Liquid biopsies in colorectal cancer screening and diagnosis

Abstract

Colorectal cancer (CRC) remains a leading cause of global cancer mortality, underscoring the urgent need for effective early detection. Current screening methods, primarily colonoscopy, are limited by invasiveness and low population adherence, while traditional serological biomarkers lack sufficient sensitivity and specificity for reliable early-stage diagnosis. This review examines recent advances in liquid biopsy components as promising noninvasive biomarkers for CRC screening and diagnosis. Key circulating analytes are discussed, including circulating tumor cells, circulating tumor DNA, tumor-educated platelets, exosomes, and circulating RNAs. These markers collectively provide a systemic, real-time molecular profile of tumors, offering valuable information on tumor presence, stage, and biological characteristics beyond that achieved by traditional single-marker approaches. Integrating multiple biomarkers into diagnostic panels shows promise for significantly improving detection accuracy compared to conventional biomarkers like carcinoembryonic antigen. However, translating this potential into clinical practice faces substantial challenges, including the standardization of techniques, validation in large prospective cohorts, and demonstration of improved patient outcomes through interventional studies. Addressing these challenges is essential for realizing the transformative potential of liquid biopsies in personalized CRC management. By enabling earlier, less invasive, and more-accurate diagnosis, liquid biopsy represents a paradigm shift in screening that could ultimately reduce CRC mortality through timely intervention.

Key Words: Colorectal cancer; Liquid biopsies; Screening; Diagnosis; Biomarkers

Core Tip: Liquid biopsy is revolutionizing colorectal cancer management by enabling noninvasive, real-time tumor profiling through analysis of circulating biomarkers. Emerging evidence supports the clinical utility of multi-analyte panels integrating circulating tumor DNA, circulating tumor cells, tumor-educated platelets, and exosomal RNAs for early detection and molecular characterization, potentially overcoming the limitations of traditional methods and guiding personalized treatment strategies.

INTRODUCTION

Colorectal cancer (CRC) is the third most common malignancy worldwide and the second leading cause of cancer death[1]. The incidence and mortality in all regions of the world continue to rise in people > 50 years old. The morbidity and mortality in males are higher than those in females[2]. In 2022, global CRC incidence and mortality were estimated at 1.926 million new cases and 904000 deaths. China accounted for 27% of both incidence (517000 cases) and mortality (240000 deaths)[3]. China’s age-standardized incidence rate (20.1 per 100000) was lower than that of the United States (27.0 per 100000) and the United Kingdom (30.9 per 100000). However, China’s mortality rate (8.6 per 100000) exceeded that of the United States (7.9 per 100000)[3]. These data clearly reveal the severity of the global burden of CRC and its significant heterogeneity across regions with varying levels of development. Screening is one of the most effective measures to reduce the incidence and mortality rates of CRC. Its prevalence is closely associated with long-term trends in incidence[4].

Multiple screening methods are currently recommended for early detection. Colonoscopy is the gold standard for the diagnosis of CRC. It enables detection and removal of polyps, particularly advanced adenomas, and has significantly higher sensitivity compared to imaging studies and fecal tests. Recent technological advances, such as high-definition chromoendoscopy and artificial intelligence (AI)-assisted polyp detection systems, solidify its position as a highly sensitive diagnostic tool[5]. The early endoscopic identification, resection, and treatment of precancerous adenoma and early-stage cancer have been shown to reduce the prevalence of CRC and the mortality rate of CRC[6]. However, its invasiveness, cumbersome preparation, and high cost limit its widespread application in areas with large populations or poor economic conditions. In addition to invasive screening methods, non-invasive screening methods have also significantly expanded clinical application scenarios. Noninvasive screening methods have also significantly expanded clinical applications. Methods such as fecal immunochemical testing (FIT) and multitarget stool DNA play a crucial role in preliminary CRC screening due to being noninvasive, convenient, and cost-effective[7,8]. Carcinoembryonic antigen (CEA) is the most commonly used serum tumor marker for CRC[9]. However, it exhibits limitations in sensitivity and specificity, particularly with low positivity rates in early-stage CRC, which restricts its value for stand-alone screening[10].

