuNPs	Breast cancer	Provides excellent selectivity, reproducibility, and early cancer detection	[63]

3. Limitations and Challenges

3.1. Pre‐Analytical and Biological Variation

Given the fragility of cfDNA, the process of collecting blood, transfer time, temperature, or other transport variations may negatively or positively affect the fragments. Mutations in blood cells resulting from clonal hematopoiesis of indeterminate potential (CHIP), particularly in older patients, could contaminate ctDNA assays that are not tumor‐informed or orthogonally validated.

3.2. Platform Differences and Standardization

The modalities currently in use, such as hybrid‐capture NGS, digital droplet PCR, and biosensor platforms, have substantial differences, including varying sensitivities, specificities, and limits of detection reporting. ctDNA testing modalities require comparisons across platforms to develop clinically actionable cutoffs, considering the limited number of reputable standard reference materials or proficiency testing at present. It is critical to have recommendations that are standardized and harmonized for specimen procurement, pretreatment, and reporting to realize reproducible investigations that can ultimately lead to inter‐laboratory comparisons.

There have been worldwide attempts to standardize ctDNA assay protocols to minimize platform‐specific variability and increase comparability across studies. Standardized reference materials (e.g., synthetic cfDNA controls and digital reference samples from the National Institute of Standards and Technology [NIST]) are crucial for benchmarking assay performance across platforms [64, 65]. Inter‐laboratory proficiency testing programs (e.g., European Molecular Genetics Quality Network [EMQN]) are also important for harmonizing testing procedures and ensuring that assay performance across laboratories is consistent [66, 67]. Consensus guidelines developed by organizations (e.g., Blood Profiling Atlas in Cancer Consortium [BloodPAC] and the International Liquid Biopsy Standardization Alliance [ILSA]) are similarly necessary to establish universal recommendations on different approaches for each component of the liquid biopsy workflow (e.g., processing of samples, library preparation, and reporting thresholds) [68, 69]. Overall, a comprehensive approach involving reference materials, proficiency testing, and consensus guidelines is essential to enable reliable and comparable clinical liquid biopsy practices, at the very least, to build evidence for improving the adoption of liquid biopsy technologies together with these standards.

3.3. Economic and Regulatory Concerns

Ultra‐sensitive assays leveraging emerging technologies rely on certain reagents, sophisticated instrumentation in the laboratory, and bespoke bioinformatics interpretations. The limitations of these modalities are their associated costs; therefore, scalability and access are limited in resource‐limited areas. Regulatory approval and reimbursement from payers is limited because of their price. Evidence of ctDNA with clinical applications exists and is not restricted to a single prospective or retrospective trial, but further evidence is needed in the form of large‐scale randomized trials to show an association between ctDNA‐based interventions and increased survival rates.

Economic factors are still a significant barrier to the widespread adoption of ultrasensitive ctDNA technologies as diagnostic tools, particularly in low‐resource contexts. Case studies reported that ctDNA had a tremendous range of costs, $199–$9124 per sample, to generate information [70]. Note that these costs depend on the platform (coverage), setting, and volume of tests. One study reported that ctDNA is a low‐cost strategy ($8541), and even at double the cost of the ctDNA it would still be the low‐cost strategy [71]. The data recorded supported the low‐cost of utilizing ctDNA as a surveillance tool in instances of possible recurrence. The greatest difference appeared in equivocal, with just repeating a ctDNA test for ctDNA‐based surveillance, the final cost would be $9041 [71]. This price level presents barriers to access and reimbursement of ctDNA, especially in countries with budgeting‐constrained healthcare systems. In addition, the downstream effort to use NGS‐based workflows requires infrastructure including sequencing platforms, trained staff, and bioinformatics, all of which may serve as challenges for smaller clinics or hospitals. Cost‐effectiveness studies have suggested that ctDNA could reduce overtreatment and hospital admissions in comparison to more conservative treatments; however, future work in health economics modeling is needed. Public‐private partnerships, tiered pricing, and decentralized testing through point‐of‐care devices may be useful in the future.

