
==== Front
Oncol Lett
Oncol Lett
OL
Oncology Letters
1792-1074
1792-1082
D.A. Spandidos

10.3892/ol.2024.14681
OL-28-5-14681
Review
Liquid biopsy: Comprehensive overview of circulating tumor DNA (Review)
Ge Qian 1
Zhang Zhi-Yun 2
Li Suo-Ni 3
Ma Jie-Qun 3
Zhao Zheng 3
1 Graduate School, Xi'an Medical University, Xi'an, Shaanxi 710000, P.R. China
2 Graduate School, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, P.R. China
3 Department of Internal Medicine, Shaanxi Provincial Cancer Hospital, Xi'an, Shaanxi 710000, P.R. China
Correspondence to: Mr. Zheng Zhao, Department of Internal Medicine, Shaanxi Provincial Cancer Hospital, 309 Yanta West Road, Yanta, Xi'an, Shaanxi 710000, P.R. China, E-mail: lacustrian@163.com zhaozheng0628@163.com
11 2024
13 9 2024
13 9 2024
28 5 54821 6 2024
29 8 2024
Copyright: © 2024 Ge et al.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Traditional tumor diagnosis methods rely on tissue biopsy, which can be invasive and unsuitable for long-term monitoring of tumor dynamics. The advent of liquid biopsy has notably improved the overall management of patients with cancer. Liquid biopsy techniques primarily involve detection of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). The present review focuses on ctDNA because of its significance in tumor diagnosis, monitoring and treatment. The use of ctDNA-based liquid biopsy offers several advantages, including non-invasive or minimally invasive collection methods, the ability to conduct repeated assessment and comprehensive insights into tumor biology. It serves crucial roles in disease management by facilitating screening of high-risk patients, dynamically monitoring therapeutic responses and diagnosis. Furthermore, ctDNA can be used to demonstrate pseudo-progression, monitor postoperative tumor status and guide adaptive treatment plans. The present study provides a comprehensive review of ctDNA, exploring its origins, metabolism, detection methods, clinical role and the current challenges associated with its application.

circulating tumor DNA
liquid biopsy
overall tumor management
Shaanxi Provincial Cancer Hospital2022SF-282 cphcf-2022-218 24YXYJ0179 The present study was supported by Shaanxi Provincial Cancer Hospital, grant no. 2022SF-282, cphcf-2022-218); and 24YXYJ0179).
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pmc1. Introduction

Currently, pathological tissue biopsy is the gold standard for diagnosing and monitoring a number of malignant tumors, such as lung cancer, stomach cancer, colorectal cancer. This method allows rapid assessment of the extent and nature of lesions, provides relatively accurate pathological classification and facilitates early detection and diagnosis. In addition, tissue biopsies conducted during treatment can reflect disease progression and treatment efficacy and provide clinicians with valuable information to tailor treatment plans (1). However, use of insufficient or inadequate tissue samples may lead to diagnostic bias, which may be exacerbated by tumor heterogeneity (2). Furthermore, repeated invasive procedures can cause discomfort for patients, particularly as the disease advances.

The emergence of liquid biopsy has effectively addressed several of these challenges. Liquid biopsy involves the molecular analysis of liquid (non-tissue) samples to evaluate physiological states (3). While blood samples are the most commonly used, other bodily fluids such as cerebrospinal fluid (CSF), saliva, pleural effusion, bile, abdominal fluid and urine can also be utilized (4). Liquid biopsy primarily focuses on analyzing circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), as well as circulating cell-free (cf)RNA, extracellular vesicles and tumor-inducing platelets (5). ctDNA has emerged as a pivotal element in clinical practice as it serves a key role in cancer diagnosis, monitoring and treatment. In clinical treatment, physicians are able manage treatment strategies by reference to ctDNA results. The detection of ctDNA pre-treatment and pre- and post-operation guide patient treatment plans (6). However, there are limitations to ctDNA analysis in liquid biopsy, including challenges in evaluating tumor pathology, detection sensitivity and the absence of standardized protocols.

2. Overview of ctDNA-based liquid biopsy

cfDNA

Circulating cfDNA refers to highly fragmented DNA released from cells into the bloodstream that circulates freely in human blood (7). First discovered in 1948 (7), cfDNA has become a major focus of medical research (8–10). It is composed of both double- and single-stranded fragments (11–13), typically 120–220 bp in length, with an average length of ~167 bp, which is associated with the nucleosome (14).

In the bloodstream, cfDNA exists in three primary forms: Free, bound to protein (such as nucleosomes and lipoproteins) or associated with extracellular vesicles, such as exosomes, apoptotic bodies and microvesicles (15,16). The majority of plasma cfDNA is found in exosomes (17). The sources of cfDNA have been a topic of debate and can be generally divided into two main categories: Cellular destruction and active cellular release (18,19). Potential sources of cfDNA include cellular byproducts released during normal physiological processes, exogenous DNA originating from dietary intake, blood transfusions or infections, release from the nervous system, secretion into the blood circulation due to factors such as stress, hereditary conditions, degeneration or disease, fetal cellular material transferred to the mother during pregnancy and systemic release due to obesity and aging (19). Plasma cfDNA from healthy individuals predominantly originates from white blood cells (55%), red blood cell progenitors (30%) and vascular endothelial (10%) and liver cells (1%) (19). In patients with cancer, however, cfDNA is mainly derived from tumor tissue and surrounding cells (18).

cfDNA is a key biomarker for various physiological and pathological conditions and is associated with factors such as aging (20) and physical or psychological stress (21). In addition, cfDNA serves as a key biomarker in several types of cancer, including non-small cell lung cancer (NSCLC) (22), liver (23), breast (24,25), pancreatic (26), oral (27) and colorectal cancer (28,29). Previous studies suggest that cfDNA may also be associated with xenotransplantation (30,31).

cfDNA encompasses short and long DNA fragments and analysis of these fragments can provide valuable insight into a health status. Increasing evidence shows that cfDNA is vital for immune regulation, tumor-associated inflammation and the maintenance of cell homeostasis (32,33). cfDNA can impact cellular function and transformation, contribute to tumor growth and metastasis and holds promise for early disease detection (32,33). In healthy individuals, there is a dynamic balance between production and clearance of cfDNA. cfDNA is primarily cleared by the liver, spleen and kidney (34,35). However, in patients with chronic inflammation or tumors, cfDNA levels significantly increase due to impaired clearance and subsequent accumulation (32).

ctDNA

ctDNA is a subset of cfDNA that consists of DNA fragments that originate from tumor tissue and potentially other sources, such as shedding cells from normal tissue, characterized by genetic alterations that mirror those of the tumor (18,36). The increase in cfDNA levels in patients with cancer is primarily attributable to the elevated levels of ctDNA (37). Typically, ctDNA fragments are ~140 bp in length and have a half-life >2 h, which makes their dynamics variable (38,39). The detection rate of ctDNA in plasma ranging from 0.01% to a majority in the cfDNA, which reflects notable heterogeneity among different types of tumor (40,41). Despite this variability, the quantity of ctDNA detected is usually high compared with that of CTCs (42). The ability to detect ctDNA is influenced by characteristics of the tumor. For example, smaller solid tumors or those with low metabolic activity may be more challenging to detect, which may lead to false-negative results (6). Notably, ctDNA levels decrease rapidly following radical tumor resection if there is no or minimal residual tumor (43).

3. ctDNA detection in biological samples: Blood vs. non-blood sources

Blood

Blood is the most widely used sample type for detection of ctDNA (44), primarily due to the minimally invasive collection process and reproducibility. However, the precise mechanisms by which ctDNA enters the bloodstream remain unclear. It is has been hypothesized that ctDNA in blood originates from three primary sources: Apoptotic or necrotic and viable tumor cells and CTCs (45). Plasma is considered the optimal sample for ctDNA analysis, as serum (excluding clotting factors) contains an increased proportion of DNA from leukocyte lysis (46). For example, Heger et al (47) developed a molecular prognostic index for central nervous system (CNS) lymphoma using plasma ctDNA and demonstrated that is was effective in predicting patient outcomes. This ultra-sensitive method can detect CNSL-derived mutations in plasma ctDNA that are highly consistent with CSF and tumor tissue. Plasma ctDNA undetectable at baseline was associated with favorable outcomes. However, detecting ctDNA in white blood cells poses challenges due to high levels of cfDNA in these cells, which leads to notable dilution of the ctDNA (48). Compared with blood samples, ctDNA is easier to detect in non-blood samples.

CSF

Acquiring brain tissue for diagnostic purposes is both challenging and high-risk due to the unique structure and function of the brain. Therefore, CSF serves an irreplaceable role. While magnetic resonance imaging (MRI) is commonly used to monitor CNS diseases (49), its predictive capability is limited. Liquid biopsy that involves the detection of ctDNA in CSF may serve as a novel detection method for CNS diseases. The unique composition of CSF, along with the protective blood-brain barrier, decreases interference from cfDNA, which results in increased concentration of ctDNA and mutated allele frequencies (MAF) in CSF. This improves the sensitivity and accuracy of ctDNA mutation detection. Consequently, CSF biopsy may be a promising diagnostic tool for the detection and monitoring of brain tumors and CNS metastases (50–52).

A study has shown that carcinoembryonic antigen and CSF ctDNA are effective biomarkers for distinguishing patients with and without brain parenchymal tumor or CNS metastases (53). CSF ctDNA analysis has demonstrated distinct mutational profiles in patients with bone marrow metastasis (53). In a 2022 case report (54), a patient with lung adenocarcinoma experienced neurological symptoms, including headache, nausea, aphasia, limb restlessness and sudden blindness during treatment. While initial MRI and CSF cytology did not indicate the presence of brain metastasis, CSF ctDNA analysis identified the same EGFR mutation as that detected in the lung tumor of the patient. Follow-up MRI scans 9 months later confirmed brain metastasis, which suggested earlier MRI and CSF cytology results were false negatives. This case underscores the potential of CSF ctDNA as an early diagnostic biomarker as it may detect brain metastasis before cytological or MRI evidence emerges.

