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Publications > Journals > Cancer Screening and Prevention> Article Full Text

  • Review Article
  • OPEN ACCESS

Liquid Biopsy: A Breakthrough Technology in Early Cancer Screening

  • Xuexin Liang,
  • Qingqing Tang,
  • Jiawei Chen and
  • Yanghui Wei* 
 Author information 

Abstract

Cancer is a leading cause of morbidity and mortality worldwide. Because many cancers are asymptomatic or produce nonspecific symptoms at an early stage, timely detection remains difficult. Liquid biopsy, a revolutionary approach in oncology, provides a minimally invasive, real-time method for cancer detection, monitoring, and characterization by examining circulating tumor components in body fluids. However, its clinical application in early cancer detection remains limited by the low abundance of tumor-derived analytes, variable assay performance, and insufficient prospective validation. This review aims to summarize current liquid biopsy technologies and their clinical applications, with particular emphasis on their potential value in early cancer detection. Liquid biopsy enables molecular profiling by analyzing circulating cell-free DNA, circulating tumor DNA, circulating tumor cells, exosomes, and other tumor-associated biomarkers in blood and other body fluids. Advances in detection technologies have facilitated the identification of somatic mutations, epigenetic alterations, and other molecular features associated with tumor development. Current evidence indicates that liquid biopsy may complement established diagnostic modalities by providing minimally invasive molecular information and permitting repeated assessment. Nevertheless, limited sensitivity in early-stage disease, inter-assay variability, and insufficient prospective validation remain major barriers to its use as a stand-alone population-screening test.

Keywords

Liquid biopsy, Cancer diagnosis, Circulating tumor cells, Cell-free DNA, Precision oncology, Minimally invasive techniques, Early cancer detection

Introduction

Cancer is a leading cause of death worldwide. According to Global Cancer Statistics 2022, approximately 20.0 million new cancer cases and 9.7 million cancer deaths occurred worldwide in 2022. The age-standardized incidence and mortality rates were 196.9 and 91.7 per 100,000, respectively.1 Global cancer incidence is projected to reach approximately 35 million cases by 2050, indicating a substantial increase in the global cancer burden. According to the 2024 cancer incidence and mortality estimates for the United States, approximately 2,001,140 new cancer cases and 611,720 cancer deaths were expected to occur.2 Given the high incidence and mortality rates, cancer imposes a substantial public health burden, ranking as the first or second leading cause of death in most countries. Early detection plays a crucial role in reducing cancer-related morbidity and mortality. However, due to the subtle early symptoms of malignant tumors and limited public awareness of early screening, many cancers are diagnosed at advanced stages. This delayed diagnosis often results in missed opportunities for timely intervention, leading to poorer prognoses. Effective early screening strategies enable the identification of suspicious lesions and precancerous conditions, significantly improving patient outcomes.3 For instance, lung cancer screening with low-dose computed tomography has been shown to reduce lung cancer mortality by 20% compared to conventional chest X-rays.4–6 Despite the benefits, current screening modalities vary in sensitivity, specificity, accessibility, and cost-effectiveness, underscoring the need for innovative approaches to enhance early cancer detection.7

Liquid biopsy is a transformative, non-invasive diagnostic approach that examines circulating tumor components in bodily fluids. Initially introduced for detecting circulating tumor cells (CTCs), the concept has expanded to include circulating tumor DNA (ctDNA) and other tumor-associated biomarkers.8 Compared to traditional tissue biopsy, which is invasive and may fail to capture tumor heterogeneity, liquid biopsy offers a safer, repeatable, and convenient alternative. It enables dynamic monitoring of tumor evolution, assessment of treatment response, and early identification of resistance mechanisms. Biomarkers detected in biological fluids such as blood, urine, saliva, and cerebrospinal fluid provide valuable insights into tumor biology, addressing some limitations of conventional diagnostic approaches (Fig. 1).9,10

Sources of human biofluids and their clinical applications: The diagram illustrates the collection sites and types of various biofluids (<italic>e.g.</italic>, blood, urine, saliva, pleural effusion, cerebrospinal fluid, etc.) from different parts of the human body. These samples provide critical insights for precision medicine and personalized therapy. By Figdraw. CSF, cerebrospinal fluid.
Fig. 1  Sources of human biofluids and their clinical applications: The diagram illustrates the collection sites and types of various biofluids (e.g., blood, urine, saliva, pleural effusion, cerebrospinal fluid, etc.) from different parts of the human body. These samples provide critical insights for precision medicine and personalized therapy. By Figdraw. CSF, cerebrospinal fluid.

This review aims to summarize current liquid biopsy technologies and their clinical applications, with particular emphasis on their potential value in early cancer detection. It focuses on the major circulating analytes, analytical approaches, and representative applications in lung, breast, colorectal, prostate, and gastric cancers. The principal challenges to clinical implementation and future priorities for improving assay performance, standardization, and integration with established diagnostic modalities are also considered.