The recent development of molecular biology and omics techniques and new serological markers, especially liquid biopsies, provides new possibilities for early screening and accurate diagnosis of CRC. Liquid biopsy involves collecting fluid samples such as peripheral blood, saliva, cerebrospinal fluid, ascitic fluid, or pleural fluid, followed by analysis of their components. Circulating tumor cells (CTCs), as key biomarkers for liquid biopsy, demonstrate significant potential in early tumor diagnosis, therapeutic efficacy evaluation, and prognostic monitoring, making them a research hotspot in the field of precision oncology. However, due to their low abundance in peripheral blood, platform variability, and complex biological characteristics such as epithelial-mesenchymal transition (EMT), the isolation and detection of CTCs still face significant challenges in terms of sensitivity, specificity, and standardization of clinical applications. This review summarizes new advances in serum biomarkers for CRC and explores their application prospects and challenges in clinical practice.

LIQUID BIOPSY

Liquid biopsy was proposed in the 1990s as a promising novel technology, and has gradually shifted from research to clinical applications[11]. Liquid biopsy has multiple advantages over tissue biopsy, including convenient sampling, effective monitoring, and suitability for longitudinal evaluation of treatment dynamics[12]. Despite its promising applications, it still needs to be compared with existing screening methods to evaluate its clinical value.

Cost-effectiveness

The cost structure of CRC screening strategies varies significantly among different methods. Colonoscopy incurs the highest direct costs, primarily due to its invasive nature involving complex workflows, including depreciation of expensive equipment, fees for specialized endoscopists, anesthetic expenses, and potential therapeutic costs such as immediate polypectomy or biopsy when lesions are detected. The indirect costs should not be overlooked, including lost work time due to preoperative intestinal preparation and postoperative recovery for patients, as well as potential social costs associated with the need for family accompaniment. In contrast, the cost structure of FIT is simple, with the lowest direct costs involving only kit expenses and basic laboratory analysis costs. The procedure is straightforward and does not require a specialized medical environment. As an emerging blood test, liquid biopsy currently incurs significantly higher direct costs compared to FIT, with expenses comparable to or slightly exceeding those of colonoscopy[13]. The high costs are primarily attributed to its technical complexity, including high-throughput sequencing of biomarkers such as circulating tumor DNA (ctDNA) and sophisticated bioinformatics analysis[14]. However, the indirect cost of liquid biopsy is low, as its sampling process only requires routine blood drawing, causing minimal disruption to patients’ work schedules and significantly enhancing convenience and compliance.

Population applicability

Colonoscopy is recommended as the screening option for all high-risk populations due to its high accuracy, particularly for high-risk individuals with family history or previous polyp history. It is considered the preferred method as it enables direct diagnosis and intervention[15]. For large-scale primary screening of general risk populations, FIT has become an ideal tool due to its simplicity, cost-effectiveness, and sensitivity[16]. Liquid biopsy, as an emerging noninvasive method, may serve as a supplementary or alternative option to enhance screening coverage for individuals who have a fear of invasive procedures, have contraindications to colonoscopy, or refuse stool sample processing, because of its convenient testing upon blood withdrawal.

Diagnostic performance

Comparison of detection sensitivity for CRC: As the gold standard for CRC screening, colonoscopy shows high diagnostic sensitivity and serves as the benchmark for evaluating the performance of other screening methods. However, its superior detection capability is dependent on the operator’s technical proficiency, the quality of intestinal preparation, and completeness of the examination; factors that may introduce variability in practical clinical applications[15].

In contrast, FIT, as a widely used noninvasive screening tool, demonstrates an overall sensitivity of 70%-80% for CRC[16]. The performance of FIT is significantly influenced by tumor hemorrhage status, with a higher risk of missed diagnosis in cases of nonbleeding or intermittent-bleeding tumors. Evidence also suggests that its sensitivity for proximal colon cancer may be lower than that for distal colon cancer[16]. Liquid biopsy, particularly blood-based testing, has demonstrated significant potential as an emerging technology. A model study indicated that blood tests meeting the Centers for Medicare and Medicaid Services minimum threshold exhibited a sensitivity of 74% for CRC[13]. However, the sensitivity of liquid biopsy for early-stage (I/II) CRC remains a challenge. It is generally lower than that for advanced cancers, primarily due to the low levels of biomarkers such as ctDNA released into the bloodstream by early-stage tumors[11]. Therefore, although liquid biopsy may have a higher detection rate for advanced-stage cancers (III/IV), its sensitivity for early-stage cancer still requires validation and optimization through large-scale prospective studies.