To the best of our knowledge, studies of ctDNA in CSF have predominantly focused on adults (55–58), with relatively few investigating its application in children (59–61). Pages et al (62) assessed tumor reliability by analyzing ctDNA in peripheral blood, CSF and urine samples from children with brain tumors; ctDNA detection in this demographic was limited by low tumor fraction (TF). In numerous cases, the actual TF was >1%, with TF >0.1% being undetectable. Furthermore, only a small percentage of high-grade tumors were available. Therefore, uncertainty persists regarding the application of liquid biopsy for pediatric tumors, indicating the need for further research.

Saliva and sputum

The clinical relevance of saliva as a diagnostic tool is uncertain. A study from 2019 (63) suggested that saliva may not serve as an adequate alternative sample for quantitative cfDNA testing due to insufficient cfDNA concentration for diagnosis of NSCLC. However, it was proposed that saliva may be beneficial as a complementary method to cytology. Wang et al (64) made notable advancements by optimizing the extraction technique for sputum samples to overcome limitations posed by large amounts of mucus components and the low yield of cancer cells. Super-amplification refractory mutation system was used to analyze EGFR mutation status in cfDNA derived from sputum samples; sputum could be a promising sample type for detecting EGFR mutations, although its use for diagnosing lung adenocarcinoma may be limited. Further research by Wang et al (65) highlighted saliva from patients with lung adenocarcinoma as a valuable alternative source for detecting the EGFR exon 20 p.T790M mutation, which is linked to resistance to EGFR targeted therapy (65). In addition, a previous study investigating head and neck cancer reported a high concordance (93%) in ctDNA detection between saliva and blood samples, as well as efficacy of ctDNA in saliva in predicting patient outcomes (66). A meta-analysis of 64 cases of malignant salivary gland carcinoma found increased levels of ctDNA and CTCs in malignant cases (67). According to the 2024 Expert Consensus, ctDNA extracted from saliva, along with serum or plasma, provides meaningful insight into tumor genetics and dynamics (68).

Pleural, peritoneal and pericardial effusion

Tumor supernatant, such as pleural, peritoneal and pericardial effusion, are in proximity to tumors and may provide distinct advantages over blood for ctDNA detection; for example, it is easier to detect ctDNA of abdominal tumor with abdominal fluid A previous study that compared mutant allele scores from 30 supernatant samples with those from paired formalin-fixed paraffin-embedded cell blocks reported a variant concordance up to 90% (69), and similar results were detected in both supernatant and FFPE samples in 74% of cases. This suggests that supernatant may serve as a viable alternative to traditional tissue biopsy (69).

Yang et al (70) used high-throughput next generation sequencing (NGS) to analyze cfDNA in 15 pleural, five abdominal and one pericardial effusion; they identified key pathogenic mutations in malignant fluid from 13 patients with metastatic tumors, potentially malignant fluid from two cases and benign fluid from one case. In another study that focused on peritoneal cancer, malignant ascites or peritoneal lavage fluid was collected for microdroplet digital PCR (ddPCR) analysis; peritoneal effusion cfDNA could predict the tumor load of peritoneal cancer and assess patient eligibility for cytoreductive surgery by calculating the MAF (71). Compared with blood, fluids such as pleural and pericardial effusion and ascites exhibit increased sensitivity for ctDNA testing and may serve as predictive biomarkers for responses to EGFR inhibitors (48).

Pancreatic fluid and bile

In pancreaticobiliary tract tumors, obtaining tissue biopsies can be challenging due to the occult nature of the disease. Given their direct contact with tumor tissue, bile and pancreatic fluid are ideal samples for liquid biopsies. Kinugasa et al (72) compared levels of ctDNA in tumor tissue with those in bile from 49 patients with gallbladder cancer; ctDNA isolation from bile was a valuable approach for diagnosing gallbladder cancer. The sensitivity of ctDNA testing (58.3%) was higher compared with that of cytology (45.8%), and there was a high mutation concordance between the two methods. Further study has demonstrated consistent KRAS mutations in ctDNA from bile, plasma and formalin-fixed paraffin-embedded samples from patients with bile duct tumors (39). Notably, only 18.8% of plasma ctDNA samples test positive for KRAS mutations, compared with a detection rate of 48.0% in bile ctDNA (39). Moreover, patients with KRAS mutations detected in bile ctDNA exhibit significantly lower survival rates compared with those with wild-type KRAS (39).

In early-stage pancreatic ductal adenocarcinoma, identification of mutations in plasma ctDNA is often challenging (73). A study in 2023 compared the detection rates of ctDNA sourced from pancreatic fluid with that in plasma; DNA concentrations and the ratios of Alu247/Alu115 were higher in pancreatic fluid compared with plasma, however, there was no difference in the mutation detection rate between pancreas and plasma (74). This limitation may be attributable to the small sample size and influence of enzymes present in pancreatic fluid, which underscores the need for further investigation.

Urine

In 1995, ctDNA was detected in urine (75), marking the beginning of research interest in this non-invasive biomarker. Thus far, two primary sources of ctDNA in urine have been identified, including debris shed directly from tumor cells within the urinary system and CTCs that are filtered through the kidney (76,77). The latter source of ctDNA tends to have a smaller molecular weight, restricted by pore size of the glomerular barrier (76). In 2008, a comparative study examined KRAS mutations in both blood and urine samples from patients with colon cancer; Although the study had a small sample size, mutation rates were similar in both fluids. However, as the sample size increased, mutation rates in urine became significantly higher compared with those in blood (78), highlighting potential of urine as a tumor marker.

A recent prospective multi-center study reported that measurement of DNA methylation in urine effectively differentiates pathological types of bladder cancer and predicts 180-day recurrence-free survival with 100% accuracy (79). This represents a breakthrough in use of urine DNA methylation for differentiation of pathological cancer types. In addition, a study on ctDNA in urine during neoadjuvant chemotherapy for bladder cancer demonstrated that monitoring tumor DNA dynamics in urine, supernatant and plasma predicts treatment response and outcome (80). Urinary ctDNA has also been shown to detect the recurrence of upper urinary tract urothelial carcinoma up to 60 days earlier than cystoscopy (81). Kim et al (82) reported that binding urinary ctDNA improves detection rate of hepatocellular carcinoma from 62 to 92% (82). Increased DNA methylation levels in urine have also been reported in patients with NSCLC.

Urine sampling is a non-invasive procedure that can be performed by non-professionals. Patients can conveniently collect samples at home, which can be sent to testing laboratories. Unlike blood, which is subject to buffering and regulatory mechanisms that may alter its properties (83), urine is excreted and may better reflect bodily abnormality. In addition, urine has a lower presence of contaminating proteins compared with blood, which simplifies DNA extraction processes (76). The ability to test large volumes of urine repeatedly enhances sensitivity of ctDNA detection (84), which makes it a promising tool in cancer diagnostics.

Semen

To the best of our knowledge, prostate-specific antigen is the only well-established tumor marker for prostate cancer. However, its specificity is limited, necessitating exploration of additional diagnostic tools. ctDNA testing has emerged as a valuable adjunct in diagnosis of prostate cancer (85–87). While ctDNA is commonly detected in advanced cancer such as pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, hepatocellular and head and neck cancer and melanoma, it is present in <50% of cases of primary brain, kidney, prostate and thyroid cancer (40).

Semen may serve as a potential sample for prostate cancer diagnostics. Significant differences in cfDNA levels have been observed (83) in semen samples from patients with prostate cancer, individuals with benign prostatic hyperplasia and healthy individuals (88). Patients with prostate cancer exhibit higher concentrations of cfDNA than healthy individuals in semen alongside a distinctive size distribution of cfDNA fragments. Notably, longer cfDNA fragments are significantly more prevalent in semen from patients with prostate cancer compared with those with benign hyperplasia or healthy individuals (69). Size and concentration of cfDNA fragments in semen is associated with tumor burden and treatment response (88). As a direct source of prostate disease-specific molecules, semen represents a promising body fluid for identification of prostate cancer biomarkers and may provide important insight for early diagnosis (Fig. 1) (89).

4. Detection methods

ctDNA provides insight for cancer diagnosis and treatment. To harness the potential of ctDNA, accurate detection methods are key. The main techniques for ctDNA detection include DNA sequencing, PCR-based methods and DNA-based hybridization strategies (90–92). At present there is no universally accepted standard of detection (93).

DNA sequencing

Directed error correction sequencing

Phallen et al (94) developed targeted error correction sequencing (TEC-Seq), which enables ultra-sensitive evaluation of sequence changes in ctDNA through large-scale parallel sequencing. TEC-Seq uses targeted capture of multiple genomic regions combined with deep sequencing of DNA fragments, facilitating the detection of 58 cancer-associated genes spanning 81 kb. A plasma analysis conducted on 44 healthy individuals reported that 16% of asymptomatic individuals exhibit genomic changes associated with clonal hematopoiesis, although none show alterations in driver genes linked to solid tumors (94). CancerSEEK assay (multicancer early detection blood test) demonstrates an accurate tissue-of-origin prediction in 83% of cases, while TEC-Seq yields cancer detection rates of 59–71%, depending on the type of cancer assessed (95). Achieving effective sensitivity in ctDNA analysis using TEC-Seq presents a notable technical challenge (94), which reflects the need for continued refinement of this approach to enhance its applicability in clinical settings.