Liquid biopsy components and analytical techniques

Components of liquid biopsy

Circulating cell-free DNA (cfDNA) and ctDNA

Circulating cfDNA refers to short DNA fragments present in bodily fluids, such as blood, primarily released from cells through biological processes such as apoptosis, necrosis, or active secretion.11,12 These fragments exist freely in plasma or serum, unencapsulated within cells or membranous vesicles. In healthy individuals, cfDNA is present at very low concentrations in the bloodstream (<10 ng/mL), with a modal fragment size of approximately 167 bp.13 In contrast, in cancer patients, the concentration of cfDNA is highly variable and often includes ctDNA, a subset of cfDNA derived from tumor cells. Notably, ctDNA fragments demonstrate distinct biophysical profiles, averaging 143 bp in length, significantly shorter than their non-malignant counterparts, and remain virtually undetectable in healthy populations.11,14

cfDNA is a promising source of biomarkers for early cancer detection, as it can be obtained non-invasively from blood, urine, or other body fluids and provides genetic (e.g., mutations) and epigenetic (e.g., methylation) information. These characteristics may provide information on tumor burden, tissue of origin, and dynamic changes in tumor biology.10 The short half-life of ctDNA in circulation enables repeated assessment of tumor dynamics.7Table 1 compares cfDNA and ctDNA in key characteristics and clinical significance.

Table 1

Comparison of cfDNA and ctDNA

CharacteristiccfDNActDNA
SourceDerived from normal cells (blood cells, healthy tissues, etc.) and tumor cellsSpecifically derived from tumor cells
ProportionTotal pool of extracellular DNA fragments in circulation; most molecules are generally derived from non-tumor cells.Tumor-derived fraction of cfDNA; its abundance varies by cancer type, disease stage, tumor burden, and assay sensitivity.
LengthFragment length is mainly around 167 bpFragment length is shorter, around 143 bp
SpecificityNot inherently tumor-specific; tumor-derived alterations may be detectable when ctDNA is present in the cfDNA pool.May contain tumor-associated genetic and epigenetic alterations.
Clinical significanceWidely used for monitoring various diseasesFocused on tumor diagnosis, classification, and treatment response monitoring

cfDNA, cell-free DNA; ctDNA, circulating tumor DNA.

CTCs

CTCs are tumor cells that detach from the primary tumor and enter the bloodstream, serving as key mediators in the metastatic cascade.15 Experimental evidence indicates that tumor cells may disseminate as early as the initial stages of tumor development.16 While the majority of CTCs survive in circulation for only 1–2.5 h before being eliminated by the immune system, a small subset evades immune surveillance and seeds secondary metastatic sites.17

Traditional high-throughput sequencing of tumor tissues, despite providing insights into overall genomic characteristics, is constrained by its reliance on bulk sample analysis. This limitation obscures the heterogeneity of tumor cells and dilutes the genomic information of low-abundance but biologically critical cell populations, such as CTCs and cancer stem cells, thereby limiting the resolution of genetic analyses. In contrast, CTCs exhibit pronounced heterogeneity, encompassing epithelial-type cells and mesenchymal-type cells that have undergone epithelial-to-mesenchymal transition. These cells demonstrate invasive capacities, stem-cell-like properties, and a dynamic ability to transition between epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition.15 Molecular characterization of CTCs is typically achieved through markers such as epithelial markers (e.g., EpCAM), mesenchymal markers (e.g., N-cadherin), and proliferation markers (e.g., Ki-67). Single-cell sequencing technologies have emerged as powerful tools for delineating the genomic, transcriptomic, and epigenomic variations in CTCs across peripheral blood, primary tumors, metastatic lesions, and metastatic lymph nodes. By mitigating the confounding effects of tumor heterogeneity, these approaches provide unprecedented insights into the mechanisms underlying tumor initiation, progression, and metastasis.18–20

Exosomes

Exosomes are lipid bilayer vesicles with a diameter ranging from 40 to 160 nm, actively released by most cells and stably circulating in body fluids. Increasing evidence indicates that various bioactive molecules, including nucleic acids, proteins, and lipids, are enriched in exosomes and can be transferred from donor cells to recipient cells, resulting in intercellular signaling.21–23 The bioactive substances in exosomes can be taken up by recipient cells, promoting tumor initiation and progression. Compared to cfDNA and CTCs, exosomes offer several advantages in liquid biopsy applications. First, their abundance in bodily fluids facilitates isolation, whereas only a small number of CTCs are typically present in blood.24 Second, exosomes may complement ctDNA because they are actively secreted by viable cells and contain diverse nucleic acids, proteins, and lipids.24 Moreover, exosomes exhibit inherent stability due to their lipid bilayer, allowing them to remain stable under appropriate physiological and storage conditions.25

However, the application of exosomes in liquid biopsy faces several challenges. Tumor-derived exosomes constitute only a small fraction of circulating extracellular vesicles, and their nanoscale size and heterogeneity make specific isolation and analysis challenging.26–28 Despite progress in exosome isolation and molecular analysis, limitations in sensitivity, specificity, purity, and throughput remain.

Analytical techniques

As shown in Figure 2, liquid biopsy primarily relies on samples such as cfDNA, ctDNA, CTCs, and exosomes, processed through steps of detection, enrichment, identification, and analysis. With technological advancements, enrichment methods have been continuously optimized, offering greater potential for liquid biopsy in non-invasive cancer screening.

Overview of liquid biopsy techniques and applications.
Fig. 2  Overview of liquid biopsy techniques and applications.

By Figdraw. cfDNA, cell-free DNA; CTC, circulating tumor cell; ctDNA, circulating tumor DNA; ELISA, enzyme-linked immunosorbent assay; ISET, isolation by size of epithelial tumor cells; PCR, polymerase chain reaction.