Specificity and false-positive issues: Colonoscopy exhibits high specificity, with false-positive results primarily stemming from endoscopists’ visual misjudgment of non-neoplastic inflammation, polyps, or other benign lesions rather than biological limitations inherent to the detection method[17]. The specificity of FIT is favorable, typically ranging between 90% and 95%[16]. However, its specificity may be influenced by factors such as upper gastrointestinal bleeding (e.g., gastric ulcer), ingestion of certain foods (e.g., red meat), or medication (e.g., nonsteroidal anti-inflammatory drugs), which may result in positive fecal hemoglobin detection unrelated to colorectal lesions[16]. The specific challenges faced in liquid biopsy are more complex. One of the primary sources of interference is clonal hematopoiesis, an age-related phenomenon where somatic mutations in hematopoietic stem cells are released into the bloodstream. These mutations do not originate from colorectal tumors but may be detected by liquid biopsy, leading to false-positive results[11]. A meta-analysis indicated that liquid biopsy demonstrates an overall specificity of approximately 89% in the diagnosis of CRC[18]. Nevertheless, the long-term stability of specificity and clinical implications of liquid biopsy in general screening populations (particularly asymptomatic average-risk populations) still require additional prospective data for validation.

Overview of liquid biopsy

In the diagnosis of CRC, the most valuable tissue specimen for liquid biopsy is blood, which is discussed below. Peripheral blood is collected for detecting CTCs, ctDNA, tumor-educated platelets (TEPs), exosomes, and circulating free RNA in the circulation[19].

CTCs

CTCs are shed from tumor tissue and released into the peripheral blood[20]. On entering the circulation, CTCs may metastasize to distant organs. Unlike primary cancer, CTCs have three different subtypes (epithelial, EMT, and stem features)[21,22]. Given the low number of CTCs, adequate quantification requires special enrichment, detection, and characterization techniques[23]. CTC detection can be achieved through immune cytology, molecular biology, or functional assays[24]. A study found that the number of CTCs in the CRC group (84.1%) was significantly higher than that in the healthy control group (9.7%)[25]. A meta-analysis evaluating the value of CTC monitoring for postoperative recurrence and metastasis in CRC demonstrated a pooled sensitivity and specificity of 0.71 each, and an area under the curve (AUC) of 0.76[26]. Another meta-analysis comparing the diagnostic performance of different liquid biopsy methods indicated that the AUC of CTC (0.9772) was superior to that of exosomes (0.9037), demonstrating optimal diagnostic value[18].

In early-stage (I/II) CRC patients, the detection rate of CTCs is the lowest, with sensitivity often < 30%, which constitutes the primary bottleneck for current CTC technology in early cancer screening[27]. A study reported that the detection rate of CTCs in stage I patients was only 25%[28]. Nevertheless, even the detection of a small number of CTCs may indicate a higher risk of recurrence, suggesting CTC detection can be used to predict recurrence in patients with stage II CRC and assist in decision-making for adjuvant chemotherapy[29]. Other studies have shown that the detection rate of CTCs is lower in stage III compared to stage IV patients[30]. In patients with metastatic CRC (mCRC), CTC detection demonstrates high diagnostic sensitivity. A study involving 218 patients with mCRC identified that those who consistently failed to detect CTCs throughout the treatment course had the best prognosis, while patients with persistent CTC positivity exhibited significantly shorter progression-free and overall survival[31]. CTC testing combined with an immunochemical fecal occult blood test and serum CEA assay improved CRC screening effectiveness[32]. The number of CTCs is correlated with tumor staging and is more consistent with clinical pathological features in left colon cancer, which may aid clinical staging and prognostic prediction[33].

CTCs represent a highly promising biomarker in the field of liquid biopsy for CRC, yet their clinical application faces significant challenges. These challenges primarily include low abundance in peripheral blood, platform variability, and complex biological characteristics such as EMT, which may lead to missed detection of certain CTC subpopulations.

CTCs have low abundance in peripheral blood, particularly in early-stage or minimal residual disease states, with only 1-10 cells per 10 mL blood. This necessitates detection methods with exceptionally high sensitivity[34]. In patients with nonmetastatic or early-stage CRC, the detection rate of CTCs is typically low[35]. Each milliliter of blood contains billions of blood cells, while the number of CTCs may be in the single digits, which imposes high demands on detection techniques[34]. This low abundance directly affects the value of CTC detection in critical clinical applications such as early screening and minimal residual disease monitoring.