NGS

Since US Food and Drug Administration (FDA) approval of the Guardant360® CDx (Guardant Health, Inc.) for use of third-generation tyrosine kinase inhibitor osimertinib in patients with NSCLC and EGFR mutations, the NGS approach has rapidly evolved (96). NGS is the preferred liquid biopsy technique for metastatic NSCLC according to the current European Society for Medical Oncology guidelines (97). This high-throughput sequencing method enables comprehensive analysis of DNA and RNA, which allows examination of the entire sequence of target genes and facilitates detection of a broader spectrum of mutations, including previously unknown variants (97,98). NGS can simultaneously sequence multiple genomes on a single platform, even when working with low concentrations of tumor DNA derived from plasma or other liquid biopsy samples (99). NGS can be tailored to analyze a variable number of regions, from a few loci to the entire exome or genome (100). Broadly, NGS targeting ctDNA uses two main approaches. The first approach involves deep sequencing of specific regions containing relevant mutations, which offers high sensitivity and specificity suitable for targeted clinical application. The second approach entails whole-exome or whole-genome sequencing, which can uncover novel genomic changes and is more suitable to basic research (98). NGS is characterized by high throughput, sensitivity and specificity, rendering NGS-based ctDNA mutation spectrum analysis superior to other technologies (98) such as TEC-Seq. However, there are challenges associated with NGS, including the potential for mislocalisation of mutations (97). In addition, this technology requires extensive data analysis, incurs notable costs and has a turnaround time of 7–14 days (101,102).

Targeted amplicon sequencing (TAm-Seq)

TAm-Seq is a labeled amplicon deep sequencing method that integrates efficient library preparation with advanced statistical analysis. This technique enables the sequencing of ~6,000 nucleotides and in-depth analysis (103). A notable implementation of TAm-Seq is InVisionFirst® (Neogenomics Laboratories).

Liquid biopsy platform, which is designed to detect both hotspot mutations and entire coding regions across 35 cancer-associated genes. Leveraging enhanced TAm-Seq techniques, it identifies low-frequency mutations in ctDNA by amplifying highly fragmented DNA (104). Further improvements of Tm-Seq involves optimization of the amplification process by splitting it into two steps. The initial step involves limited cycle pre-amplification using all primer sets to capture the starting molecules present in the template. This is followed by a single amplification step to purify and isolate the target sequence. This refined approach enables detection of cancer mutations in ctDNA at allelic frequencies as low as 2% with sensitivity and specificity of >97% (105). TAm-Seq method demonstrates its utility in clinical settings by routinely detecting ctDNA not only at the time of diagnosis, but also post-treatment (106).

Deep sequencing personalized cancer analysis (CAPP-Seq)

Developed by Newman et al (107), CAPP-Seq is an economical and ultra-sensitive method for quantifying ctDNA. This method integrates a low DNA initiation mass library preparation strategy with a multiphase bioinformatics approach to generate a ‘selector’ of biotinylated DNA oligonucleotides designed to target tumor regions with recurrent mutations. To monitor ctDNA, the selector is initially applied to tumor DNA to identify unique cancer-specific genetic aberrations. Subsequently, it is used on ctDNA to quantify these aberrations (107). Among the various NGS-based ctDNA analysis methods, CAPP-Seq has the lowest background error rate and detection limit (107), demonstrating greater sensitivity compared with TAm-Seq (106). Originally intended for analysis of NSCLC (107), CAPP-Seq has since been successfully applied to a variety of other types of cancer, including esophageal (108) and ovarian cancer (109), mantle cell lymphoma (110), bladder cancer (111), head and neck squamous cell carcinoma (112) and melanoma (113). However, despite its high sensitivity, CAPP-Seq has higher cost compared with TAm-Seq and may not offer advantages for routine screening and surveillance (106).

PCR

Real-time quantitative PCR (qPCR) qPCR can be used for endometrial cancer (114), non-small cell lung cancer (115), colorectal cancer (116). qPCR-based tests have received approval from US FDA and European Medicines Agency for detection, activation or identification of resistance to EGFR targeted therapies in NSCLC (117). Despite its widespread use, the simple nature of these tests may increase the risk of false positives when qPCR is used in isolation (97).

Microdroplet ddRCR

Microdroplet ddPCR, also known as third-generation PCR, uses sample allocation, restricted dilution and statistical data processing based on Poisson distributions to accurately and reliably quantify nucleic acids. This technique divides mixed nucleic acid molecules and PCR solution into small droplets. By using microfluidic loops and surfactant chemistry, sample DNA is randomly assigned to isolated droplets, generating 20,000 droplets. The template is amplified and product is detected based on specific fluorescent labeling (97,118,119). ddPCR is well-suited for studying specific single-gene hotspot mutations that may be found in CSF samples, achieving a limit of detection as low as 0.01%/reaction (119,120). It can measure mutations that constitute 0.01% of a sample (39), offering greater sensitivity compared with qPCR (97). However, compared with NGS, ddPCR has narrower reference range (90.0% of operable mutations) and does not cover certain variants, such as estrogen receptor 1 mutation (121). In addition, ddPCR requires specialized personnel to operate, which adds to overall complexity and operational costs (39,118,122).

Beads, emulsion, amplification, magnetics (BEAMing)

BEAMing is a digital PCR method that enhances the capability of ddPCR by incorporating pre-amplifications of DNA using conventional PCR and target-specific primers (97). PCR products amplified by BEAMing molecules are linked to single magnetic beads and the mutation sites extended via fluorescent probes or primers. By counting fluorescently labelled beads, BEAMing allows the quantitative detection of mutant alleles (119,123). Taniguchi et al (123) used BEAMing to monitor disease progression in patients with lung cancer undergoing EGFR targeted therapy, which effectively determined the proportion of T790M-positive alleles in cancer cells, regardless of potential contamination from normal cell DNA.

Thermal coupling index expansion

An ultra-fast monitoring method known as thermal coupling exponential amplification test has been recently reported (124). This technique combines exponential amplification reaction (EXPAR) with Thermus thermophilus argonaute-coupling), from the thermophilic bacterium Thermus thermophilus, to quickly and accurately detect ctDNA in ~16 min (124). A previous study on tumor threshold changes in mouse models (seven Kirsten rat sarcoma-2 virus (KRAS) point mutations) indicated that this method holds significant potential for monitoring tumor load and evaluating chemotherapy response (121). TtAgo-CEAR assay leverages rapid, specific cleavage function of TtAgo and the high amplification efficiency of EXPAR to identify common hotspot mutations in KRAS (124).

DNA hybridization

Traditional methods for detecting and quantifying ctDNA, such as PCR and NGS, are well-established but have limitations (125–127). These methods are not suitable for detecting short ctDNA fragments (<100 bp). Moreover, they can be costly, require complex instrumentation, time-consuming due to multiple reaction steps and prone to false positives (128). By contrast, hybrid chain reaction is an isothermal, enzyme-free amplification technique that allows indefinite amplification of signals. This method provides advantages for the detection of small molecules and shows potential for ctDNA detection (129). In 2021, researchers successfully employed a hydrogel-based hybrid chain reaction to amplify small amounts of exosomal microRNA from urine samples, achieving a 35-fold increase in detection sensitivity and effectively distinguishing patients with prostate cancer from normal controls (130). A novel device known as the hybrid chain reactor-driven laboratory fiber optic device has been introduced for ultra-fast and sensitive detection of ctDNA in whole blood. This method is time-efficient, straightforward and cost-effective, as it enables real-time monitoring of ctDNA changes (128) and represents a promising direction for advancing detection capability.

5. Clinical role of ctDNA

Screening and management of patients with cancer

ctDNA serves a key role in the screening and early diagnosis of various solid types of tumors, particularly among asymptomatic individuals. Phallen et al (94) demonstrated a strong correlation between plasma somatic mutations and tumor changes in patients with stage I or II colorectal, ovarian and breast cancer. This suggested that ctDNA analysis may be instrumental in both early detection and ongoing disease management. In a study of esophageal adenocarcinoma, baseline ctDNA levels were used to identify patients with locally advanced disease at higher risk of relapse (106). This highlights potential of ctDNA not only as a biomarker for early diagnosis but also as a prognostic tool for tailoring treatment and monitoring disease progression.

Dynamic longitudinal monitoring to evaluate prognosis

The role of ctDNA as a prognostic marker has gained recognition in recent years (131–133). ctDNA is detected in various solid tumors, with its concentration associated with the stage of the disease (134). In a prospective phase II clinical trial, ctDNA was dynamically monitored every three treatment cycles in five patients with solid tumors undergoing immune checkpoint inhibitor (ICI) treatment. The ctDNA levels were associated with tumor status, demonstrating predictive value both at baseline and following treatment (135). The presence of ctDNA following surgery is strongly indicative of tumor recurrence (6). Gale et al (136) demonstrated that ctDNA could identify residual lesions and predict recurrence in patients with NSCLC. Pre- and post-treatment ctDNA testing is shown to identify patients with NSCLC at high risk for recurrence (136). Monitoring ctDNA levels at baseline, during neoadjuvant and adjuvant therapy and after radical therapy allows clinicians to assess drug response and refine treatment regimens. This dynamic longitudinal monitoring ultimately improves prognostic evaluation and informs clinical decision-making, contributing to improved patient management and outcome.

