Advances and applications of liquid biopsy approaches in hepatopancreatobiliary (HPB) cancers: a literature review

Introduction

Hepatopancreatobiliary (HPB) cancers, including hepatocellular carcinoma (HCC), pancreatic ductal adenocarcinoma (PDAC), and cholangiocarcinoma (CCA), make up some of the most fatal malignancies around the world due to their asymptomatic nature in early stages, aggressive progression, and limited treatment options for advanced disease. PDAC, in particular, is projected to be the second leading cause of cancer-related death by the year 2030 (1). Similarly, HCC and CCA tend to present late in the disease course for most patients, often ruling out curative resection (2,3).

Despite advances in imaging and biomarkers, early detection and longitudinal monitoring of (HPB) cancers remain suboptimal. Computed tomography (CT) and magnetic resonance imaging (MRI) lack the sensitivity necessary for reliable detection of early-stage disease (4,5). Serum biomarkers such as alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA 19-9) demonstrate limited sensitivity and specificity and are often elevated in benign disease (6,7). Tissue biopsy, while the gold standard, is invasive, susceptible to sampling bias, and unsuitable for real-time disease monitoring (8,9).

Clinical limitations of traditional tissue biopsies result in delayed diagnosis and missed opportunities for timely curative intervention, hence, there is growing interest in liquid biopsy technology, which offers minimally invasive access to biomarkers such as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), exosomes, microRNAs (miRNAs), and circulating tumor cells (CTCs) (10,11). These biomarkers can be obtained through plasma, serum, or bile and analyzed using an array of methods, including next-generation sequencing (NGS), digital polymerase chain reaction (dPCR), and methylation profiling, among others (12-15). They may eventually complement, or in some clinical scenarios, even substitute, tissue-based assays.

In the context of HPB cancers, liquid biopsy has emerged as a promising tool across four key areas, which are synthesized in this review. First, it offers potential for improved early detection of malignancies, ideally at stages where curative interventions are still feasible. Second, liquid biopsy has demonstrated the ability to identify cancer-specific targets that can be used as diagnostic markers and therapeutic guides. Third, using liquid biopsy to track changes in ctDNA over time can provide insight into treatment response and patterns of resistance, which may help to better inform therapy selection and time. Finally, it plays a growing role in disease surveillance and the detection of minimal residual disease (MRD) and can identify recurrence before it becomes clinically apparent.

While many studies have demonstrated the feasibility and potential clinical benefits of liquid biopsy across HPB cancers, widespread use across the disease continuum remains limited due to variability in assay platforms, low yield of ctDNA in early-stage disease, and a lack of studies and trials providing prospective validation (8,16,17). This literature review synthesizes current evidence from 58 peer-reviewed studies published within the past decade, focusing on the role of ctDNA and cfDNA liquid biopsy in early detection, disease surveillance, cancer-specific targets, and treatment monitoring in HPB malignancies. We summarize the biomarker types, sample sources, detection methods, diagnostic performance metrics, and highlight the ongoing challenges and future direction for clinical adoption. We present this article in accordance with the Narrative Review reporting checklist (available at https://cco.amegroups.com/article/view/10.21037/cco-2025-aw-135/rc).

Methods

To identify relevant literature for this review, a search was performed across four databases: PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, and Google Scholar. The search included articles published between April 2016 and July 2025. The keywords used were: “liquid biopsy”, “ctDNA”, “cfDNA”, “pancreatic”, “hepatobiliary”, “liver”, “cholangiocarcinoma”, “gallbladder”, “early detection”, “screening”, “surveillance”, “minimal residual disease”, and “monitoring”. The search was limited to peer-reviewed studies. The exclusion criteria included preprints, conference abstracts, and editorials. No language restrictions were applied.

The study selection process was conducted in two phases. In the first phase, titles and abstracts were screened for relevance. Articles that passed this initial review underwent phase two, a full-text evaluation, as noted in Table 1.

Studies were included if they focused on HPB cancers and evaluated liquid biopsy methods such as ctDNA, cfDNA, exosomes, CTCs, or miRNAs, for early detection, diagnosis, surveillance, MRD, or treatment response monitoring.

Ultimately, 58 studies met the inclusion criteria and were included in this review. Articles were stratified into four application domains: cancer-specific targets, early detection, treatment monitoring, and disease surveillance and MRD. Of these, 36 are review articles, 17 are prospective studies, and five are retrospective studies.

Key findings

Early detection of HPB cancers using liquid biopsy

Liquid biopsy approaches using cfDNA and ctDNA have shown strong potential in the early detection of HPB malignancies. Among the 58 studies included in this review, 39 evaluated diagnostic performance in early-stage or resectable disease, including PDAC, HCC, and CCA.