3.4. ctDNA Ultralow‐Level Detection Opportunities and Challenges

3.4.1. LOD Comparison to Existing Methods

DNA sequence variants with allele frequencies of ≥ 5% were routinely detected using NGS. As all current NGS platforms have an average intrinsic error of at least 0.2%, NGS struggles to report single‐nucleotide variants (SNVs) with variant allele frequencies (VAFs) below 1% [72]. A comparative analysis of the major enabling platforms for ultrasensitive detection is presented in Table 2. Performance parameters, such as sensitivity, specificity, turnaround time, strengths, and limitations, were included in the analysis.

Table 2.

Comparison of ultrasensitive ctDNA detection technologies.

Technology platform	Sensitivity (LOD)	Specificity	Turnaround time	Strengths	Limitations
Droplet digital PCR (ddPCR)	0.01%–0.1% VAF	High	4 h to over a day	▪ High sensitivity and precision ▪ Absolute quantification ▪ Cost‐effectiveness for targeted analysis	▪ Limited multiplexing ▪ Requires prior knowledge of mutations ▪ Inability to detect all genomic alterations ▪ Potential for false positives at low VAFs
CRISPR‐based ctDNA assays	0.01% VAF	Extremely high	Under an hour	▪ Noninvasive ▪ Ultra‐sensitivity ▪ High specificity ▪ Rapid and simplicity	▪ Need for amplification ▪ Risk of off‐target effects ▪ Quantitative challenges
PhasED‐Seq	0.000000661% VAF	High	Within 7 days	▪ Superior sensitivity ▪ Reduced background noise ▪ Enhanced clinical utility	▪ Requirement for primary tumor biopsy ▪ Dependence on phased variants ▪ Potential for false negatives ▪ Less common in solid tumors
Electrochemical biosensors (DNA‐Au@MNP)	3.3 aM, 5 fM	High	20 min	▪ High sensitivity ▪ Rapid and real‐time detection ▪ Cost‐effective and portable	▪ Need for clinical validation ▪ Challenge of low VAF ▪ Dependence on known mutations ▪ Sample preparation complexity
SV‐based dPCR	0.001%–0.01% VAF	High	Within hours	▪ High analytical sensitivity and accuracy ▪ High specificity for SVs ▪ Cost‐effective for monitoring ▪ Quick turnaround time	▪ Requires a prior tumor tissue analysis ▪ Risk of false negatives ▪ Misses full variant landscape
Magnetic nano‐electrodes	∼3 aM	High	∼7 min	▪ Noninvasive ▪ Rapid analysis ▪ High specificity	▪ Limited multiplexing ▪ Technical complexity ▪ Challenges in early‐stage disease ▪ Heterogeneity and off‐target binding
Short fragment enrichment NGS	0.01%–0.1% VAF	High	1–2 weeks	▪ Higher sensitivity for low VAFs ▪ Minimally invasive	▪ Low ctDNA concentration ▪ Biological noise ▪ Limited scope for some variants

3.5. Specificity at Very Low Variant Allele Frequencies

To achieve specificity for ultralow VAFs, contemporary ctDNA assays utilize a number of strategies. SV‐based assays can inherently improve specificity owing to their reliance on the breakpoints of tumors that do not exist in normal cfDNA. Additionally, error suppression techniques, such as unique molecular identifiers (UMIs) and orthogonal validation techniques, are helpful in distinguishing tumor‐derived variants from background noise and sequencing errors. PhasED‐seq approaches increase specificity by requiring the presence of multiple linked mutations on the same DNA fragment.