Effectively identifying false advances

Response Evaluation Criteria in Solid Tumors (RECIST1.1) guidelines are key for assessing tumor progression (137). However, they also have limitations, particularly concerning pseudo-progression, which refers to the transient appearance of space-occupying lesions and edema following treatment. This can often mimic disease progression on radiographical imaging but typically resolves or changes after 4–8 weeks of follow-up (138). In clinical practice, clinicians rely on RECIST guidelines to evaluate disease progression and make treatment decisions. However, ctDNA detection can offer earlier and more accurate indication of disease status compared with imaging, potentially reducing follow-up time and offering better guidance for clinical treatment. Similarly, the emerging concept of ‘hyperprogression’ describes a rapid acceleration in tumor growth that can be induced by ICIs (139). To the best of our knowledge, no studies have reported the association between ctDNA and hyperprogressive disease. Future research should explore this association and its implications.

Detecting molecular residual disease (MRD)

Detection of ctDNA provides extensive information and enables analysis of minimal residual lesions. Early detection of residual ctDNA following local radical treatment can indicate MRD and identify patients at higher risk of recurrence or metastasis (140). Due to limited sensitivity of CTC detection, it is rarely used for MRD evaluation (141). Over the past two decades, early detection of MRD in children with acute lymphoblastic leukemia has significantly improved risk stratification, enhanced treatment for high-risk patients and decreased treatment intensity for those at low risk (142). The potential for MRD detection is established in other malignancies, including acute myeloid (143,144) and chronic lymphocytic leukemia (145), NSCLC (146,147), multiple myeloma (148–151), breast cancer (152), melanoma (153), head and neck squamous cell carcinoma (154), follicular lymphoma (155), urothelial carcinoma (156) and colorectal cancer (157).

Guiding treatment escalation and de-escalation

A promising application of ctDNA is its ability to inform decisions regarding treatment escalation and de-escalation (158). Patients with a positive MRD result may be candidates for intensified adjuvant therapy, while those with a negative MRD result may potentially benefit from a reduction in treatment intensity (43). It has been suggested that a key treatment endpoint for colorectal cancer should be complete clearance of ctDNA (43). Currently, a multicenter, prospective, randomized clinical trial is underway to evaluate efficacy of ctDNA-guided adjuvant chemotherapy strategies compared to standard care (158). This aims to assess whether ctDNA-guided treatment adjustments yield superior outcomes in terms of three-year disease-free survival for patients with high-risk stage II and III colorectal cancer (Fig. 2) (158).

6. Limitations

A notable challenge associated with ctDNA-based liquid biopsy is limited detection capability. The mutation abundance of ctDNA is often lower compared with that in localized tumor tissues, and its detectability is influenced by factors including tumor type and load, anatomical location, cellular turnover and disease stage (141). In the context of early cancer detection, ctDNA levels are particularly low, often causing MAF to fall below detection limits of current methods (159). Thus, improving sensitivity of existing detection methods is key. Employing a combination of DNA analysis from liquid biopsy and tissue samples may improve the overall sensitivity and diagnostic accuracy (160).

Discrepancies in results can arise from the diverse standards and interpretations employed by different ctDNA testing methods and laboratories. There is need for the establishment of standardized testing protocols and interpretative guidelines. It is also important to select the appropriate sampling methods, as improper collection of cfDNA from body fluids can lead to missed detection of ctDNA even with appropriate testing methods (100).

Another barrier is the high cost associated with ctDNA detection technologies and equipment, which hampers comprehensive clinical monitoring and may affect treatment decisions. Furthermore, there is currently no evidence to suggest that ctDNA can fully replace traditional pathological testing. Addressing these challenges is key for refining the role of ctDNA in clinical practice.

7. Outlook

ctDNA may serve a key role tumor treatment. Despite existing challenges, ongoing research may advance the utility of ctDNA in clinical practice. As improvements in detection sensitivity, standardization of testing protocols and cost reduction are realized, ctDNA may enhance patient care by guiding treatment decisions, monitoring therapeutic response and improving outcomes.

Acknowledgements

Not applicable.

Availability of data and materials

Not applicable.

Authors' contributions

QG and ZYZ conceived the study. QG, ZYZ, SL, JM and ZZ wrote and reviewed the manuscript. Data authentication is not applicable. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Figure 1. ctDNA samples. In addition to blood samples, ctDNA can be detected in pleural, peritoneal and pericardial effusion, cerebrospinal fluid, saliva and sputum, pancreatic fluid and bile, urine and semen. ct, circulating tumor.

Figure 2. ctDNA in patients with cancer. ctDNA detection may facilitate diagnosis and treatment strategies. ct, circulating tumor.
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References