Analytical techniques for cfDNA/ctDNA

The analysis of ctDNA has historically relied on polymerase chain reaction (PCR)-based assays or next-generation sequencing (NGS) combined with specialized bioinformatics tools. Digital PCR, initially proposed by Kinzler and Vogelstein, introduced the concept of single-molecule amplification, enabling the evaluation of rare mutant alleles amidst a background of abundant wild-type alleles.29 The first high-throughput digital PCR method, known as BEAMing (i.e., beads, emulsions, amplification, and magnetics), integrated PCR with flow cytometry, facilitating highly sensitive detection of known mutations.30 This method demonstrated strong concordance with tissue biopsy results and successfully validated the utility of digital PCR in detecting ctDNA from metastatic diseases and monitoring therapeutic responses. Numerous studies have established the analytical and clinical validity of droplet digital PCR.31 However, droplet digital PCR is currently limited to analyzing one potential mutation per reaction, necessitating large quantities of cfDNA for the analysis of multiple mutations. Compared with NGS, digital PCR provides a relatively rapid and straightforward approach for targeted analysis of known variants, although its multiplexing capacity is more limited.32

The maturation of NGS technologies has introduced orders-of-magnitude higher multiplexing capabilities for cfDNA analysis. With the advent of molecular barcoding for individual DNA molecules and novel bioinformatics pipelines, NGS has evolved to overcome limitations associated with PCR amplification or NGS-induced errors, achieving limits of detection comparable to or even exceeding those of digital PCR. NGS provides a deeper and more comprehensive analytical approach to identifying genetic alterations.33 Unlike digital PCR, which uses molecular probes to query known mutations, NGS enables the relatively unbiased discovery of genetic perturbations, as sequencing inherently identifies all base pairs in a given DNA molecule. However, the inherent nature of strand synthesis and PCR amplification makes NGS prone to sequencing errors. Despite these limitations, the ability of NGS to detect a wide range of genetic changes with high multiplexing potential underscores its value in cfDNA-based diagnostics.

Additionally, methylation alterations are closely associated with tumorigenesis. These changes often persist in a stable manner and can occur earlier than genomic mutations.34 cfDNA also reflects tissue-specific methylation signatures, providing a means to identify the tissue of origin.12,35 Numerous studies have focused on developing high-performance multi-cancer early detection (MCED) tests utilizing cfDNA methylation patterns.

To capture these methylation patterns, various methods based on distinct technologies have been developed to selectively enrich genome-wide CpG regions, followed by high-throughput NGS. For example, cell-free methylated DNA immunoprecipitation and sequencing enables the detection of methylation patterns from small amounts of cfDNA.36 This approach employs immunoprecipitation with antibodies specifically recognizing methylated cytosine residues to enrich methylated DNA fragments. Cell-free methylated DNA immunoprecipitation and sequencing has been effectively used to differentiate plasma samples from pancreatic cancer, lung cancer, and acute myeloid leukemia from those of other cancer types.

In addition to genome-wide methylation sequencing, targeted methylation sequencing has emerged as a powerful and more commonly used approach for MCED tests, showing remarkable results. These methods typically use a set of DNA probes or primers to capture methylation patterns of target genomic regions via NGS. The target regions are often selected based on comparisons between non-cancer samples and samples from each type of target cancer. Consequently, the panel size can be highly flexible, allowing for cost-effective deep sequencing depending on the number of target regions.12 Ample research has demonstrated the accuracy and sensitivity of targeted methylation sequencing. Klein et al.37 showcased a highly targeted methylation detection method capable of detecting over 50 types of cancer and pinpointing their tissue of origin. Overall, targeted methylation sequencing represents a promising approach for MCED, offering high sensitivity and specificity while maintaining cost-effectiveness and flexibility.

CTC isolation and analysis

The extremely low proportion of CTCs in the blood presents a significant challenge for accurately isolating these cells from the vast number of blood cells. CTCs can be enriched and captured based on either physical or biological properties. Physical methods leverage differences between CTCs and blood cells in size, density, deformability, and electrical properties. For example, the Oncoquick system utilizes density gradients to separate red and white blood cells, while the Apostream system employs dielectrophoresis in microfluidic chambers to isolate CTCs.38,39 These approaches are cost-effective and preserve cell viability but are generally limited by low efficiency, poor purity, and lack of specificity. Furthermore, they may fail to capture CTCs that share similar physical characteristics with white blood cells. In contrast, biological property-based methods rely on antibody-antigen interactions. Some negative-enrichment platforms deplete CD45-positive white blood cells to enrich unlabeled CTCs.40 In contrast, the CellSearch system uses immunomagnetic EpCAM-based enrichment and identifies CTCs as cytokeratin-positive, DAPI-positive, and CD45-negative cells.41 However, the high heterogeneity of CTC surface antigens can result in under-detection of EpCAM-low CTCs, leading to inaccuracies. Physical property-based methods, by comparison, are unaffected by such antigen variability.

Microfluidics and nanotechnology have further enhanced CTC sorting capabilities. For example, CTC-chip technology isolates viable CTCs from whole blood without pre-labeling or extensive sample processing, improving cell viability and purity.15,42 Advances such as the NP-HB CTC-Chip, which combines herringbone microfluidics with gold nanoparticles for chemical ligand exchange reactions, allow for efficient isolation and safe release of viable CTCs for further analysis.43 Despite their high capture efficiency and cell viability, these microfluidic platforms face barriers to clinical implementation due to high initial costs, lengthy setup times, bulky instrumentation, and limited capacity for single-cell molecular analysis.