There are multiple CTC detection platforms in the market based on different principles, with varying enrichment, identification, and counting methods, leading to incompatibility of detection results across platforms and severely hindering the unified interpretation and standardized application of clinical data[36]. For instance, platforms based on immune affinity (such as the CellSearch system) and those utilizing biophysical properties (e.g., size filtration and density gradient centrifugation) show significant differences in capture efficiency and the subpopulations of CTCs captured, rendering direct comparison among studies challenging[37]. CTCs are enriched and identified based on epithelial cell markers such as epithelial cell adhesion molecules (EpCAMs). However, increasing evidence suggests that CTCs exhibit high heterogeneity, which manifests at multiple levels, including genotype, phenotype, and function[38]. Of particular note, a subset of CTCs retains epithelial characteristics and acquires stem-cell-like properties, forming what is termed the epithelial-stem cell hybrid subtype[38]. This subtype is considered the more aggressive component within the CTC population, as it may evade conventional killing and has enhanced colonization and tumorigenic potential[39]. EMT is a core biological process driving phenotypic heterogeneity of CTCs in CRC. During this process, tumor cells downregulate the expression of epithelial markers (such as EpCAM) while upregulating the expression of stromal markers (such as vimentin), thereby acquiring migratory and invasive capabilities[40]. CTCs with EMT characteristics are considered to possess greater stem-cell-like properties and metastatic potential. However, conventional EpCAM-based capture methods (such as the CellSearch system) fail to isolate this critical cell subset, leading to detection bias and potential omission of the most aggressive CTCs[41].

ctDNA

Cell-free DNA, present in circulating plasma, is believed to derive primarily from apoptosis of normal cells of hematopoietic lineage[42]. The ctDNA represents a small fraction of cell-free DNA and is released by tumor cells into the blood and tissue. ctDNA has a short half-life, which makes it more advantageous than traditional biopsy markers[19]. The ctDNA is a widely applicable liquid biopsy method with high sensitivity and specificity, and can be used for early diagnosis, therapeutic monitoring, detecting minimal residual disease, guiding adjuvant therapy, and predicting the prognosis of CRC[43-47]. The ctDNA can objectively reflect the intratumoral heterogeneity characteristics of CRC[48]. A proof-of-concept study involving patients with CRC liver metastases demonstrated that intrapatient heterogeneity, including tumor heterogeneity within primary lesions and matched metastatic lesions, combined with ctDNA variability, has potential translational significance[48]. Although ctDNA fragment analysis holds value in tumor detection, there is a lack of direct and consistent correlation between fragment size distribution and the degree of intratumoral heterogeneity[49].

The ability of ctDNA detection technology to reflect heterogeneity fundamentally depends on two core variables: The nucleic acid release level from tumor cells and the sensitivity of the detection technology[50]. Tumor size and proliferative capacity are key factors associated with ctDNA release in CRC[51]. Additionally, secretory and consensus molecular subtype 3 of CRC exhibit lower ctDNA release levels, whereas tumors with microsatellite instability demonstrate higher ctDNA levels[50]. The ctDNA levels are associated with clinical and pathological features of cancer, including staging, localization, vascularization, and response to treatment[51].

Although ctDNA testing demonstrates high sensitivity in advanced CRC, its application in early-stage (particularly stage I) disease remains challenging due to lower sensitivity[52]. The plasma concentration of ctDNA after pretreatment of stage I CRC patients was significantly lower than that of stage II/III patients[53]. Another study involving patients with stage I-IV CRC demonstrated that the detection model based on ctDNA methylation markers exhibited only 79.4% sensitivity in stage I patients, which was significantly lower than the 96.2% sensitivity observed in stage IV patients[52]. A multimodal analysis confirmed that detection sensitivity was 73.9% for stage I cancer, while it increased to 88.3% for nonmetastatic stage IIIA disease[54]. Detection of ctDNA in patients with mCRC demonstrates high sensitivity, primarily attributed to the substantial tumor burden and extensive clonal diversity. In stage IV disease, the overall tumor volume significantly increases, often accompanied by multiorgan metastases, leading to high rates of tumor cell necrosis and apoptosis, which release a large amount of ctDNA fragments into the bloodstream[55]. However, the specificity of ctDNA detection faces the critical challenge of clonal hematopoiesis. Mutated fragments released into the plasma by hematopoietic cells are difficult to distinguish from true tumor-derived mutations in ctDNA detection based on targeted sequencing, particularly when the mutation allele frequency is low, which can easily lead to false-positive interpretation[56,57]. The ctDNA methylation can identify individuals at high risk for CRC and shows promising application[52,58,59]. The most promising circulating markers identified among nucleic acids were SDC2-methylated DNA and SEPT9-methylated DNA[60]. The SEPT9 methylation of ctDNA exhibits a strong correlation with early-stage CRC lesions[61], while methylation of SFRP1, SFRP2, and SDC2 demonstrates diagnostic sensitivity exceeding 90% for CRC[62,63]. The SEPT9, SDC2, and BCAT1 are significantly upregulated in CRC patients, with SEPT9 demonstrating high sensitivity and specificity of 83.7% and 93.9%, respectively[58]. Detection of SEPT9 and SDC2 methylation shows potential in CRC screening, but the issue of false positives primarily stems from nonspecificity at the biological level. Clonal hematopoietic-related mutations are a significant cause of false-positive results in blood tests. These mutations occur with advancing age and may involve hematopoietic cells carrying methylation alterations in genes such as SEPT9. These methylated DNA fragments entering the bloodstream can be detected by highly sensitive assays even in the absence of colorectal tumors, leading to non-tumor-related positive signals[64]. SEPT9 methylation has been reported in various other malignant tumors, such as esophageal cancer and head and neck squamous cell carcinoma[65]. This broad cross-reactivity implies that when these biomarkers are detected to be methylated, the signal may originate from benign or malignant diseases in organs outside the colorectum, thereby constituting false positives.