The majority of studies employed NGS, quantitative PCR (qPCR), droplet digital PCR (ddPCR), or methylation-specific techniques. Sensitivities for ctDNA-based early detection ranged from 63% to 91%, with several studies reporting areas under the curve (AUCs) greater than 0.90 in patient groups with early-stage PDAC or CCA (18-20). For example, in a prospective cohort study, Ben-Ami et al. found an AUC of 0.94 for the detection of stage 1 pancreatic cancer when combining the use of cfDNA methylation profiles and protein biomarkers in plasma (13). Moreover, Lapitz et al.’s prospective study identified serum/extracellular vesicle (EV) protein panels [C-reactive protein (CRP)/ferritin light chain protein (FRIL) and CRP/fibrinogen/FRIL] that accurately detect early-stage CCA, which was validated by enzyme-linked immunosorbent assay (ELISA) in serum with AUCs exceeding 0.80 (3).

In PDAC, fragmentation patterns, nucleosome footprint (FT), and copy number alteration (CNA) have improved early detection and prognosis of PDAC when combined with conventional mutation testing (21). Blood tests that combine multiple biomarkers, such as KRAS mutations, DNA methylation patterns, and fragmentomics, have shown potentially higher accuracy in detecting PDAC (Table 2). In a case-control study, Cohen et al. reported that an assay leveraging both KRAS mutations and protein biomarkers simultaneously achieved an AUC of ~0.95 (22). Reese et al. reviewed several similar approaches, noting their potential, but highlighting the need for additional, larger-scale studies (31).

In HCC, cfDNA and ctDNA profiling have identified early TERT promoter, TP53, and CTNNB1 mutations, along with hypermethylation of RASSF1A, APC, and GSTP1, which serve as early markers of hepatocarcinogenesis (18,38) (Table 3). Hu et al. note that using TERT promoter in combination with AFP and DCP protein markers can identify early-stage HCC with 100% sensitivity (18). Additionally, Park et al.’s review found that cfDNA-based platforms such as HelioLiver^TM, Oncoguard^® Liver, and cfMethyl-Seq achieved an AUC of greater than or equal to 0.90 for early-stage detection of HCC (39).

In CCA, several studies noted that cfDNA obtained from bile offers improved sensitivity compared to plasma. Arechederra et al. found that bile cfDNA sequencing detected malignancy with 96% sensitivity and 100% specificity in patients with biliary strictures, compared with 27% sensitivity with cfDNA obtained from plasma (12).

In summary, cfDNA and ctDNA assays, especially those incorporating epigenetic or fragmentomic features, have demonstrated encouraging diagnostic performance in early-stage disease across multiple cancer types. While plasma is still the most common sample type, bile cfDNA appears to have several advantages in CCA. Further prospective validation is warranted to discern the optimal clinical approach.

Cancer specific targets

HPB cancers share overlapping clinical features but have distinct genomic and epigenomic differences that can be used as targets for liquid biopsies.

PDAC often involves gene mutations, which can be detected in ctDNA and tumor cells in the bloodstream (Table 2). Botrus et al. studied 282 patients with advanced or metastatic PDAC and found that 90% had at least one mutation, with KRAS (88%), TP53 (60%), SMAD4, and CDKN2A being the most common. 48% of patients were found to have mutations in BRCA1/2, ATM, or PIK3CA, which are therapeutically relevant. This demonstrates that ctDNA profiling can identify actionable targets even in advanced disease (23). In another study of 104 PDAC patients, Botrus et al. showed that ctDNA mutations in KRAS and TP53 were associated with significantly worse progression-free and overall survival. Patients with two or more detectable ctDNA alterations had shorter overall survival (11.5 vs. 24.2 months). A dominant clone allele frequency (DCAF) greater than 0.45% also predicted poorer outcomes. Clearance of KRAS or TP53 mutations during treatment correlated with improved survival, underscoring ctDNA’s role as a dynamic prognostic and monitoring biomarker (24). Furthermore, epigenetic methods, such as DNA methylation profiling, have shown especially high sensitivity. In a retrospective cohort study, Zhao et al. identified six specific markers of hypermethylation in cfDNA linked to genes including KCNA3, PRRX1, CCNA1, TRIM58, and NR2FI-AS1. This model delivered an overall sensitivity of 88.7% for detecting PDAC. It showed a 78% sensitivity in stage I PDAC, outperforming mutation-based assays, and improved to over 95% sensitivity when combined with CA 19-9 (the traditional serum biomarker for PDAC) (20). Integrated assays combining cfDNA methylation with protein biomarkers or immune-related signatures hold promise for enhanced diagnostic accuracy (11).