3.6. Prospective Validation in Independent Cohorts

Multiple ultra‐rapid ctDNA platforms have been prospectively validated in large, independent cohorts of patients. For example, in a cohort of 95 patients with early stage breast cancer, SV‐informed ctDNA assays detected ctDNA at baseline in 96% of patients [15]. Multicancer validation has been performed in colorectal cancer, NSCLC, and lymphoid tissues, supporting the effectiveness of these assays across tumor types. Broader validation of pan‐cancer population studies remains an important objective for future research.

3.7. Prognostic or Predictive Value of Early ctDNA Detection

Numerous studies have demonstrated the prognostic potential of ctDNA detection, as reported in colorectal and breast cancer studies [36, 73, 74]. Contemporaneously, quantitative detection of ctDNA following treatment is highly associated with increased recurrence risk [75], while clearance of ctDNA during treatment is associated with significantly higher overall survival and progression‐free survival [76]. The dynamic detection of ctDNA has predicted the response earlier than imaging in NSCLC and lymphomas [40, 77], making it an effective predictive tool for personalized therapy.

3.8. Lead Time Compared to Clinical or Radiological Recurrence

ctDNA can reveal molecular relapse several months before clinical or radiologic progression. For example, ctDNA is highly sensitive and specific; it can detect a molecular relapse a month before clinical symptoms [78]. ctDNA detection has been shown to occur, on average, 4 months before radiological relapse, which supports the use of ctDNA as a marker of disease recurrence [79]. A separate study noted that serial monitoring of ctDNA determined that molecular progression was evident before RECIST progression [39]. The average timing was reported to be 11 months after the diagnosis of clinically overt metastases, and the specificity was reported to be as high as 100% [80]. Another study determined that, on average, ctDNA was reported 5.5 months before evidence of imaging progression [81].

3.9. Turnaround Time and Clinical Compatibility

The turnaround time for ctDNA assays differs by platform, ranging from 1 to 7 days, depending on sample logistics and sequencing depth. Traditional gene detection technology has been reported to be unable to achieve real‐time, low‐cost, and portable measurement of ctDNA, whereas electrochemical biosensors are reported to be low‐cost, highly specific, sensitive, and portable [82]. DNA‐Au@MNP‐based sensors have been reported to provide a viable method for a 20‐min response time, and for minimally invasive detection of early‐stage cancer [20]. This DNA‐Au@MNP‐based sensor can selectively detect short‐ and long‐stranded DNA targets [20]. It has a wide dynamic range (2 aM to 20 nM) for the 22 nucleotide DNA target with an ultra‐low detection limit of 3.3 aM [20]. For a 101 nucleotide ctDNA target, a dynamic range of 200 nM to 20 nM was achieved with a detection limit of 5 fM [20]. Another study reported that an electrochemical biosensor has a wide detection range from 10 fM to 20 pM, with a very high detection limit for ctDNA down to 2.3 fM [83]. The high degree of linear correlation over a wide range of ctDNA concentrations further validates the quantitative capability of the biosensor, which is significant for monitoring cancer dynamics and treatment responses [83]. A study utilizing nanoparticle surface‐localized genetic amplification with Fe3O4−Au core−shell nanoparticles demonstrated ultrasensitive ( ~ 3 aM) and rapid ( ~ 7 min) detection of metastatic breast cancer ctDNA in vitro [27]. These findings support the feasibility and routine use of ctDNA in clinical practice to support clinical decision making, especially for monitoring and therapeutic adjustments.

3.10. Controlling Pre‐Analytical Variables

ctDNA workflows have recently incorporated preanalytical standardization to help mitigate false‐negative results and ensure sample quality. The in vitro degradation of ctDNA and the subsequent release of contaminating genomic DNA from lysed blood cells should be avoided [84]. Streck Cell‐Free DNA blood collection tubes (cfDNA BCTs) may provide advantages over standard K₂EDTA tubes but have only been verified in healthy individuals. The above study confirmed the applicability of clinical oncology specimens collected and stored in cfDNA BCTs for up to 3 days to facilitate reliable cfDNA and mutation analyses [84]. Clonal hematopoietic (CH) mutations detected in plasma cfDNA analyses should be cautiously evaluated for their potential pathological significance [85]. A previous study presented evidence supporting that enrichment of cfDNA fragments in the 90–150 bp range can enhance ctDNA detection [31].