Compared with FIT, blood-based SEPT9 methylation testing typically demonstrates lower or comparable overall sensitivity for CRC detection, with a range of 70%-80%, whereas FIT sensitivity generally ranges between 70% and 85%[66]. In contrast, fecal-based SDC2 methylation testing demonstrates higher sensitivity, particularly with advantages in the detection of early-stage cancer[67]. For the detection of advanced adenomas, FIT generally exhibits low sensitivity, ranging from 20% to 40%[68]. Blood SEPT9 testing also demonstrates limited sensitivity for advanced adenoma, with a range of 20%-35%[68]. In terms of specificity, SEPT9 and SDC2 methylation detection, along with FIT, can achieve > 90% accuracy in healthy populations[67]. However, it should be noted that FIT may be more susceptible to false-positive results due to upper gastrointestinal bleeding, certain foods, or medications.

TEPs

Tumor cells can affect the RNA information and protein levels of platelets through various signaling molecules or receptors, leading to the formation of TEPs. TEPs are involved in the progression and spread of various solid tumors, and spliced TEP RNA can provide information about the presence, location, and molecular characteristics of cancer[69], which may make it potentially useful for predicting tumors[70,71]. In a retrospective cohort study, transcriptome sequencing of platelets isolated from 132 early- and late-stage CRC patients and 190 controls identified 921 genes with the greatest contribution to classification. The constructed diagnostic model achieved an area under the receiver operating characteristic curve of 0.928 in the training set and 0.92 in the internal validation set, demonstrating significantly higher diagnostic accuracy compared to clinically commonly used serum biomarkers CEA and carbohydrate antigen 19-9[72].

TEPs can activate the coagulation cascade, leading to the formation of platelet-rich clots around CTCs. This process can promote CTC survival[73]. However, directly observing and accurately quantifying this TEP-CTC binding event in clinical blood samples poses significant technical challenges. Existing CTC enrichment techniques, particularly positive capture methods based on EpCAM (such as the CellSearch system), are primarily designed for free, EpCAM-expressing epithelial-derived CTCs. This method has significant limitations. On the one hand, CTCs tightly encapsulated by platelets or undergoing EMT may downregulate EpCAM expression, thereby being missed during capture. On the other hand, the capture process may disrupt the fragile natural binding state between CTCs and platelets, leading to underestimation or misjudgment of this critical interaction event[74,75]. Most TEP studies in CRC patients adopted a retrospective design, which constitutes a fundamental methodological limitation. These studies typically relied on archived blood samples and clinical data for post hoc analysis, a model that inevitably introduces the risk of selection bias and information bias[76]. For instance, a study aimed at evaluating TEP long-chain noncoding RNA as a diagnostic biomarker for CRC enrolled 75 patients and 42 healthy controls, which was a small exploratory study[77]. It was essentially a hypothesis-generating study, and its conclusions should be regarded as preliminary and exploratory rather than confirmatory evidence[76]. Furthermore, the TEP study lacked rigorous external validation data, which prevents us from accurately assessing the calibration of the constructed predictive model, as well as its discriminative ability in broader populations[76]. A study on TEP RNA profiling for differentiating CRC from noncancerous intestinal diseases reported high AUCs in the internal validation set and a small external validation set. However, the scale and representativeness of the external validation set still require expansion[72].

In addition, the RNA sequencing and analysis workflow used by TEP lacks standardization. At the experimental operational level, significant variations exist among different laboratories in protocols spanning blood collection, platelet separation and purification, RNA extraction, and library construction. These differences directly affect the yield and quality of platelet RNA, as well as the final transcriptome profiles[76]. Clinical samples inherently possess unique characteristics, potentially accompanied by hemolysis or coagulation, yet there is currently a lack of optimized standard operating procedures for such low-quality samples and low-input RNA. Furthermore, consensus has not been reached regarding bioinformatics analysis workflows. Variations across studies in reference genome selection, sequence alignment tools, differential expression analysis methods, and data normalization strategies directly hinder direct comparison and integration of results between studies[76]. In summary, the clinical application of TEP is currently limited.