CTC detection methods include fluorescence microscopy, fluorescence spectrophotometry, flow cytometry, surface-enhanced Raman scattering, and electrical impedance. Morphological analysis using immunocytochemistry (hereinafter referred to as ICC) with antibodies against cytokeratins is a widely applied approach for qualitative and quantitative CTC analysis.44 To address the labor-intensive nature of conventional immunofluorescence, automated technologies such as laser scanning cytometers have been developed to screen highly enriched CTCs more efficiently. Automated high-definition image analysis platforms have been applied to the detection and characterization of CTCs in several cancer types.45–47

PCR-based methods offer higher sensitivity and specificity for CTC detection. RT-qPCR, for instance, can specifically target tumor-related genes while excluding non-cancerous blood cells. This method can detect extremely low concentrations of CTCs, such as one CTC among more than 106 leukocytes. By extracting total RNA from CTCs, RT-qPCR amplifies tumor-specific gene sequences, such as cancer-associated markers (EpCAM, mucin1, ERBB2), EMT-related transcription factors (Twist1, Snail, PI3Kα, Akt-2), and stem cell markers (CD34, CD133, ALDH1).48

The isolated and enriched CTCs are valuable for downstream analyses, including genomics, transcriptomics, proteomics, and cell culture. These approaches enable in-depth investigations into the molecular characteristics and biological functions of CTCs, contributing to advances in precision medicine, cancer progression studies, and therapeutic strategies.

Application of liquid biopsies in early cancer diagnosis

Many solid tumors are classified from localized to advanced stages, commonly using stages I–IV, with stage 0 used for selected in situ lesions. Compared to late-stage cancers (III/IV), early-stage cancers (I/II) are associated with lower treatment costs and better prognosis.49 The majority of patients diagnosed with stage I cancer (approximately 70%) undergo surgery as part of their treatment. When feasible, surgery offers patients with stage I cancer the best chance of cure and may cause fewer side effects than chemotherapy or radiotherapy.10 Therefore, early cancer detection significantly reduces treatment costs and improves patients’ expected survival and quality of life. However, due to the subtle nature of early cancer symptoms and the difficulty of detecting them with conventional diagnostic methods, the rate of early cancer diagnosis remains low. Current liquid biopsy assays still have limited sensitivity in some early-detection settings, particularly for stage I cancers, and their specificity requires further validation in real-world screening populations.50 As illustrated in Figure 3, various types of cancer release biomarkers such as RNAs, DNA, CTCs, and exosomes into the bloodstream, which can be utilized for liquid biopsy.

Liquid biopsy workflow in cancer detection and analysis.
Fig. 3  Liquid biopsy workflow in cancer detection and analysis.

The image illustrates the process of liquid biopsy for cancers such as lung, breast, colorectal, prostate, and gastric cancers. Tumor-derived biomarkers, including RNAs, DNA, exosomes, and circulating tumor cells, are released from tissue lesions into the bloodstream. These biomarkers are collected through blood sampling, followed by advanced testing and analysis to aid in cancer diagnosis, monitoring, and personalized treatment. By Figdraw. CTC, circulating tumor cell.

In recent years, many countries have implemented cancer screening programs for asymptomatic high-risk populations, based on risk factors such as age, gender, family history, and epidemiological data, with the aim of reducing mortality and incidence. For example, mammography is widely used for population-based breast cancer screening, while ultrasonography is generally used as an adjunct in selected populations.51 For lung cancer, low-dose computed tomography screening has significantly reduced mortality rates.52 For gastric cancer, endoscopy with histopathological confirmation is central to diagnosis.53 For individuals at high risk of hepatocellular carcinoma, surveillance commonly uses ultrasonography, with or without alpha-fetoprotein testing.54 A prospective study has also evaluated a blood-based CTC assay for the detection of colorectal adenomas and CRC.55 However, established cancer-specific screening and diagnostic modalities vary in cost, accessibility, invasiveness, radiation exposure, and diagnostic performance.10 Multi-cancer tests may complement existing cancer-specific screening programs by detecting multiple cancer types in a single test.10,35 Further prospective evaluation is needed before their routine use in population screening.10 In symptomatic patients with nonspecific symptoms and a low initial suspicion of cancer, blood-based tests may also be evaluated as diagnostic triage tools; this use should be distinguished from population screening of asymptomatic individuals.10

Tissue biopsy is considered the “gold standard” for tumor profiling in cancer diagnosis, and in most cases, tissue biopsy is required to determine the specific type of cancer.10,56 Depending on the tumor location and clinical context, tissue may be obtained by needle or core biopsy, endoscopic biopsy, incisional biopsy, or excisional biopsy.10 Open surgical biopsy can provide adequate tissue but carries increased risks of infection and bleeding. Moreover, a single tissue specimen may not fully capture the spatial and temporal heterogeneity of tumors or their clonal evolution.56 This limitation reduces its utility for repeated tumor monitoring.

Liquid biopsy involves analyzing tumor-related biomarkers circulating in body fluids, such as blood. Early-stage tumors are often small, meaning that the levels of tumor-related biomarkers released into the circulation may be very low. As a result, biomarkers detected at this stage may not necessarily be derived directly from cancer cells, whereas systemic, non-tumor-derived markers may be more abundant.57,58 Liquid biopsy can be performed relatively rapidly and can provide genomic, proteomic, and metabolomic information. Pan-omics approaches combining tumor-derived and non-tumor-derived information may improve early cancer detection.59,60 Additionally, liquid biopsy is generally less invasive and easier to repeat than tissue biopsy, although its cost and analytical reliability vary across assays and clinical settings.49,61 Unlike formalin-fixed tissue specimens, liquid biopsy avoids fixation-related artifacts, but its results may still be affected by pre-analytical factors such as sample collection, processing time, and storage conditions.33 These factors should be considered when interpreting liquid biopsy results and comparing findings across studies. Table 2 summarizes liquid biopsy-based analytical techniques applied to various cancers and associated biofluids.