In HCC, recurrent mutations in TP53 and CTNNB1 are often found in ctDNA and indicate activation of cell cycle and Wnt/B-catenin pathways (18,38). Alterations in the TERT promoter occur early in hepatocarcinogenesis and serve as a potential biomarker for molecular surveillance through cfDNA analysis (Table 3). Park et al. noted that cfDNA, CTC, and EV assays demonstrate strong tissue-liquid concordance for known HCC driver mutations (TERT, TP53, CTNNB1), methylation signatures, and chromatin-remodeling genes, such as ARID1A and AXIN1 (39).

In CCA, some important targets for treatment include FGFR2 fusions and IDH1/2 mutations, as well as inactivation of genes like ARID1A and BAP1 (Table 4). While these can be detected using plasma-based assays, recent research suggests that analyzing cfDNA from bile is more sensitive. In a prospective study by Arechederra et al., bile cfDNA sequencing identified malignancy with 96% sensitivity versus only 27% using plasma. This prospective study also reliably detected common mutations in KRAS, TP53, FGFR3, IDH2, ERBB2/3, and GNAS (12). Additional studies have shown that cfDNA from bile is technically stable and consistently detects KRAS mutations, validating its reliability for routine clinical use (53). The concordance between tissue biopsy and liquid biopsy is also an important factor to measure. A multicenter cfDNA profiling study of 1,671 patients with advanced CCA reported clinically actionable alterations in 44% of cases, including FGFR2 fusions (1.4%), IDH1 mutations (9.1%), BRAF V600E (1.3%), ERBB2 amplifications (4.9%), and PIK3CA mutations (6.8%). Importantly, this work demonstrated strong tissue-liquid concordance for key mutations such as IDH1 (87%) and BRAF V600E (100%), but lower concordance for FGFR2 fusions (18%), which reflects the technical limitations of fusion detection in cfDNA. CfDNA also revealed secondary mutations in the FGFR2 kinase domain associated with acquired resistance to FGFR2 inhibitors and showed that higher pre-treatment cfDNA variant-allele fractions correlated with a worse prognosis (47). Another study by Uson et al. analyzed ctDNA from 67 patients with advanced CCA treated with platinum-based chemotherapy. A DCAF greater than 3% was found to be associated with significantly shorter overall survival (10.8 vs. 18.8 months) and progression-free survival. The most common DCAF mutations were TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1, mirroring tissue-based genomic profiles (48).

In PDAC, HCC, and CCA, liquid biopsies aid in identifying key genetic changes (Tables 2-4). In PDAC, these include KRAS, TP53, SMAD4, CDKN2A; in HCC, they include TERT, TP53, CTNNB1, along with cfDNA methylation signatures; and in CCA, they include FGFR2 fusions, IDH1/2 mutations, ERBB2 alterations, BRAF mutations, and inactivation of ARID1A and BAP1. Additionally, cfDNA methylation patterns improve diagnostic accuracy across these malignancies. These cancer-specific molecular profiles provide a foundation for early detection and therapeutic guidance in HPB malignancies.

Treatment monitoring

Liquid biopsy has also shown significant promise for treatment monitoring in HPB cancers, enabling real-time assessment of therapeutic efficacy, MRD, and the identification of any new resistance mutations that may develop during treatment.

In PDAC, dynamic changes in ctDNA levels correlate closely with treatment response. A decrease in ctDNA KRAS mutations during chemotherapy has been associated with radiographic response and longer progression-free survival, while an increase in ctDNA levels can signal early disease progression (11). Post-resection ctDNA detection identifies patients at high risk for relapse, with positive ctDNA results postoperatively linked to significantly shorter disease-free and overall survival. When ctDNA is combined with CA 19-9, it enhances prognostic accuracy and often detects recurrence before it appears on scans (8,11,21,25). Beyond mutations, multi-feature cfDNA prognostic models that integrate fragmentation patterns, end motifs, and FTs have further improved the ability to assess the risk of recurrence (11).

In addition, the changes in ctDNA during chemotherapy have also been studied as a predictor of treatment response. In unresectable PDAC, Bahado-Singh et al.’s prospective cohort study demonstrated that declining cfDNA levels correlated with radiographic response and an improvement in progression-free survival, while rising cfDNA suggested early resistance (25).