3.11. Cost‐Effectiveness and Scalability

Even though developing ultrasensitive assays from inception has significant costs, novel methods that leverage cartridge‐based microfluidics, automation, and AI‐assisted interpretation have begun to reduce the cost of the assays. Currently, point‐of‐care (POC) platforms are being developed along with multiplexed assays (such as CRISPR‐based detection). These point‐of‐care platforms and multiplexed assays can be used in decentralized locations. If economies of scale and regulatory approval are achieved, the tests may be affordable and available in a range of healthcare facilities.

3.12. Addressing Tumor Heterogeneity and Clonal Evolution

Tumor heterogeneity represents one of the fundamental characteristics of cancer and has relevant implications for the tumor response to chemotherapy, prognosis, and risk of relapse. ctDNA is released from tumor cells into the bloodstream and contains specific genetic information about the tumor, which may include mutations, gene rearrangements, and epigenetic changes. ctDNA provides a noninvasive mechanism to monitor tumor evolution and treatment response, overcoming the constraints of conventional tissue biopsies. Serial ctDNA sampling allows for the dynamic monitoring of clonal evolution over time, particularly in relation to therapy. The analysis of ctDNA with respect to the levels or patterns of mutations can provide clinicians with information about whether resistance mutations are being detected and an opportunity to refine treatment. For example, in NSCLC, the emergence of the epidermal growth factor receptor (EGFR) T790M resistance mutation can be detected in ctDNA weeks before clinical deterioration to directly inform timely therapeutic changes [86]. Likewise, detection of the KRAS G12C mutation in ctDNA may also indicate resistance to therapy and assist in determining the best alternative therapy. ctDNA assessment may identify a range of resistance mechanisms, including increased MET copy number and proposed resistance mechanisms in NSCLC [87].

4. Distinguishing MRD From Clonal Hematopoiesis

Discrimination between MRD and clonal hematopoiesis of indeterminate potential (CHIP)‐related mutations is performed using a tumor‐informed assay, which compares ctDNA in plasma to known tumor mutations. CHIP mutations (e.g., DNMT3A, TET2, and ASXL1) are frequently detected in cfDNA and may interfere with ctDNA analyses [85]. In addition to mutations, ctDNA fragment size, end motifs, and methylation patterns may also be useful in distinguishing ctDNA from nontumor DNA and ascertaining the tissue of origin. ctDNAs derived from tumors exhibit characteristic fragmentation patterns and methylation profiles, enhancing the precision of identification. In addition to variant detection, fragmentomic analysis improves the sensitivity of MRD detection, particularly when ctDNA levels are low.

4.1. Comparison With Traditional Biomarkers and Imaging

Existing evidence suggests that ctDNA is a better marker of residual disease and early recurrence than carcinoembryonic antigen (CEA) [88]. In breast cancer, ctDNA was found to be more accurate at identifying early recurrences than carbohydrate antigen 15‐3 (CA 15‐3) [88]. Other potential advantages of ctDNA over protein biomarkers when monitoring a patient with cancer include its shorter half‐life in plasma, the ability to predict the likely response to particular therapies, and the identification of mechanisms of therapy resistance [88]. Additionally, in the case of colorectal cancer, ctDNA is superior to CEA in detecting relapse [89]. In metastatic GI cancer and lymphoma, ctDNA dynamics can predict the treatment response more accurately than radiological imaging studies [43, 90].