Table 2

Summary of reported liquid biopsy-based analytical techniques in various cancers

CancerBiofluidAnalytePotential analytical techniques
LungPlasma; Pleural fluidEVs; CTCs; ctDNA; miRNAPCR; qRT-PCR; ARMS-PCR; CAPP-Seq; EFIRM; NGS; Methylation-specific PCR (MSP); ISET; CellSearch; Nano-quantum dots microarray
BreastPlasma; SalivactDNA; CTCs; cfmiRNABEAMing; ddPCR; qRT-PCR; TEC-Seq; Personalized ultradeep sequencing; Large NGS panels; Nanotube CTC chip; CTC-iChip; Gene-expression microarray
ColorectalPlasma; Serum; SalivactDNA; CTCs; EV RNA; EV proteins; TEPsCellMax (CMx) platform; CellSearch; Safe-SeqS; NGS; BEAMing; ddPCR; qPCR; ALU-qPCR; Electrochemical sensing
ProstatePlasma; SerumctDNA; CTCs; EVs; ctRNA; ucfRNAqPCR; Bisulfite PCR; qRT-PCR; ELISA
GastricPlasma; Gastric juice; Saliva; UrinectDNA; CTCs; EVs; ctRNA; miRNA; TEPsddPCR; NGS; MSP; cfMeDIP-seq; RNA-seq

ALU-qPCR, ALU quantitative polymerase chain reaction; ARMS-PCR, amplification refractory mutation system polymerase chain reaction; BEAMing, beads, emulsion, amplification, and magnetics; CAPP-Seq, cancer personalized profiling by deep sequencing; cfMeDIP-seq, cell-free methylated DNA immunoprecipitation sequencing; cfmiRNA, cell-free microRNA; CMx, CellMax; CTC, circulating tumor cell; CTC-iChip, circulating tumor cell iChip; ctDNA, circulating tumor DNA; ctRNA, circulating tumor RNA; ddPCR, droplet digital polymerase chain reaction; EFIRM, electric field-induced release and measurement; ELISA, enzyme-linked immunosorbent assay; EV, extracellular vesicle; ISET, isolation by size of tumor cells; miRNA, microRNA; MSP, methylation-specific polymerase chain reaction; NGS, next-generation sequencing; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RNA-seq, RNA sequencing; Safe-SeqS, Safe-Sequencing System; TEC-Seq, targeted error correction sequencing; TEP, tumor-educated platelet; ucfRNA, urinary cell-free RNA.

Lung cancer

Lung cancer was the most frequently diagnosed cancer and the leading cause of cancer death worldwide in 2022, accounting for 12.4% of new cancer cases and 18.7% of cancer deaths.1

Liquid biopsy has shown significant progress in the clinical application and technological research of CTCs in lung cancer. CTCs were first described by Ashworth in 1869.62 In a case series of patients with early-stage lung adenocarcinoma, CTC counts increased significantly in all seven patients with tumor progression, and postoperative CTC levels or changes were higher in patients with tumor progression than in those with disease-free survival.63 NGS analysis of CTCs from patients with early-stage lung cancer revealed that more than 50% harbored mutations in four commonly affected genes, namely, NOTCH1, IGF2, EGFR, and PTCH1.64 In the same study, liquid chromatography-mass spectrometry analysis identified 100 differential metabolites, among which 10 showed potential clinical value for the diagnosis of CTC-positive early-stage lung cancer.64 These findings suggest that NGS analysis of CTCs and serum metabolomics may provide candidate biomarkers for the early diagnosis of lung cancer. Furthermore, in an exploratory study of 168 patients with chronic obstructive pulmonary disease without clinically detectable lung cancer, CTCs were detected in five patients. Annual low-dose computed tomography surveillance subsequently identified lung nodules 1–4 years after CTC detection in all five patients, leading to histopathological diagnosis of early-stage lung cancer.65 However, these findings were based on a very small CTC-positive subgroup and require validation in larger prospective studies.

Regarding plasma circulating DNA, one study demonstrated that cfDNA levels in patients with resectable non-small cell lung cancer (NSCLC) were significantly higher than those in patients with chronic respiratory inflammation and healthy controls.66 In a CAPP-Seq study, ctDNA was detected in plasma samples from 100% of patients with stage II–IV NSCLC and 50% of patients with stage I NSCLC.67 These findings highlight the reduced sensitivity of ctDNA detection in stage I disease and the need for more sensitive or multimodal detection approaches. In the same study, ctDNA levels were highly correlated with tumor volume, distinguished residual disease from treatment-related imaging changes, and enabled earlier assessment of treatment response than radiographic approaches.67

Beyond CTCs and ctDNA, exosome-derived microRNAs (miRNAs) can be analyzed to differentiate between lung adenocarcinoma and pulmonary granuloma patients. miRNA analysis can also be used to distinguish lung cancer patients from healthy individuals. Further research has confirmed that plasma exosomes containing miR-30e-3p, miR-30a-3p, miR-181-5p, and miR-361-5p are specific diagnostic biomarkers for adenocarcinoma, while those containing miR-15b-5p, miR-10b-5p, and miR-320b can serve as specific diagnostic biomarkers for squamous cell carcinoma.68

Despite these advances, challenges remain, including the lack of standardized detection methods, limited sensitivity in early-stage lung cancer, and the need for validation in large prospective studies. Clinical studies have evaluated ctDNA analysis for molecular profiling and treatment monitoring in lung cancer, although further evidence is required before its broad clinical implementation.5

Breast cancer (BC)

BC is the most commonly diagnosed cancer among women worldwide. In 2022, female breast cancer accounted for approximately 11.6% of all new cancer cases and 6.9% of cancer deaths globally.1