In HCC, the amount of methylation in cfDNA serves as a key predictor of patient outcomes following locoregional and systemic therapy. Hypermethylation of tumor suppressors such as RASSF1A, APC, GSTP1 has been associated with early recurrence and reduced overall survival after ablation and resection (38). Similarly, longitudinal monitoring has shown that persistent or rising cfDNA methylation levels following therapy can signal disease progression earlier than radiographic modalities, underscoring its potential as a real-time biomarker of therapeutic efficacy (38).

In CCA, ctDNA has enabled monitoring of resistance to targeted therapies, particularly in patients receiving FGFR inhibitors. Serial ctDNA profiling during treatment has identified the emergence of multiple secondary mutations of FGFR2 kinase domain, including the gatekeeper mutation V564F. These mutations signal therapeutic resistance and can be detected months before the progression becomes visible using traditional radiographic methods (49). Polyclonal FGFR2 mutations, which often occur simultaneously with BAP1, CDKN2A/B, and TP53 alterations, are linked with poorer outcomes. However, it has been shown that transitioning patients to next-generation irreversible FGFR inhibitors, such as futibatinib, using ctDNA as a guide, may be effective in overcoming resistance (49).

Taken together, these findings demonstrate that liquid biopsy can extend beyond diagnosis to function as a dynamic monitoring tool. By capturing patient responses to chemotherapy in PDAC, methylation changes in HCC, and the development of resistance in CCA, cfDNA and ctDNA profiling allow healthcare providers to intervene earlier, assess risk more effectively, and tailor treatments to the individual needs of patients who are battling HPB malignancies.

Disease surveillance and MRD detection

In addition to early diagnosis, cfDNA and ctDNA are more frequently being used for disease monitoring, stratification of recurrence risk, and MRD detection after definitive therapy. Of the 58 included studies, 13 assessed ctDNA or cfDNA for longitudinal surveillance following surgical resection, local ablative therapy, or systemic treatment.

Postoperative ctDNA positivity was consistently associated with early recurrence. In PDAC, Poruk et al. reported that patients with detectable ctDNA after resection had significantly shorter recurrence-free survival, with ctDNA detecting relapse with an average of 4.5 months before imaging (15). In HCC, Hu et al. found that the presence of cfDNA on methylation after local therapy was a strong predictor of early progression and correlated with reduced overall survival (18).

Bile cfDNA has also demonstrated strong potential for MRD assessment in CCA. CtDNA is cfDNA released by tumors undergoing necrosis or apoptosis (18). Choi et al. notes that recurrent ctDNA mutations in the perioperative setting were associated with early recurrence and worse survival outcomes (54). Similarly, Wang et al. found that methylation changes in bile cfDNA predicted clinical relapse in up to 83% of patients with CCA (50).

Detection of MRD in low-volume settings remains challenging. Several studies employed ultra-deep sequencing or tumor-informed panels to improve sensitivity, although these are not yet commonly used in clinical settings. Nevertheless, cfDNA and ctDNA detection identified recurrence earlier than traditional imaging across cancer types, sometimes by several months. While these findings suggest potential for guiding personalized post-treatment surveillance, broader validation is needed before routine adoption.

Discussion

Liquid biopsy has emerged as a useful tool in the diagnosis and surveillance of HPB cancers, malignancies that are typically defined by late-stage detection and poor prognoses. This review incorporates findings from 58 studies evaluating ctDNA and cfDNA as non-invasive biomarkers for early detection, minimal residual disease and disease monitoring, molecular target identification, and treatment monitoring.

When compared with traditional diagnostic modalities, liquid biopsy offers several distinct advantages while also posing some notable limitations. CT and MRI provide essential anatomical information but lack molecular resolution and often can only detect disease after substantial tumor development, especially in early-stage HPB cancers (4,5). Serum biomarkers, including AFP and CA 19-9, are widely available but show a limited sensitivity and specificity in early-stage disease and perform poorly for real-time treatment monitoring (6,7). Liquid biopsy, on the other hand, allows for detection of tumor-derived molecular alterations earlier than radiographic progression, captures distinct tumor cell populations, and enables serial sampling to assess treatment response and emerging resistance, all of which support its rising role in early detection and disease monitoring (9,55).

When it comes to early detection, plasma-based ctDNA and cfDNA assays, particularly those using NGS, methylation profiling, and fragmentomic analyses, have shown strong diagnostic performance. Multiple studies have reported AUC values exceeding 0.90, including assays combining cfDNA methylation panels with protein biomarkers for stage 1 pancreatic cancer, and cfDNA-based approaches for early-stage CCA (3). CfDNA obtained from bile demonstrated higher detection rates than cfDNA obtained from plasma in patients with CCA, which reflects its proximity to tumor tissue and its potential utility as a complementary biomarker (3,12).