1 Schoop R Roode LM de Boer LL Dashtbozorg B Framework for deep learning based Multi-Modality image registration of snapshot and pathology images IEEE J Biomed Health Inform Aug 16 2024 (Epub ahead of print) 10.1109/JBHI.2024.3444908 39150810
2 Pritzker K Nieminen HJ Needle biopsy adequacy in the era of precision medicine and value-based health care Arch Pathol Lab Med 143 1399 1415 2019 10.5858/arpa.2018-0463-RA 31100015
3 Nikanjam M Kato S Kurzrock R Liquid biopsy: Current technology and clinical applications J Hematol Oncol 15 131 2022 10.1186/s13045-022-01351-y 36096847
4 Tivey A Church M Rothwell D Dive C Cook N Circulating tumour DNA-looking beyond the blood Nat Rev Clin Oncol 19 600 612 2022 10.1038/s41571-022-00660-y 35915225
5 Alix-Panabieres C Pantel K Liquid biopsy: From discovery to clinical application Cancer Discov 11 858 873 2021 10.1158/2159-8290.CD-20-1311 33811121
6 Cohen SA Liu MC Aleshin A Practical recommendations for using ctDNA in clinical decision making Nature 619 259 268 2023 10.1038/s41586-023-06225-y 37438589
7 Mandel P Metais P Nuclear acids in human blood plasma C R Seances Soc Biol Fil 142 241 243 1948 (In French) 18875018
8 Hobbs KJ Cooper BL Dembek K Sheats MK Investigation of extracted plasma cell-free DNA as a biomarker in foals with sepsis Vet Sci 11 346 2024 39195800
9 Zhang LX Jiang YZ Qiu LJ Huang DP Quantitative detection and integrality analysis of plasma circulating Cell-free DNA in multiple myeloma Zhongguo Shi Yan Xue Ye Xue Za Zhi 32 1106 1111 2024 (In Chinese) 39192405
10 Joshi J Raval A Desai U Upadhyay V Bhavsar M Shah K Rawal R Panchal H Shah F EGFR mutation analysis in Non-small cell lung carcinoma patients: A liquid biopsy approach Indian J Clin Biochem 36 51 58 2021 10.1007/s12291-019-00864-7 33505127
11 Volik S Alcaide M Morin RD Collins C Cell-free DNA (cfDNA): Clinical significance and utility in cancer shaped by emerging technologies Mol Cancer Res 14 898 908 2016 10.1158/1541-7786.MCR-16-0044 27422709
12 Tan EM Schur PH Carr RI Kunkel HG Deoxybonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus J Clin Invest 45 1732 1740 1966 10.1172/JCI105479 4959277
13 Koffler D Agnello V Winchester R Kunkel HG The occurrence of single-stranded DNA in the serum of patients with systemic lupus erythematosus and other diseases J Clin Invest 52 198 204 1973 10.1172/JCI107165 4629907
14 Giacona MB Ruben GC Iczkowski KA Roos TB Porter DM Sorenson GD Cell-free DNA in human blood plasma: Length measurements in patients with pancreatic cancer and healthy controls Pancreas 17 89 97 1998 10.1097/00006676-199807000-00012 9667526
15 Szilagyi M Pos O Marton E Buglyo G Soltesz B Keseru J Penyige A Szemes T Nagy B Circulating cell-free nucleic acids: Main characteristics and clinical application Int J Mol Sci 21 6827 2020 10.3390/ijms21186827 32957662
16 Biro O Fothi A Alasztics B Nagy B Orban TI Rigo JJ Circulating exosomal and Argonaute-bound microRNAs in preeclampsia Gene 692 138 144 2019 10.1016/j.gene.2019.01.012 30659946
17 Fernando MR Jiang C Krzyzanowski GD Ryan WL New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes PLoS One 12 e0183915 2017 10.1371/journal.pone.0183915 28850588
18 Aucamp J Bronkhorst AJ Badenhorst C Pretorius PJ The diverse origins of circulating cell-free DNA in the human body: A critical re-evaluation of the literature Biol Rev Camb Philos Soc 93 1649 1683 2018 10.1111/brv.12413 29654714
19 Moss J Magenheim J Neiman D Zemmour H Loyfer N Korach A Samet Y Maoz M Druid H Arner P Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease Nat Commun 9 5068 2018 10.1038/s41467-018-07466-6 30498206
20 Teo YV Capri M Morsiani C Pizza G Faria A Franceschi C Neretti N Cell-free DNA as a biomarker of aging Aging Cell 18 e12890 2019 10.1111/acel.12890 30575273
21 Hummel EM Hessas E Muller S Beiter T Fisch M Eibl A Wolf OT Giebel B Platen P Kumsta R Moser DA Cell-free DNA release under psychosocial and physical stress conditions Transl Psychiatry 8 236 2018 10.1038/s41398-018-0264-x 30374018
22 Ai B Liu H Huang Y Peng P Circulating cell-free DNA as a prognostic and predictive biomarker in non-small cell lung cancer Oncotarget 7 44583 44595 2016 10.18632/oncotarget.10069 27323821
23 Kim DY Cho EH Kim JS Chie EK Kang HC Plasma Circulating Cell-free DNa in advanced hepatocellular carcinoma patients treated with radiation therapy In Vivo 37 2306 2313 2023 10.21873/invivo.13333 37652507
24 Gianni C Palleschi M Merloni F Di Menna G Sirico M Sarti S Virga A Ulivi P Cecconetto L Mariotti M De Giorgi U Cell-Free DNA Fragmentomics: A promising biomarker for diagnosis, prognosis and prediction of response in breast cancer Int J Mol Sci 23 14197 2022 10.3390/ijms232214197 36430675
25 Al SN Messaoudi SA Babu SR Chaudhary AB Alsharm AA Alrefaei AF Kadasah S Abu-Elmagd M Assidi M Buhmeida A Utility of circulating Cell-free DNA in assessing microsatellite instability and loss of Heterozygosity in breast cancer using human identification approach Genes (Basel) 13 590 2022 10.3390/genes13040590 35456396
26 Bahado-Singh RO Turkoglu O Aydas B Vishweswaraiah S Precision oncology: Artificial intelligence, circulating cell-free DNA, and the minimally invasive detection of pancreatic cancer-A pilot study Cancer Med 12 19644 19655 2023 10.1002/cam4.6604 37787018
27 Lin LH Chang KW Kao SY Cheng HW Liu CJ Increased plasma circulating Cell-Free DNA could be a potential marker for oral cancer Int J Mol Sci 19 3303 2018 10.3390/ijms19113303 30352977
28 Fang Q Yuan Z Hu H Zhang W Wang G Wang X Genome-wide discovery of circulating cell-free DNA methylation biomarkers for colorectal cancer detection Clin Epigenetics 15 119 2023 10.1186/s13148-023-01518-5 37501075
29 Eskander NS Mansour L Abdelaal A Saad E Mohamed D Circulating cell free DNA integrity index as a biomarker for response to chemotherapy in patients with metastatic colorectal carcinoma Asian Pac J Cancer Prev 23 339 348 2022 10.31557/APJCP.2022.23.1.339 35092403
30 Heidrich I Pantel K Liquid biopsy: Blood-based analyses of circulating cell-free DNA in xenografts EMBO Mol Med 14 e16326 2022 10.15252/emmm.202216326 35903952
31 Kroeze A Cornelissen AS Pascutti MF Verheij M Bulder I Klarenbeek S Ait SA Hazenberg MD Nur E van der Schoot CE Cell-free DNA levels are increased in acute graft-versus-host disease Eur J Haematol 109 271 281 2022 10.1111/ejh.13806 35617105
32 Kustanovich A Schwartz R Peretz T Grinshpun A Life and death of circulating cell-free DNA Cancer Biol Ther 20 1057 1067 2019 10.1080/15384047.2019.1598759 30990132
33 Stewart CM Tsui D Circulating cell-free DNA for non-invasive cancer management Cancer Genet 228 229 169 179 2018
34 Yu SC Lee SW Jiang P Leung TY Chan KC Chiu RW Lo YM High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing Clin Chem 59 1228 1237 2013 10.1373/clinchem.2013.203679 23603797
35 Butler TM Spellman PT Gray J Circulating-tumor DNA as an early detection and diagnostic tool Curr Opin Genet Dev 42 14 21 2017 10.1016/j.gde.2016.12.003 28126649
36 Jahr S Hentze H Englisch S Hardt D Fackelmayer FO Hesch RD Knippers R DNA fragments in the blood plasma of cancer patients: Quantitations and evidence for their origin from apoptotic and necrotic cells Cancer Res 61 1659 1665 2001 11245480
37 Miller AM Karajannis MA Current role and future potential of CSF ctDNA for the diagnosis and clinical management of pediatric central nervous system tumors J Natl Compr Canc Netw 20 1363 1369 2022 36509077
38 Diehl F Schmidt K Choti MA Romans K Goodman S Li M Thornton K Agrawal N Sokoll L Szabo SA Circulating mutant DNA to assess tumor dynamics Nat Med 14 985 990 2008 10.1038/nm.1789 18670422
39 Han JY Ahn KS Kim TS Kim YH Cho KB Shin DW Baek WK Suh SI Jang BC Kang KJ Liquid biopsy from Bile-Circulating tumor DNA in patients with biliary tract cancer Cancers (Basel) 13 4581 2021 10.3390/cancers13184581 34572808
40 Bettegowda C Sausen M Leary RJ Kinde I Wang Y Agrawal N Bartlett BR Wang H Luber B Alani RM Detection of circulating tumor DNA in early- and late-stage human malignancies Sci Transl Med 6 224ra24 2014 10.1126/scitranslmed.3007094 24553385
41 Diehl F Li M Dressman D He Y Shen D Szabo S Diaz LJ Goodman SN David KA Juhl H Detection and quantification of mutations in the plasma of patients with colorectal tumors Proc Natl Acad Sci USA 102 16368 16373 2005 10.1073/pnas.0507904102 16258065
42 Dawson SJ Tsui DW Murtaza M Biggs H Rueda OM Chin SF Dunning MJ Gale D Forshew T Mahler-Araujo B Analysis of circulating tumor DNA to monitor metastatic breast cancer N Engl J Med 368 1199 1209 2013 10.1056/NEJMoa1213261 23484797
43 Sato S Nakamura Y Oki E Yoshino T Molecular residual Disease-guided adjuvant treatment in resected colorectal cancer: Focus on CIRCULATE-Japan Clin Colorectal Cancer 22 53 58 2023 10.1016/j.clcc.2022.12.001 36567192
44 Yi K Wang X Filippov SK Zhang H Emerging ctDNA detection strategies in clinical cancer theranostics Smart Med 2 e20230031 2023 10.1002/SMMD.20230031 39188296
45 Cheng F Su L Qian C Circulating tumor DNA: A promising biomarker in the liquid biopsy of cancer Oncotarget 7 48832 48841 2016 10.18632/oncotarget.9453 27223063