To date, biomarkers based on cfDNA, ctDNA, CTCs, and miRNA have been described in numerous studies. Some studies have identified cfDNA as an early detection biomarker for BC based on the analysis of DNA damage and DNA methylation changes. Li et al.69 were the first to assess the methylation status of the EGFR and PPM1E promoters in plasma using next-generation bisulfite sequencing. Their study found significantly higher methylation levels in patients with BC than in healthy controls, highlighting the potential of cfDNA methylation as an early detection biomarker.69 In addition to cfDNA methylation, circulating miRNA profiles have also shown potential for the early detection of BC.70

The detection of CTCs as a non-invasive biomarker for the early diagnosis of BC has yielded promising results. Kruspe et al.71 developed a rapid, highly sensitive diagnostic method for detecting CTCs based on nuclease-activated probe technology, which can distinguish patients with breast cancer from healthy controls using blood samples.

Among circulating miRNA approaches, Shimomura et al.70 evaluated the expression profiles of miRNAs in the serum of BC patients and healthy women. A combination of five miRNAs (miR-1246, miR-1307-3p, miR-4634, miR-6861-5p, and miR-6875-5p) was found to be helpful in detecting BC (with a sensitivity of 97.3%, specificity of 82.9%, and accuracy of 89.7%) and individuals with early-stage BC (with a sensitivity of 98.0% for in situ carcinoma).

While liquid biopsy technologies for BC are promising, challenges remain in terms of sensitivity and specificity. For example, the detection of CTCs in early-stage BC patients remains difficult, and more research is needed to enhance the sensitivity of these methods.72 Current research suggests that the use of liquid biopsy, in conjunction with positron emission tomography/computed tomography, is not an alternative but rather a complementary analytical approach for diagnosing various types of malignant tumors, locations, and stages of disease. However, further studies are needed to assess the clinical utility, risks, and cost-effectiveness of these tests.73

Colorectal cancer (CRC)

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide, accounting for approximately 9.6% of new cancer cases and 9.3% of cancer deaths in 2022.1 Early detection of CRC is crucial, as early-stage CRC is associated with substantially better survival than advanced disease.

Tsai et al.55 demonstrated the potential utility of CTCs for the early detection of colorectal neoplasia. In their prospective study, the CellMax platform detected CRC with an overall sensitivity of 95.2% and an area under the receiver operating characteristic curve of 0.940; the specificity among participants with a normal colonoscopy was 82.1%.55 The sensitivity was 89.2%, 97.9%, 95.7%, and 97.4% for stage I, II, III, and IV CRC, respectively, and CTC counts were significantly associated with increasing disease severity.55 However, the diagnostic performance of CTC assays varies substantially across detection platforms and study populations. Further standardization and prospective validation are therefore required before CTC-based assays can be routinely used for early CRC detection.74

cfDNA is an emerging potential biomarker for guiding early CRC screening. In a multicenter cohort study, 88.5% of patients with stages I–III CRC tested positive for ctDNA.75 Wu et al.76 identified a novel cfDNA methylation model based on 11 methylation biomarkers to improve the detection of early CRC. Additionally, methylation markers like EYA4, GRIA4, and ITGA4 in metastatic CRC patients have shown promising results for monitoring tumor burden and treatment efficacy.77 Moreover, promoter hypermethylation of SEPT9 in cfDNA has been investigated as a blood-based biomarker for CRC detection.78–80

Because exosomal miRNAs are detectable in circulation and may show CRC-associated expression patterns, they have been investigated as candidate biomarkers for CRC detection and prognosis. Serum exosomal miR-19a and miR-92a, members of the miR-17-92 cluster, were upregulated in patients with both early- and advanced-stage CRC, and high exosomal miR-19a expression was associated with recurrence and poorer prognosis.81 Furthermore, Wang et al.82 reported that miR-125a-3p and miR-320c were highly upregulated in plasma exosomes from patients with early-stage colon cancer.

Despite the promising potential of liquid biopsy in CRC, challenges such as variability in detection sensitivity, particularly for early-stage cancer, remain. Standardization of methods and larger clinical validation studies are essential to improve reliability and accuracy. Combining liquid biopsy with other diagnostic techniques, like colonoscopy, may offer a more comprehensive approach for early detection, with future advancements focusing on enhancing sensitivity and minimizing false negatives, especially in high-risk populations.

Prostate cancer (PCa)

PCa is a prevalent malignant tumor and the second most commonly diagnosed cancer among men worldwide, accounting for approximately 7.3% of all new cancer cases and 4.1% of cancer deaths.1

DNA methylation-based liquid biopsy has demonstrated significant diagnostic potential in early-stage prostate cancer detection, offering a non-invasive approach for identifying epigenetic alterations associated with tumorigenesis. Studies have indicated that hypermethylation at specific CpG sites of RARB2 and GSTP1 can be utilized for the diagnosis of PCa.83 Additionally, the analysis of miRNA expression profiles has increasingly been employed for the early diagnosis of PCa. Mitchell et al.84 were the first to confirm the presence of miRNAs in the plasma of PCa patients. In 2018, Liu et al.85 analyzed serum samples from two active-surveillance cohorts and developed a three-miRNA score comprising miR-24, miR-223, and miR-375 to predict disease reclassification. The three-miRNA score combined with PSA may help predict disease reclassification among patients with low-risk PCa undergoing active surveillance.85,86 As for CTCs, the ISET-CTC-ICC method has been found to have a positive predictive value of 99% and a negative predictive value of 97% in studies. However, the rarity of CTCs in the bloodstream limits their use for diagnosis.87

In addition to blood, urine is also considered a suitable source for liquid biopsies in the early diagnosis of prostate cancer.88 From urine samples, various analytes can be isolated and detected, among which cfDNA/RNA, CTCs, and extracellular vesicles contribute to the clinical diagnosis and treatment of patients with urogenital system malignancies. A retrospective study by Casadio et al.89 demonstrated that the integrity of urine DNA could distinguish between PCa patients and healthy individuals with an accuracy of approximately 80%. However, Salvi et al.90 reported that urinary cell-free DNA integrity showed lower sensitivity and specificity than PSA in their study.