4.2. Evidence for Improved Outcomes via ctDNA‐Guided Interventions

Preliminary evidence supports the adjustment of therapies based on ctDNA. The detection of ctDNA after surgery and before adjuvant treatment is associated with a high risk of distant metastasis [91]. A ctDNA‐guided treatment approach for patients with stage II colon cancer reduces the use of adjuvant chemotherapy without compromising recurrence‐free survival [92]. Another trial showed that ctDNA‐guided risk stratification led to reduced use of adjuvant chemotherapy without compromising the risk of recurrence [93]. Furthermore, ctDNA detection after adjuvant treatment was significantly associated with the recurrence‐free interval (RFI) [94]. In comparison to patients receiving unmatched therapy, those who received matched targeted therapy based on ctDNA profiling had significantly improved overall survival [95].

5. Future Directions

Overall, ctDNA testing utilizes multiplex detection technology to detect ctDNAs. The CRISPR‐Cas12a and Cas13a systems utilize trans‐cleavage mechanisms that allow for detection methods that do not require amplification, while simultaneously being able to target several nucleic acid species in a single reaction. In addition, advances in integrated microfluidic methods in conjunction with nanomaterials will allow cartridge‐based assays to be designed for decentralized testing. However, as is true for all social changes, artificial intelligence (AI) will eventually reflect the use of machine learning algorithms, for example, detection and high confidence that true tumor‐derived variants will surpass nonspecific technical noise or CHIP artifacts, especially as they can be generated from multidimensional features (e.g., sequence, fragmentation, and methylation). ctDNA testing could be combined with other liquid biopsies (e.g., exosomal RNA/proteins and tumor‐educated platelets), with the hope that these resultant analytes will be representative of some composite measures that overcome the weakness of any one modality. ctDNA and/or other analytes could pave the way for multi‐analyte precision oncology research.

To address the above‐mentioned limitations, the following are possible outcomes [1]: adoption of standard blood collection and specimen storage protocols to decrease pre‐analytical variability [2]; approaches that use tumor‐informed or CHIP filtering algorithms to distinguish true ctDNA from clonal hematopoiesis [3]; global collaborations that develop reference standards and proficiency testing for ctDNA assays; and [4] reimbursement schemes that support the equitable implementation of high‐cost ultra‐sensitive diagnostics in clinical workflows.

6. Conclusion

The advent of ultrasensitive ctDNA detection has enabled liquid biopsy to rapidly become an important component of cancer care, creating unprecedented opportunities for early detection of malignancy, continuous monitoring of treatment response, and surveillance for disease recurrence. In elevating the ability to detect ctDNA to ultralow variant allele frequencies, these emerging ctDNA technologies remove one of the last barriers to practice change in precision oncology and hold promise for detecting disease progression months ahead of clinical or radiological confirmation of disease. Structural variant‐based assays, electrochemical biosensors, magnetic nanoelectrodes, and enrichment methods based on short fragments have led to rapid advances in assay performance, particularly in terms of sensitivity and turnaround time. Challenges remain in terms of normalizing methods among laboratories and platforms, inter‐platform variability, and affordability that can hinder the adoption of ctDNA assays in practice. Prospective and multicenter studies of moderate to large sizes will be necessary to establish prognostic and predictive values among ctDNA assays across different cancers. Coupling ctDNA assays with clinical predictors, artificial intelligence for error suppression, and multi‐analyte liquid biopsies will further increase specificity and clinical utility. Equally important is the development of scalable, affordable, and decentralized platforms that provide equitable access, particularly in resource‐limited settings. If these challenges can be addressed, ctDNA assessment will become routine in oncology and will influence strategies concerning the early detection of cancer and personalized treatment approaches globally. Ultimately, the successful implementation of ctDNA technologies in clinics will result in a seismic shift in the diagnosis, monitoring, and promotion of survivorship concepts in cancer.

Author Contributions

Md Mohiuddin: conceptualization, writing – review and editing, and resources.

Conflicts of Interest

The author declares no conflicts of interest.

Transparency Statement

The lead author, Md Mohiuddin, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Data Availability Statement

Data availability is not applicable to this article as no new data were created or analyzed in this study.