46 Lee TH Montalvo L Chrebtow V Busch MP Quantitation of genomic DNA in plasma and serum samples: Higher concentrations of genomic DNA found in serum than in plasma Transfusion 41 276 282 2001 10.1046/j.1537-2995.2001.41020276.x 11239235
47 Heger JM Mattlener J Schneider J Godel P Sieg N Ullrich F Lewis RI Bucaciuc-Mracica T Schwarz RF Ruess D Entirely noninvasive outcome prediction in central nervous system lymphomas using circulating tumor DNA Blood 143 522 534 2023 10.1182/blood.2023022020
48 Werner B Warton K Ford CE Transcending Blood-Opportunities for alternate liquid biopsies in oncology Cancers (Basel) 14 1309 2022 10.3390/cancers14051309 35267615
49 Seyhan AA Circulating liquid biopsy biomarkers in glioblastoma: Advances and challenges Int J Mol Sci 25 7974 2024 10.3390/ijms25147974 39063215
50 Zheng MM Li YS Jiang BY Tu HY Tang WF Yang JJ Zhang XC Ye JY Yan HH Su J Clinical utility of cerebrospinal fluid Cell-Free DNA as liquid biopsy for leptomeningeal metastases in ALK-Rearranged NSCLC J Thorac Oncol 14 924 932 2019 10.1016/j.jtho.2019.01.007 30659989
51 Wu J Liu Z Huang T Wang Y Song MM Song T Long G Zhang X Li X Zhang L Cerebrospinal fluid circulating tumor DNA depicts profiling of brain metastasis in NSCLC Mol Oncol 17 810 824 2023 10.1002/1878-0261.13357 36495130
52 De Mattos-Arruda L Mayor R Ng C Weigelt B Martinez-Ricarte F Torrejon D Oliveira M Arias A Raventos C Tang J Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma Nat Commun 6 8839 2015 10.1038/ncomms9839 26554728
53 Wang Y Luo N Gao Y Wu Y Qin X Qi Y Sun T Tao R Qi C Liu B Yuan S The joint detection of CEA and ctDNA in cerebrospinal fluid: An auxiliary tool for the diagnosis of leptomeningeal metastases in cancer J Cancer Res Clin Oncol 149 1679 1690 2023 10.1007/s00432-022-04053-7 35583828
54 Bai Y Yu Q Liu N Liu J Wang D Liu X Yuan S Case report: Cerebrospinal fluid-derived circulating tumor DNA diagnoses and guides the treatment of a lung adenocarcinoma case with leptomeningeal metastasis Front Oncol 12 944963 2022 10.3389/fonc.2022.944963 36518317
55 van der Wel J Boelens MC Jebbink M Smulders SA Maas KW Luitse M Compter A Boltjes R Sol N Monkhorst K Osimertinib-induced DNA resistance mutations in cerebrospinal fluid of EGFR mutated NSCLC patients developing leptomeningeal metastases: ORA-LM study Neuro Oncol Aug 7 2024 doi: 10.1093/neuonc/noae138 (Epub ahead of print) 10.1093/neuonc/noae138 39110039
56 Azad TD Nanjo S Jin MC Chabon JJ Kurtz DM Chaudhuri AA Connolly ID Hui AB Liu CL Merriott D Quantification of cerebrospinal fluid tumor DNA in lung cancer patients with suspected leptomeningeal carcinomatosis NPJ Precis Oncol 8 121 2024 10.1038/s41698-024-00582-1 38806586
57 Valerius AR Webb MJ Hammad N Sener U Malani R Cerebrospinal fluid liquid biopsies in the evaluation of adult gliomas Curr Oncol Rep 26 377 390 2024 10.1007/s11912-024-01517-6 38488990
58 Dai L Liu Z Zhu Y Ma L Genome-wide methylation analysis of circulating tumor DNA: A new biomarker for recurrent glioblastom Heliyon 9 e14339 2023 10.1016/j.heliyon.2023.e14339 36967887
59 Kojic M Maybury MK Waddell N Koufariotis LT Addala V Millar A Wood S Pearson JV Hansford JR Hassall T Efficient detection and monitoring of pediatric brain malignancies with liquid biopsy based on patient-specific somatic mutation screening Neuro Oncol 25 1507 1517 2023 10.1093/neuonc/noad032 36757207
60 Izquierdo E Proszek P Pericoli G Temelso S Clarke M Carvalho DM Mackay A Marshall LV Carceller F Hargrave D Droplet digital PCR-based detection of circulating tumor DNA from pediatric high grade and diffuse midline glioma patients Neurooncol Adv 3 vdab013 2021 34169282
61 Li J Zhao S Lee M Yin Y Li J Zhou Y Ballester LY Esquenazi Y Dashwood RH Davies P Reliable tumor detection by whole-genome methylation sequencing of cell-free DNA in cerebrospinal fluid of pediatric medulloblastoma Sci Adv 6 eabb5427 2020 10.1126/sciadv.abb5427 33067228
62 Pages M Rotem D Gydush G Reed S Rhoades J Ha G Lo C Fleharty M Duran M Jones R Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine Neuro Oncol 24 1352 1363 2022 10.1093/neuonc/noab299 34984433
63 Ding S Song X Geng X Liu L Ma H Wang X Wei L Xie L Song X Saliva-derived cfDNA is applicable for EGFR mutation detection but not for quantitation analysis in non-small cell lung cancer Thorac Cancer 10 1973 1983 2019 10.1111/1759-7714.13178 31441578
64 Wang Z Zhang L Li L Li X Xu Y Wang M Liang L Jiao P Li Y He S Sputum Cell-Free DNA: Valued surrogate sample for detection of EGFR mutation in patients with advanced lung adenocarcinoma J Mol Diagn 22 934 942 2020 10.1016/j.jmoldx.2020.04.208 32407801
65 Wang Z Li X Zhang L Xu Y Wang M Liang L Jiao P Li Y He S Du J Sputum cell-free DNA: Valued surrogate sample for the detection of EGFR exon 20 p.T790M mutation in patients with advanced lung adenocarcinoma and acquired resistance to EGFR-TKIs Cancer Med 10 3323 3331 2021 10.1002/cam4.3817 33932095
66 Ferrier ST Tsering T Sadeghi N Zeitouni A Burnier JV Blood and saliva-derived ctDNA is a marker of residual disease after treatment and correlates with recurrence in human papillomavirus-associated head and neck cancer Cancer Med 12 15777 15787 2023 10.1002/cam4.6191 37526056
67 Britze TE Jakobsen KK Gronhoj C von Buchwald C A systematic review on the role of biomarkers in liquid biopsies and saliva samples in the monitoring of salivary gland cancer Acta Otolaryngol 143 709 713 2023 10.1080/00016489.2023.2238757 37534452
68 Gupta S Singh B Abhishek R Gupta S Sachan M The emerging role of liquid biopsy in oral squamous cell carcinoma detection: Advantages and challenges Expert Rev Mol Diagn 24 311 331 2024 10.1080/14737159.2024.2340997 38607339
69 Perrone ME Alvarez R Vo TT Chung MW Chhieng DC Paulson VA Colbert BG Q Konnick E Huang EC Validating cell-free DNA from supernatant for molecular diagnostics on cytology specimens Cancer Cytopathol 129 956 965 2021 10.1002/cncy.22491 34265180
70 Yang SR Mooney KL Libiran P Jones CD Joshi R Lau HD Stehr H Berry GJ Zehnder JL Long SR Targeted deep sequencing of cell-free DNA in serous body cavity fluids with malignant, suspicious, and benign cytology Cancer Cytopathol 128 43 56 2020 10.1002/cncy.22205 31751001
71 Leick KM Kazarian AG Rajput M Tomanek-Chalkley A Miller A Shrader HR Mccarthy A Coleman KL Kasi PM Chan C Peritoneal Cell-free tumor DNA as biomarker for peritoneal surface malignancies Ann Surg Oncol 27 5065 5071 2020 10.1245/s10434-020-08832-9 32648179
72 Kinugasa H Nouso K Ako S Dohi C Matsushita H Matsumoto K Kato H Okada H Liquid biopsy of bile for the molecular diagnosis of gallbladder cancer Cancer Biol Ther 19 934 938 2018 10.1080/15384047.2018.1456604 29580156
73 Takai E Totoki Y Nakamura H Morizane C Nara S Hama N Suzuki M Furukawa E Kato M Hayashi H Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer Sci Rep 5 18425 2015 10.1038/srep18425 26669280
74 Levink I Jansen M Azmani Z van Ijcken W van Marion R Peppelenbosch MP Cahen DL Fuhler GM Bruno MJ Mutation analysis of pancreatic juice and plasma for the detection of pancreatic cancer Int J Mol Sci 24 13116 2023 10.3390/ijms24065097 37685923
75 Fitzgerald JM Ramchurren N Rieger K Levesque P Silverman M Libertino JA Summerhayes IC Identification of H-ras mutations in urine sediments complements cytology in the detection of bladder tumors J Natl Cancer Inst 87 129 133 1995 10.1093/jnci/87.2.129 7707384
76 Jain S Lin SY Song W Su YH Urine-based liquid biopsy for nonurological cancers Genet Test Mol Biomarkers 23 277 283 2019 10.1089/gtmb.2018.0189 30986103
77 Su YH Wang M Block TM Landt O Botezatu I Serdyuk O Lichtenstein A Melkonyan H Tomei LD Umansky S Transrenal DNA as a diagnostic tool: Important technical notes Ann N Y Acad Sci 1022 81 89 2004 10.1196/annals.1318.014 15251944
78 Su YH Wang M Brenner DE Norton PA Block TM Detection of mutated K-ras DNA in urine, plasma, and serum of patients with colorectal carcinoma or adenomatous polyps Ann N Y Acad Sci 1137 197 206 2008 10.1196/annals.1448.027 18837947
79 Xiao Y Ju L Qian K Jin W Wang G Zhao Y Jiang W Liu N Wu K Peng M Non-invasive diagnosis and surveillance of bladder cancer with driver and passenger DNA methylation in a prospective cohort study Clin Transl Med 12 e1008 2022 10.1002/ctm2.1008 35968916
80 Christensen E Nordentoft I Birkenkamp-Demtroder K Elbaek SK Lindskrog SV Taber A Andreasen TG Strandgaard T Knudsen M Lamy P Cell-Free urine and plasma DNA mutational analysis predicts neoadjuvant chemotherapy response and outcome in patients with muscle-invasive bladder cancer Clin Cancer Res 29 1582 1591 2023 10.1158/1078-0432.CCR-22-3250 36780195
81 Tamura D Abe M Hiraki H Sasaki N Yashima-Abo A Ikarashi D Kato R Kato Y Maekawa S Kanehira M Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma Cancer Sci 115 529 539 2023 10.1111/cas.16025 38083992
82 Kim AK Hamilton JP Lin SY Chang TT Hann HW Hu CT Lou Y Lin YJ Gade TP Park G Urine DNA biomarkers for hepatocellular carcinoma screening Br J Cancer 126 1432 1438 2022 10.1038/s41416-022-01706-9 35046521