Exosomes

Exosomes, secreted by cells into the surrounding microenvironment, are a subtype of membrane vesicles with a diameter of 40-200 nm. Exosomes can transport multiple substances, including proteins, lipids, mRNA, micro RNA (miRNA), long noncoding RNA (lncRNA), and DNA; maintain cellular homeostasis; remove cellular debris; and facilitate intercellular communication[78,79]. Increasing evidence suggests that extracellular vesicles play an important role in cancer development. Exosome-derived miR-548am-5p promotes CRC progression[80]. Multiple miRNAs in CRC-derived exosomes, including miR-1915, miR-1308, miR-1290, miR-1268, miR-1246, miR-1229, miR-1224, miR-638, miR-483-5p, miR-223, miR-181d, miR-181b, miR-150, miR-23a, miR-21, miR-205-5p, miR-377-3p, miR-381-3p, and let-7, can serve as biomarkers[81-84]. A study constructed a diagnostic model by detecting four miRNAs (miR-15b, miR-16, miR-21, and miR-31) in serum exosomes. The model demonstrated a sensitivity of 95.06% and specificity of 94.44% in distinguishing healthy controls from CRC patients[85]. The lncRNAs carried by exosomes (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) are significantly upregulated in the plasma of patients with CRC and may serve as noninvasive biomarkers for early diagnosis of CRC[86]. The combination of miR-654-5p, miR-126, miR-10b, and miR-144 has diagnostic value for CRC[87]. The miR-99b-5p and miR-409-3p combined with CEA displayed higher diagnostic power than any single miRNA[88]. These studies indicate that RNA carried by exosomes, alone or in combination with other biomarkers, has practical value for diagnosing CRC. Comparative information is shown in Table 1.

Table 1 Comparison of exosomal biomarkers for colorectal cancer diagnosis.

Ref.	Biomarkers	Expression in CRC vs controls	Diagnostic performance (sensitivity/specificity/AUC)	Study size (CRC patients/HCs)	Validation status and key findings
Li et al[80], 2023	Exosomal miR-548am-5p	Upregulated	Not mentioned	Clinical tissues: 18 pairs of CRC and adjacent nontumor tissues	This study elucidates the oncogenic function of exosomal miR-548am-5p rather than establishing its clinical diagnostic value
Ogata-Kawata et al[81], 2014	Seven-miRNA Panel (e.g., let-7a, miR-1246, miR-23a)	Upregulated	Reported high sensitivity in ROC analysis. Specific AUC for the panel is not provided in the excerpt, but individual miRNAs like miR-1246 showed high diagnostic accuracy	Discovery: 88 CRC/11 HC; validation: 13 CRC	Preliminary validation in an independent set; miRNA levels decreased post-surgery, indicating tumor origin; the study provides early but promising evidence
Zhao et al[83], 2025	miR-205-5p	Downregulated	AUC for CRC vs HC: 0.873	157 CRC/135 HC/20 benign	Single-cohort study with a relatively large sample; expression was lower in CRC and early-stage patients, and increased postoperatively. Shows potential as a diagnostic biomarker
Wang et al[84], 2022	miR-377-3p and miR-381-3p	Downregulated	Combined (CRC vs HC): AUC = 0.886miR-377-3p: AUC = 0.826; miR-381-3p: AUC = 0.843	175 CRC/172 HC	Single-cohort study; the combination showed improved diagnostic performance; expression was downregulated in early-stage CRC
Han et al[85], 2021	Panel: MiR-15b, miR-16, miR-21, miR-31	Upregulated	CRC vs HC: Sensitivity: 95.06%, specificity: 94.44% CRC vs Adenoma: Sensitivity: 85.19%, specificity: 82.09%	Training: 123 CRC/150 HC; validation: 81 CRC/90 HC	Validated in an independent cohort; the panel demonstrated high and consistent diagnostic accuracy in both training and validation sets
Hu et al[86], 2018	Six exosomal lncRNAs (e.g., LNCV6_116109)	Upregulated	Individual AUCs ranged from 0.650 to 0.770	50 CRC/50 HC	Proof-of-concept study; provides preliminary AUC values for individual lncRNAs, suggesting their potential as noninvasive biomarkers; requires further validation
Du et al[87], 2022	Panel: MiR-654-5p, miR-126, miR-10b, miR-144	miR-654-5p, -126, -10b upregulated; miR-144 downregulated	The diagnostic model based on this 4-miRNA signature achieved an AUC of 0.913 in the clinical validation cohort	88 CRC/11 HCclinical; validation: 100 CRC/120 HC	Validated in an independent clinical cohort; the signature was identified via machine learning and showed high diagnostic potential in validation
Zhang et al[88], 2024	miR-99b-5p and miR-409-3p[88]	Upregulated	For early CRC: miR-99b-5p alone: AUC = 0.735, sensitivity: 72.7%, specificity: 78.3%miR-99b-5p + miR-409-3p: AUC = 0.741, sensitivity: 77.3%, specificity: 78.3% + CEA: AUC = 0.812, sensitivity: 90.9%, specificity: 65.2%	68 CRC/27 HC	Single-cohort study; the study highlights the value of combining exosomal miRNAs with the traditional protein marker CEA to significantly boost sensitivity for early CRC detection