Despite the potential of liquid biopsy in prostate cancer detection, several obstacles remain, including the limited sensitivity of ctDNA and the low abundance of CTCs, which complicate early diagnosis. Urinary biomarkers, while promising, are also still under investigation and need further validation for clinical use. However, combining liquid biopsy with established methods like PSA testing and imaging could enhance early detection and ongoing monitoring of PCa. Future research should aim at improving sensitivity, particularly for detecting early-stage tumors and assessing treatment response.

Gastric cancer

Gastric cancer is one of the most common cancers worldwide. In 2022, approximately 968,000 new cases and 660,000 deaths occurred, accounting for 4.9% of all new cancer cases and 6.8% of cancer deaths.1 Early detection plays a crucial role in improving prognosis, as late-stage gastric cancer is generally associated with poor survival.

CTCs have shown potential in the diagnosis of gastric cancer, but their sensitivity needs improvement.91 In one study, CTCs were isolated from 7.5-mL blood samples obtained from 116 patients with gastric cancer and 31 healthy controls using a centrifugal microfluidic system. Using a threshold of at least two CTCs per 7.5 mL, the assay achieved a sensitivity of 85.3%, specificity of 90.3%, positive predictive value of 97.1%, and negative predictive value of 62.2% for distinguishing patients with gastric cancer from healthy controls.92 A recent study indicated that combining protein tyrosine kinase 7 (PTK7) with epithelial cell adhesion molecule (EpCAM) improved the capture of heterogeneous gastric cancer cells. The dual-targeting approach was also applied to peripheral blood samples from patients with gastric cancer.93

Regarding cfDNA, Kim et al.94 reported that plasma cfDNA levels were higher in patients with gastric cancer than in healthy controls and decreased significantly after surgical resection. Zhong et al.95 found, through receiver operating characteristic analysis, that cfDNA had greater diagnostic performance than conventional biomarkers such as CA19-9, CA125, and alpha-fetoprotein in patients with advanced gastric cancer. Methylated cfDNA and ctDNA are significant research topics. A genome-wide methylation analysis based on 1,781 gastrointestinal tumor and adjacent normal tissue methylation profiles was conducted, followed by validation with 300 cfDNA samples. The results suggested that gastrointestinal cancers can be distinguished by differentially methylated regions obtained from blood samples.96 Additionally, studies have shown that promoter methylation of tumor suppressor genes in circulating cfDNA, including PCDH10, RASSF1A, RUNX3, and RPRM, occurs more frequently in patients with gastric cancer than in non-cancer controls, indicating that these alterations may serve as potential diagnostic biomarkers.97,98

While liquid biopsy shows promise for gastric cancer detection, challenges such as low CTC sensitivity, especially in early-stage disease, and the need for further validation of cfDNA remain. Additionally, cfDNA methylation studies require more extensive clinical validation before becoming reliable biomarkers for routine screening. Combining liquid biopsy with imaging techniques like endoscopy or CT scans could improve early detection. Future research should focus on enhancing sensitivity, particularly for early-stage gastric cancer, and confirming the clinical utility of cfDNA- and CTC-based biomarkers.

Clinical translation, challenges, and future perspectives

Liquid biopsy has emerged as a promising tool in early cancer diagnosis, offering a minimally invasive alternative to traditional diagnostic methods. By analyzing cfDNA, ctDNA, CTCs, and miRNAs, liquid biopsy permits repeated molecular assessment and may capture aspects of tumor heterogeneity. These features support its evaluation as a complementary approach for early cancer detection. Selected registered studies evaluating liquid biopsy for cancer detection and monitoring are summarized in Table 3.

Table 3

Summary of selected registered studies evaluating liquid biopsy for cancer detection and monitoring

Study descriptionCancer typeSampleStudy typeAnalyteTrial identifier
Fluid biopsy for the diagnosis of lung cancerLung cancerBloodObservationalCTCNCT04162678
Liquid biopsy in lung cancerLung cancerBloodObservationalCTC; ctDNANCT03479099
A study to compare tissue and liquid Biopsies in people with different types of cancerNSCLC; CRC; PDACBloodInterventionalctDNANCT05708599
Liquid biopsy for early non-small lung cancer detectionNSCLCBloodObservationalctDNANCT05462795
Assessment of early-detection based on liquid biopsy in lung cancerLung cancerBloodObservationalctDNA; cfDNA; CTCNCT04817046
Early breast cancer detection based on liquid biopsies and micrornasBreast cancerPlasmaObservationalMicroRNANCT06439940
Exploring a breast cancer early screening model Based on cfDNABreast cancerPlasmaObservationalcfDNANCT06016790
Evaluation of circulating tumor cells (CTC) Relevance in breast cancer follow-up using the ScreenCell device (PROBE-CTC)Breast cancerBloodInterventionalCTCNCT06807502
Early detection of five common cancers using the ctDNA analysing testBreast cancer; Liver cancer; Gastric cancer; Colorectal cancer; Lung cancerBloodObservationalctDNANCT05227261
Serial circulating tumor DNA (ctDNA) monitoring during adjuvant capecitabine in early triple-negative breast cancerBreast cancerBloodInterventionalctDNANCT04768426
Early onset colorectal cancer detectionColorectal cancerBloodObservationalMicroRNANCT06342401
Exploratory study of a novel based rbcDNA liquid biopsy technique for colorectal cancer early detectionColorectal cancerBlood; StoolObservationalrbcDNANCT05875584
Assessment of early-detection based on liquid biopsy in hepatobiliary cancer malignanciesHepatobiliary cancerSerumObservationalctDNA; RNANCT04835675
Assessment of early-detection based on liquid biopsy in gastric cancerGastric cancerSerumObservationalctDNA; cfDNANCT05224596
Stomach cancer exosome-based detectionGastric cancerSerumObservationalmiRNA; cfDNANCT06342427
Early-stage detection of liver, biliary tract and pancreatic cancersLiver Cancer; Biliary Tract Cancer; Pancreatic CancerSerumObservationalcfDNA; microRNANCT06139042