83 Adrogue HJ Madias NE Assessing Acid-base status: Physiologic versus physicochemical approach Am J Kidney Dis 68 793 802 2016 10.1053/j.ajkd.2016.04.023 27590096
84 Dermody SM Bhambhani C Swiecicki PL Brenner JC Tewari M Trans-renal cell-free tumor DNA for Urine-based liquid biopsy of cancer Front Genet 13 879108 2022 10.3389/fgene.2022.879108 35571046
85 Alahdal M Perera RA Moschovas MC Patel V Perera RJ Current advances of liquid biopsies in prostate cancer: Molecular biomarkers Mol Ther Oncolytics 30 27 38 2023 10.1016/j.omto.2023.07.004 37575217
86 Fonseca NM Maurice-Dror C Herberts C Tu W Fan W Murtha AJ Kollmannsberger C Kwan EM Parekh K Schonlau E Prediction of plasma ctDNA fraction and prognostic implications of liquid biopsy in advanced prostate cancer Nat Commun 15 1828 2024 10.1038/s41467-024-45475-w 38418825
87 Tolmeijer SH Boerrigter E Van Erp NP Mehra N Using early on-treatment circulating tumor DNA measurements as response assessment in metastatic castration resistant prostate cancer Oncotarget 15 421 423 2024 10.18632/oncotarget.28599 38953903
88 Ponti G Maccaferri M Manfredini M Micali S Torricelli F Milandri R Del PC Ciarrocchi A Ruini C Benassi L Quick assessment of cell-free DNA in seminal fluid and fragment size for early non-invasive prostate cancer diagnosis Clin Chim Acta 497 76 80 2019 10.1016/j.cca.2019.07.011 31301282
89 Ponti G Maccaferri M Percesepe A Tomasi A Ozben T Liquid biopsy with cell free DNA: New horizons for prostate cancer Crit Rev Clin Lab Sci 58 60 76 2021 10.1080/10408363.2020.1803789 32805148
90 Yu B Ma W Biomarker discovery in hepatocellular carcinoma (HCC) for personalized treatment and enhanced prognosis Cytokine Growth Factor Rev Aug 24 2024 doi: 10.1016/j.cytogfr.2024.08.006 (Epub ahead of print) 10.1016/j.cytogfr.2024.08.006 39191624
91 Zhu L Xu R Yang L Shi W Zhang Y Liu J Li X Zhou J Bing P Minimal residual disease (MRD) detection in solid tumors using circulating tumor DNA: A systematic review Front Genet 14 1172108 2023 10.3389/fgene.2023.1172108 37636270
92 Li S Li H Li X Zhu M Li H Xia F Hybridization Chain Reaction-amplified electrochemical DNA-based sensors enable calibration-free measurements of nucleic acids directly in whole blood Anal Chem 93 8354 8361 2021 10.1021/acs.analchem.1c01436 34061504
93 Ho HY Chung KK Kan CM Wong SC Liquid biopsy in the clinical management of cancers Int J Mol Sci 25 8594 2024 10.3390/ijms25168594 39201281
94 Phallen J Sausen M Adleff V Leal A Hruban C White J Anagnostou V Fiksel J Cristiano S Papp E Direct detection of early-stage cancers using circulating tumor DNA Sci Transl Med 9 eaan2415 2017 10.1126/scitranslmed.aan2415 28814544
95 Bittla P Kaur S Sojitra V Zahra A Hutchinson J Folawemi O Khan S Exploring Circulating tumor DNA (CtDNA) and its role in early detection of cancer: A systematic review Cureus 15 e45784 2023 37745752
96 Yu W Hurley J Roberts D Chakrabortty SK Enderle D Noerholm M Breakefield XO Skog JK Exosome-based liquid biopsies in cancer: Opportunities and challenges Ann Oncol 32 466 477 2021 10.1016/j.annonc.2021.01.074 33548389
97 Kemper M Krekeler C Menck K Lenz G Evers G Schulze AB Bleckmann A Liquid Biopsies in Lung Cancer Cancers (Basel) 15 1430 2023 10.3390/cancers15051430 36900221
98 Lin C Liu X Zheng B Ke R Tzeng CM Liquid biopsy, ctDNA diagnosis through NGS Life (Basel) 11 890 2021 34575039
99 Fernandes M Cruz-Martins N Souto MC Guimaraes S Pereira RJ Justino A Pina MJ Magalhaes A Queiroga H Machado JC Clinical application of Next-generation sequencing of plasma Cell-free DNA for genotyping untreated advanced Non-small cell lung cancer Cancers (Basel) 13 2707 2021 10.3390/cancers13112707 34070940
100 Roberto TM Jorge MA Francisco GV Noelia T Pilar RG Andres C Strategies for improving detection of circulating tumor DNA using next generation sequencing Cancer Treat Rev 119 102595 2023 10.1016/j.ctrv.2023.102595 37390697
101 Grada A Weinbrecht K Next-generation sequencing: Methodology and application J Invest Dermatol 133 e11 2013 10.1038/jid.2013.248 23856935
102 Cheng ML Pectasides E Hanna GJ Parsons HA Choudhury AD Oxnard GR Circulating tumor DNA in advanced solid tumors: Clinical relevance and future directions CA Cancer J Clin 71 176 190 2021 10.3322/caac.21650 33165928
103 Ma M Zhu H Zhang C Sun X Gao X Chen G ‘Liquid biopsy’-ctDNA detection with great potential and challenges Ann Transl Med 3 235 2015 26539452
104 Gale D Lawson A Howarth K Madi M Durham B Smalley S Calaway J Blais S Jones G Clark J Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA PLoS One 13 e0194630 2018 10.1371/journal.pone.0194630 29547634
105 Forshew T Murtaza M Parkinson C Gale D Tsui DW Kaper F Dawson SJ Piskorz AM Jimenez-Linan M Bentley D Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA Sci Transl Med 4 136ra68 2012 10.1126/scitranslmed.3003726 22649089
106 Cabalag CS Yates M Corrales MB Yeh P Wong SQ Zhang BZ Fujihara KM Chong L Hii MW Dawson SJ Potential clinical utility of a targeted circulating tumor DNA Assay in esophageal adenocarcinoma Ann Surg 276 e120 e126 2022 10.1097/SLA.0000000000005177 35737908
107 Newman AM Bratman SV To J Wynne JF Eclov NC Modlin LA Liu CL Neal JW Wakelee HA Merritt RE An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage Nat Med 20 548 554 2014 10.1038/nm.3519 24705333
108 Azad TD Chaudhuri AA Fang P Qiao Y Esfahani MS Chabon JJ Hamilton EG Yang YD Lovejoy A Newman AM Circulating tumor DNA analysis for detection of minimal residual disease after chemoradiotherapy for localized esophageal cancer Gastroenterology 158 494 505 2020 10.1053/j.gastro.2019.10.039 31711920
109 Noguchi T Sakai K Iwahashi N Matsuda K Matsukawa H Yahata T Toujima S Nishio K Ino K Changes in the gene mutation profiles of circulating tumor DNA detected using CAPP-Seq in neoadjuvant chemotherapy-treated advanced ovarian cancer Oncol Lett 19 2713 2720 2020 32218822
110 Jung D Jain P Yao Y Wang M Advances in the assessment of minimal residual disease in mantle cell lymphoma J Hematol Oncol 13 127 2020 10.1186/s13045-020-00961-8 32972438
111 Satyal U Srivastava A Abbosh PH Urine biopsy-liquid gold for molecular detection and surveillance of bladder cancer Front Oncol 9 1266 2019 10.3389/fonc.2019.01266 31803629
112 Taylor K Zou J Magalhaes M Oliva M Spreafico A Hansen AR Mcdade SS Coyle VM Lawler M Elimova E Circulating tumour DNA kinetics in recurrent/metastatic head and neck squamous cell cancer patients Eur J Cancer 188 29 38 2023 10.1016/j.ejca.2023.04.014 37182343
113 Aoude LG Brosda S Ng J Lonie JM Belle CJ Patel K Koufariotis LT Wood S Atkinson V Smithers BM Circulating tumor DNA: A promising biomarker for predicting recurrence in patients with BRAF-Negative melanoma J Mol Diagn 25 771 781 2023 10.1016/j.jmoldx.2023.06.014 37544359
114 Grassi T Harris FR Smadbeck JB Murphy SJ Block MS Multinu F Schaefer KJ Zhang P Karagouga G Liu MC Personalized tumor-specific DNA junctions to detect circulating tumor in patients with endometrial cancer PLoS One 16 e0252390 2021 10.1371/journal.pone.0252390 34111149
115 Mansson CT Vad-Nielsen J Meldgaard P Nielsen AL Sorensen BS EGFR transcription in non-small-cell lung cancer tumours can be revealed in ctDNA by cell-free chromatin immunoprecipitation (cfChIP) Mol Oncol 15 2868 2876 2021 10.1002/1878-0261.13093 34453867
116 Jafri H Mushtaq S Baig S Bhatty A Siraj S Comparison of KRAS gene in circulating tumor DNA levels vs histological grading of colorectal cancer patients through liquid biopsy Saudi J Gastroenterol 29 371 375 2023 10.4103/sjg.sjg_85_23 37602638
117 FDA Summary of Safety and Effectiveness Data (SSED) P150047. Cobas EGFR Mutation Test v2® 2016
118 Biglari N Soltani-Zangbar MS Mohammadian J Mehdizadeh A Abbasi K ctDNA as a novel and promising approach for cancer diagnosis: A focus on hepatocellular carcinoma EXCLI J 22 752 780 2023 37720239
119 Hickman RA Miller AM Arcila ME Cerebrospinal fluid: A unique source of circulating tumor DNA with broad clinical applications Transl Oncol 33 101688 2023 10.1016/j.tranon.2023.101688 37196447
120 Rimelen V Ahle G Pencreach E Zinniger N Debliquis A Zalmai L Harzallah I Hurstel R Alamome I Lamy F Tumor cell-free DNA detection in CSF for primary CNS lymphoma diagnosis Acta Neuropathol Commun 7 43 2019 10.1186/s40478-019-0692-8 30885253
121 Venetis K Pepe F Pescia C Cursano G Criscitiello C Frascarelli C Mane E Russo G Taurelli SB Troncone G ESR1 mutations in HR+/HER2-metastatic breast cancer: Enhancing the accuracy of ctDNA testing Cancer Treat Rev 121 102642 2023 10.1016/j.ctrv.2023.102642 37864956
122 Teh SY Lin R Hung LH Lee AP Droplet microfluidics Lab Chip 8 198 220 2008 10.1039/b715524g 18231657
123 Taniguchi K Uchida J Nishino K Kumagai T Okuyama T Okami J Higashiyama M Kodama K Imamura F Kato K Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas Clin Cancer Res 17 7808 7815 2011 10.1158/1078-0432.CCR-11-1712 21976538
124 Fang J Yuan C Luo X He Z Fu W A Thermus thermophilus argonaute-coupling exponential amplification assay for ultrarapid analysis of circulating tumor DNA Talanta 266 125034 2024 10.1016/j.talanta.2023.125034 37597338