CRC: Colorectal cancer; AUC: Area under the curve; HC: Healthy control; ROC: Receiver operating characteristic; CEA: Carcinoembryonic antigen; miRNA: Micro RNA.

RNA

Non-coding RNAs are abnormally expressed in CRC. Various non-coding RNAs are associated with CRC, such as miRNAs, lncRNAs, and circular RNAs (circRNAs)[89,90]. A combination of four miRNAs (miR-193a-5p, miR-210, miR-513a-5p, and miR-628-3p) yielded an AUC of 0.92 (95% confidence interval: 0.85-0.96) for identifying early-onset CRC in a training cohort[91]. Another study reported significant upregulation of miR-155-5p, miR-21-5p, and miR-191-5p, and downregulation of miR-16-5p, directly after surgery. In paired follow-up samples, miR-106a-5p and miR-16-5p displayed the most significant upregulation, and miR-21-5p showed the most significant downregulation[92]. Extensive research confirms that miRNAs play an important role in the pathogenesis of CRC[93]. The lncRNAs are also involved in CRC[94] and showed utility as prognostic markers[95,96]. The lncRNAs FOXD2-AS1, NRIR, XLOC_009459, ASB16-AS1, AFAP1-AS, SNHG16, SNHG11, and PGM5-AS1 are potential diagnostic biomarkers for early-stage CRC[97-101]. Widespread circRNA downregulation in early-stage CRC has been demonstrated[102]. The circRNAs have tissue specificity and are expressed stably in blood and saliva, implying potential as biomarkers[103]. The circ-FMN2, circ-LMNB1, circ-ZNF609, hsa_circ_101303, and circ_001659 could be useful serum biomarkers for CRC diagnosis and prognosis[104-106]. Circular RNA chaperonin containing TCP1 subunit 3 is a promising biomarker for poor prognosis in colorectal adenocarcinoma, independently predicting tumor recurrence[107].

Combined application of different liquid biopsy components

Recent research trends indicate that single biomarker types often exhibit limitations in sensitivity or specificity. Therefore, integrating ctDNA methylation markers with circulating RNA or exosome profiles through multiomics joint analysis enables synergistic capture of tumor signals from epigenetic, transcriptional, and proteomic dimensions. This approach holds promise for overcoming the limitations of single biomarkers and significantly enhancing the accuracy and reliability of screening[108]. For instance, combining SEPT9 gene methylation with miR-92a expression, or NDRG4 methylation with exosomal miR-23a, demonstrated significantly higher AUC values for detecting colorectal adenomas and early CRC compared to any single biomarker[58]. In terms of translational applications, some blood testing products based on multi-markers (including methylation and proteins) have entered clinical trials or attracted regulatory attention. For example, the Epi proColon test based on SEPT9 methylation has obtained relevant approvals, but commercially available products that truly integrate multiomics information, such as ctDNA methylation and exosomal RNA, are still in the research and validation stages[109]. A large prospective study conducted on an average-risk population demonstrated that a multiomics liquid biopsy platform achieved detection sensitivity of 90% and specificity of 89% for CRC, with particularly strong recognition capabilities for early-stage (I/II) cancers[110]. These achievements mark steady progress in the field toward clinical application. The following section compares CTCs, ctDNA, TEPs, exosomes, and RNA in terms of detection methods, representative biomarkers, advantages and limitations, clinical evidence level, and study size and validation cohort (Table 2).

Table 2 Comparison of components in liquid biopsy.