cfDNA, cell-free DNA; CRC, colorectal carcinoma; CTC, circulating tumor cells; ctDNA, circulating tumor DNA; NSCLC, non-small cell lung cancer; PDAC, pancreatic ductal adenocarcinoma; rbcDNA, red blood cell-derived circulating DNA.

However, the journey from a promising diagnostic tool to standard clinical practice is fraught with challenges. The sensitivity and specificity of liquid biopsy assays vary across different cancer types and stages, necessitating further optimization to ensure reliability. For instance, the rarity of CTCs in the bloodstream, as observed in gastric cancer, and the heterogeneity of ctDNA present significant hurdles that require innovative solutions. In our view, these challenges are not insurmountable but will require significant innovation in both technology and understanding of tumor biology. For example, advancements in amplification technologies and improved methods for profiling ctDNA could potentially overcome some of these limitations. Still, addressing the rarity of CTCs may require a more tailored approach, such as focusing on specific subtypes or utilizing multi-modal approaches to increase detection rates.

The field is also in dire need of standardized protocols for sample collection, processing, and analysis to ensure the reproducibility and comparability of results across different studies and clinical settings. This is a critical issue that could hinder the broader adoption of liquid biopsy in clinical practice. From our perspective, establishing standard operating procedures for liquid biopsy assays is essential for ensuring consistency and reliability. Furthermore, collaborations between academic researchers, clinicians, and industry partners will be crucial in developing these standards. The cost-effectiveness of liquid biopsy in the context of early cancer detection also warrants thorough evaluation, particularly in light of the potential for widespread screening programs.

It is important to highlight that liquid biopsy demonstrates substantial potential in precision oncology, not only serving as a tool for early cancer diagnosis but also providing critical value in prognostic prediction, personalized therapeutic guidance, and recurrence monitoring. Continuous advancements in detection technologies have significantly enhanced the analytical accuracy of biomarkers such as cfDNA and CTCs, thereby improving the clinical reliability of liquid biopsy. Further research is warranted to address existing challenges and optimize methodologies, which will ultimately advance the development of precision medicine.

Looking ahead, the integration of liquid biopsy with existing diagnostic modalities, such as imaging and tissue biopsy, could enhance the accuracy and comprehensiveness of cancer diagnosis. Future research should focus on the discovery of novel biomarkers, the development of more sensitive and specific assays, and the conduct of large-scale clinical trials to validate the clinical utility of liquid biopsy.

This narrative review did not use a prespecified systematic search or formal quality-assessment process, and relevant studies may therefore have been missed. The included evidence also varied substantially in cancer type, disease stage, analyte, assay platform, and outcome definition, limiting direct comparison of reported diagnostic performance. In addition, the review focused on selected cancers and representative technologies rather than providing an exhaustive assessment of all liquid biopsy applications.

Conclusions

Liquid biopsy shows substantial promise as a minimally invasive adjunct for early cancer detection and molecular characterization. It permits repeated sampling and may capture aspects of spatial and temporal tumor heterogeneity. However, limited sensitivity in early-stage disease, variation among analytical platforms, and insufficient prospective evidence remain major barriers to routine population screening. Future studies should prioritize assay standardization, clinically relevant prospective validation, and careful integration with established imaging, endoscopic, and tissue-based diagnostic pathways.

Declarations

Acknowledgement

None.

Funding

No funding was received for the preparation of this review.

Conflict of interest

The authors declare no conflicts of interest related to this manuscript.

Authors’ contributions

Conceptualized the review topic, defined research questions, designed the framework, and wrote the main body of the manuscript (XL); assisted with literature retrieval and synthesis, supplemented critical references, and prepared the figures and tables (QT, JC); and critically revised and edited the final version (YW). All authors approved the final manuscript.

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Liang X, Tang Q, Chen J, Wei Y. Liquid Biopsy: A Breakthrough Technology in Early Cancer Screening. Cancer Screen Prev. 2025;4(1):40-52. doi: 10.14218/CSP.2024.00031.
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Article History
Received Revised Accepted Published
December 30, 2024 February 19, 2025 March 12, 2025 March 25, 2025
DOI http://dx.doi.org/10.14218/CSP.2024.00031
  • Cancer Screening and Prevention
  • pISSN 2993-6314
  • eISSN 2835-3315
  • © 2026 Xia & He Publishing Inc.
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Liquid Biopsy: A Breakthrough Technology in Early Cancer Screening

Xuexin Liang, Qingqing Tang, Jiawei Chen, Yanghui Wei
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