125 Cappello F Angerilli V Munari G Ceccon C Sabbadin M Pagni F Fusco N Malapelle U Fassan M FFPE-Based NGS approaches into clinical practice: The limits of glory from a pathologist viewpoint J Pers Med 12 102642 2022 10.3390/jpm12050750
126 Mantilla WA Sanabria-Salas MC Baldion AM Sua LF Gonzalez DM Lema M NGS in lung, breast, and unknown primary cancer in colombia: A multidisciplinary consensus on challenges and opportunities JCO Glob Oncol 7 1012 1023 2021 10.1200/GO.21.00046 34185572
127 Lin YH Liao XJ Chang W Chiou CC Ultrafast DNA amplification using microchannel Flow-through PCR device Biosensors (Basel) 12 303 2022 10.3390/bios12050303 35624604
128 Xu J Han X Xu W Liu J Zhu A Song D Long F Development of a hybridization chain reaction-powered lab-on-fiber device for ultrafast point-of-care testing of circulating tuor DNA in whole blood Talanta 259 124475 2023 10.1016/j.talanta.2023.124475 37004394
129 Wu J Lv J Zheng X Wu ZS Hybridization chain reaction and its applications in biosensing Talanta 234 122637 2021 10.1016/j.talanta.2021.122637 34364446
130 Kim J Shim JS Han BH Kim HJ Park J Cho IJ Kang SG Kang JY Bong KW Choi N Hydrogel-based hybridization chain reaction (HCR) for detection of urinary exosomal miRNAs as a diagnostic tool of prostate cancer Biosens Bioelectron 192 113504 2021 10.1016/j.bios.2021.113504 34298498
131 Papakonstantinou A Gonzalez NS Pimentel I Sunol A Zamora E Ortiz C Espinosa-Bravo M Peg V Vivancos A Saura C Prognostic value of ctDNA detection in patients with early breast cancer undergoing neoadjuvant therapy: A systematic review and meta-analysis Cancer Treat Rev 104 102362 2022 10.1016/j.ctrv.2022.102362 35219090
132 Wang D Zhao P Lu T Ren J Zhu L Han X Zhang G Dong X Ma H Yu M Cai H ctDNA as a prognostic biomarker in resectable CLM: Systematic review and meta-analysis Open Life Sci 18 20220615 2023 10.1515/biol-2022-0615 37250841
133 Wei J Feng J Weng Y Xu Z Jin Y Wang P Cui X Ruan P Luo R Li N Peng M The prognostic value of ctDNA and bTMB on immune checkpoint inhibitors in human cancer Front Oncol 11 706910 2021 10.3389/fonc.2021.706910 34660274
134 Markou A Tzanikou E Lianidou E The potential of liquid biopsy in the management of cancer patients Semin Cancer Biol 84 69 79 2022 10.1016/j.semcancer.2022.03.013 35331850
135 Bratman SV Yang S Iafolla M Liu Z Hansen AR Bedard PL Lheureux S Spreafico A Razak AA Shchegrova S Personalized circulating tumor DNA analysis as a predictive biomarker in solid tumor patients treated with pembrolizumab Nat Cancer 1 873 881 2020 10.1038/s43018-020-0096-5 35121950
136 Gale D Heider K Ruiz-Valdepenas A Hackinger S Perry M Marsico G Rundell V Wulff J Sharma G Knock H Residual ctDNA after treatment predicts early relapse in patients with early-stage non-small cell lung cancer Ann Oncol 33 500 510 2022 10.1016/j.annonc.2022.02.007 35306155
137 Borcoman E Kanjanapan Y Champiat S Kato S Servois V Kurzrock R Goel S Bedard P Le Tourneau C Novel patterns of response under immunotherapy Ann Oncol 30 385 396 2019 10.1093/annonc/mdz003 30657859
138 Young JS Al-Adli N Scotford K Cha S Berger MS Pseudoprogression versus true progression in glioblastoma: What neurosurgeons need to know J Neurosurg 139 748 759 2023 10.3171/2022.12.JNS222173 36790010
139 Zheng J Zhou X Fu Y Chen Q Advances in the study of hyperprogression of different tumors treated with PD-1/PD-L1 antibody and the mechanisms of its occurrence Cancers (Basel) 15 1314 2023 10.3390/cancers15041314 36831655
140 Vellanki PJ Ghosh S Pathak A Fusco MJ Bloomquist EW Tang S Singh H Philip R Pazdur R Beaver JA Regulatory implications of ctDNA in Immuno-oncology for solid tumors J Immunother Cancer 11 e005344 2023 10.1136/jitc-2022-005344 36796877
141 Mahuron KM Fong Y Applications of liquid biopsy for surgical patients with cancer: A review JAMA Surg 159 96 103 2024 10.1001/jamasurg.2023.5394 37910091
142 Juarez-Avendano G Mendez-Ramirez N Luna-Silva NC Gomez-Almaguer D Pelayo R Balandran JC Molecular and cellular markers for measurable residual disease in acute lymphoblastic leukemia Bol Med Hosp Infant Mex 78 159 170 2021 34167145
143 Li Y Solis-Ruiz J Yang F Long N Tong CH Lacbawan FL Racke FK Press RD NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia Blood Cancer J 13 59 2023 10.1038/s41408-023-00833-7 37088803
144 Gutman JA Winters A Kent A Amaya M Mcmahon C Smith C Jordan CT Stevens B Minhajuddin M Pei S Higher-dose venetoclax with measurable residual disease-guided azacitidine discontinuation in newly diagnosed acute myeloid leukemia Haematologica 108 2616 2625 2023 10.3324/haematol.2023.282681 37051756
145 Munir T Cairns DA Bloor A Allsup D Cwynarski K Pettitt A Paneesha S Fox CP Eyre TA Forconi F Chronic lymphocytic leukemia therapy guided by measurable residual disease N Engl J Med 390 326 337 2024 10.1056/NEJMoa2310063 38078508
146 Zhang JT Liu SY Gao W Liu SM Yan HH Ji L Chen Y Gong Y Lu HL Lin JT Longitudinal undetectable molecular residual disease defines potentially cured population in localized non-small cell lung cancer Cancer Discov 12 1690 1701 2022 10.1158/2159-8290.CD-21-1486 35543554
147 Jung HA Ku BM Kim YJ Park S Sun JM Lee SH Ahn JS Cho JH Kim HK Choi YS Longitudinal monitoring of circulating tumor DNA from plasma in patients with curative resected Stages I to IIIA EGFR-Mutant Non-Small cell lung cancer J Thorac Oncol 18 1199 1208 2023 10.1016/j.jtho.2023.05.027 37308037
148 Costa LJ Chhabra S Medvedova E Dholaria BR Schmidt TM Godby KN Silbermann R Dhakal B Bal S Giri S Daratumumab, Carfilzomib, Lenalidomide, and dexamethasone with minimal residual disease Response-Adapted therapy in newly diagnosed multiple myeloma J Clin Oncol 40 2901 2912 2022 10.1200/JCO.21.01935 34898239
149 San-Miguel J Avet-Loiseau H Paiva B Kumar S Dimopoulos MA Facon T Mateos MV Touzeau C Jakubowiak A Usmani SZ Sustained minimal residual disease negativity in newly diagnosed multiple myeloma and the impact of daratumumab in MAIA and ALCYONE Blood 139 492 501 2022 10.1182/blood.2020010439 34269818
150 Costa LJ Chhabra S Medvedova E Dholaria BR Schmidt TM Godby KN Silbermann R Dhakal B Bal S Giri S Minimal residual disease response-adapted therapy in newly diagnosed multiple myeloma (MASTER): Final report of the multicentre, single-arm, phase 2 trial Lancet Haematol 10 e890 e901 2023 10.1016/S2352-3026(23)00236-3 37776872
151 D'Agostino M Bertuglia G Rota-Scalabrini D Belotti A More S Corradini P Oliva S Ledda A Grasso M Pavone V Predictors of unsustained minimal residual disease negativity in multiple myeloma (MM) Patients Blood 143 2023 doi:10.1182/blood.2023022080
152 Medford AJ Moy B Spring LM Hurvitz SA Turner NC Bardia A Molecular residual disease in breast cancer: Detection and therapeutic interception Clin Cancer Res 29 4540 4548 2023 10.1158/1078-0432.CCR-23-0757 37477704
153 Patel RP Somasundram PM Smith LK Sheppard KE Mcarthur GA The therapeutic potential of targeting minimal residual disease in melanoma Clin Transl Med 13 e1197 2023 10.1002/ctm2.1197 36967556
154 Honore N van Marcke C Galot R Helaers R Ambroise J van Maanen A Mendola A Dahou H Marbaix E Van Eeckhout P Tumor-agnostic plasma assay for circulating tumor DNA detects minimal residual disease and predicts outcome in locally advanced squamous cell carcinoma of the head and neck Ann Oncol 34 1175 1186 2023 10.1016/j.annonc.2023.09.2004 37879442
155 Pott C Jurinovic V Trotman J Kehden B Unterhalt M Herold M Jagt RV Janssens A Kneba M Mayer J Minimal residual disease status predicts outcome in patients with previously untreated follicular lymphoma: A prospective analysis of the Phase III GALLIUM study J Clin Oncol 42 550 561 2024 10.1200/JCO.23.00838 38096461
156 Yang K Hu H Wu J Wang H Guo Z Yu W Yao L Ding F Zhou T Wang W Letter to the Editor: Clinical utility of urine DNA for noninvasive detection and minimal residual disease monitoring in urothelial carcinoma Mol Cancer 22 25 2023 10.1158/1538-7445.AM2023-25 36739413
157 Mo S Ye L Wang D Han L Zhou S Wang H Dai W Wang Y Luo W Wang R Early detection of molecular residual disease and risk stratification for stage I to III colorectal cancer via circulating tumor DNA Methylation JAMA Oncol 9 770 778 2023 10.1001/jamaoncol.2023.0425 37079312
158 Slater S Bryant A Chen HC Begum R Rana I Aresu M Peckitt C Zhitkov O Lazaro-Alcausi R Borja V ctDNA guided adjuvant chemotherapy versus standard of care adjuvant chemotherapy after curative surgery in patients with high risk stage II or stage III colorectal cancer: A multi-centre, prospective, randomised control trial (TRACC Part C) BMC Cancer 23 257 2023 10.1186/s12885-023-10699-4 36941575
159 Armakolas A Kotsari M Koskinas J Liquid biopsies, novel approaches and future directions Cancers (Basel) 15 1579 2023 10.3390/cancers15051579 36900369
160 Xie J Yao W Chen L Zhu W Liu Q Geng G Fang J Zhao Y Xiao L Huang Z Zhao J Plasma ctDNA increases tissue NGS-based detection of therapeutically targetable mutations in lung cancers BMC Cancer 23 294 2023 10.1186/s12885-023-10674-z 37004022