	CTCs	ctDNA	TEPs	Exosomes	RNA
Detection methods	CellSearch system, immunomagnetic bead enrichment, membrane filtration method	dPCR, next-generation sequencing, methylation sequencing, ARMS-PCR	Supercritical centrifugation, size exclusion chromatography, immune affinity capture	Ultra-high speed centrifugation, SEC, commercial extraction kits, microfluidic technology	qRT-PCR, dPCR, transcriptome sequencing
Representative biomarkers	EpCAM, CK8/18/19; CD44, CD133, ALDH1	KRAS, NRAS, BRAF, TP53; SEPT9, SDC2, VIM; MSI/MMR status	EGFR, EpCAM; vesicle-associated miRNAs	CD9, CD63, CD81; miR-92a, EGFR	miRNA, circRNA, lncRNA
Advantages	Obtain comprehensive tumor cell information, directly reflecting phenotype, stemness, and metastatic potential	The detection technology is mature with high standardization and excellent sensitivity	Stability superior to free nucleic acids with stable content	Long blood half-life and high stability	High sensitivity with a wide range of biomarkers
Limitations	Low abundance in peripheral blood, platform variability, and complex biological characteristics such as EMT	Early CRC exhibits extremely low abundance	No unified gold standard for separation and purification	Lack of unified separation criteria makes purity control difficult	Free RNA is highly susceptible to degradation, requiring stringent sample processing protocols
Clinical evidence level	Moderate to high level of evidence	High-quality evidence	Limited clinical evidence	Preclinical translational phase	Moderate evidence
Study size and validation cohort	Prospective large cohort studies on early CRC are insufficient	Predominantly prospective cohort	Lack of multicenter prospective validation	Multicenter validation cohort scarcity	The multicenter independent validation cohort requires refinement

CTC: Circulating tumor cell; ctDNA: Circulating DNA; TEP: Tumor-educated platelet; dPCR: Digital polymerase chain reaction; ARMS-PCR: Amplification-refractory mutation system polymerase chain reaction; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; SEC: Size exclusion chromatography; EpCAM: Epithelial cell adhesion molecule; ALDH1: Aldehyde dehydrogenase 1; MSI: Microsatellite instability; MMR: Mismatch repair; EGFR: Epidermal growth factor receptor; circRNA: Circular RNA; lncRNA: Long noncoding RNA; EMT: Epithelial mesenchymal transition; CRC: Colorectal cancer; miRNA: Micro RNA.

DISCUSSION

This review synthesizes the current evidence on the collective potential of diverse components of liquid biopsy, including CTCs, ctDNA, TEPs, exosomes, and circulating RNAs, to transform the early detection and diagnosis of CRC. The convergence of these technologies represents a paradigm shift, moving beyond the limitations of invasive colonoscopy and the suboptimal sensitivity and specificity of traditional serological markers like CEA. The fundamental advantage of liquid biopsy is its ability to provide a systemic, real-time molecular portrait of a tumor that overcomes spatial heterogeneity. Each analyzed component offers a unique and complementary window into tumor biology.

However, translating this promising research into routine clinical practice faces significant challenges. In early-stage CRC, the low abundance of ctDNA presents a major technical barrier to sensitive and reliable detection. The significant variability in exosome isolation methods compromises the reproducibility and comparability of liquid biopsy results. These two limitations jointly restrict the clinical application of liquid biopsy in early CRC diagnosis and translational research. Technical standardization is paramount. Preanalytical variables, extraction methods, and detection platforms (especially for low-abundance targets) require rigorous harmonization to ensure reproducibility and comparability across studies and laboratories. Analytical validation of the reported multianalyte signatures in large, independent, and prospectively collected cohorts is essential to confirm their sensitivity, specificity, and generalizability. The clinical utility of these biomarkers must be proved through interventional trials, leading to improved patient outcomes, such as reduced cancer mortality through earlier detection or better therapeutic decisions, compared to the current standard of care. Finally, cost-effectiveness analysis is needed to justify the integration of potentially complex multianalyte liquid biopsy panels into population-based screening programs, particularly in resource-limited settings.

CONCLUSION

Liquid biopsy for CRC screening and diagnosis is no longer a futuristic concept but a rapidly maturing field grounded in robust molecular evidence. The future likely will not involve a single best biomarker but rather integrated multianalyte panels. Such a comprehensive approach could dramatically increase sensitivity for early-stage detection while providing robust specificity. By addressing the current challenges of standardization and validation, liquid biopsies hold the potential to become a cornerstone of personalized CRC management, enabling earlier, less invasive, and more accurate diagnosis, ultimately improving survival rates.