Cancer-specific molecular targets in HPB malignancies are becoming increasingly actionable, with implications for both diagnosis and treatment. In PDAC, recurrent mutations in KRAS, TP53, SMAD4, and CDKN2A are highly detectable in cfDNA, while methylation-based assays have shown improved diagnostic sensitivity (8,20,32). It is important to note that some biomarkers present technical challenges in liquid biopsy. For example, Pietrasz et al. report that some studies find no KRAS mutation in ctDNA in early-stage PDAC, which may demonstrate functional heterogeneity within a single tumor, or that not all tumor cells within a single tumor release mutant KRAS ctDNA at all (26). In HCC, detectable alterations include mutations in TERT promoter, TP53, and CTNNB1, alongside cfDNA methylation signatures like RASSF1A, APC, and GSTP1, which have shown promise as early detection and prognostic tools (18,38). In CCA, emerging targets like FGFR2 fusions, IDH1/2 mutations, and loss of ARID1A/BAP1 are becoming more clinically relevant. Notably, bile-derived cfDNA has demonstrated better sensitivity than plasma in detecting these alterations (12,53).

Treatment monitoring is another emerging application of liquid biopsy. In PDAC, changes in ctDNA levels during chemotherapy, as well as detection of MRD following surgical resection, have shown prognostic value and correlation with clinical outcomes (11). In HCC, elevated cfDNA methylation burden has been associated with increased risk of recurrence and disease progression following both local therapy and systemic immunotherapy (38). In CCA, serial ctDNA profiling of patients undergoing FGFR inhibitor therapy has enabled earlier detection of resistance mutations, such as the FGFR2 V564F, which is often detectable several months before radiographic progression can be seen. These findings support the potential of liquid biopsy to guide dynamic treatment strategies and ultimately improve longitudinal management of HPB malignancies (49).

Despite the promising results, challenges to broader clinical adoption of ctDNA-based assays remain. In early-stage disease, when ctDNA makes up a small percentage of the total cfDNA, the ability to detect low-frequency variants poses a significant challenge (27,56). Further, distinguishing tumor-derived ctDNA from background interference from clonal hematopoiesis of indeterminate potential (CHIP) may result in false-positive findings if not adequately controlled for during analysis (14,55,57,58). In addition, assay heterogeneity, limited standardization of ctDNA thresholds, and a lack of uniform reporting across studies make broader adoption into patient care more difficult (14,58). Although bile-derived ctDNA has demonstrated superior sensitivity for CCA compared with plasma-based assays, bile sampling is invasive and operator-dependent, which limits its utility in general clinical practice (12,53).

Importantly, while the diagnostic and surveillance potential of ctDNA appears promising, few studies enrolled asymptomatic individuals or prospective surveillance cohorts at high risk for HPB cancers. Most of the available evidence comes from retrospective analyses or small prospective cohorts, emphasizing the need for larger, multicenter validation trials.

Liquid biopsy is likely to be the most useful when incorporated into existing clinical workflows rather than being used on its own as a diagnostic tool. Potential scenarios include perioperative MRD assessment, to guide adjuvant therapy decisions (15,26), longitudinal monitoring of treatment response during systemic therapy (25), and early detection of acquired resistance during targeted therapy (9,49). After treatment, serial monitoring of ctDNA can allow for detection of molecular relapse long before radiographic progression, allowing earlier clinical intervention (55), and ultimately better survival outcomes.

In the future, synthesizing and coordinating assay platforms, developing more cost-effective panels, and integrating ctDNA testing into clinical practice will be important. Research on the adoption of ctDNA-guided treatment, especially for MRD, will further define the clinical benefits of these biomarkers.

Conclusions

Liquid biopsy using cfDNA and ctDNA represents a minimally invasive approach with growing relevance in the management of HPB cancers. Its primary applications include early detection, disease surveillance, molecular target identification, and treatment monitoring, with growing evidence supporting its role in enabling more personalized and dynamic patient care. Despite promising diagnostic performance, broader clinical adoption remains limited by variability in assay platforms, lack of standardization, and insufficient prospective validation. Moving forward, well-designed clinical trials are needed to establish the utility of liquid biopsy across diverse patient populations, alongside cost-effectiveness assessments to support routine implementation. With additional innovation and validation, liquid biopsy has significant potential to transform the detection, surveillance, and treatment of these high-mortality malignancies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-aw-135/rc

Peer Review File: Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-aw-135/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://cco.amegroups.com/article/view/10.21037/cco-2025-aw-135/coif). M.J.B. reports grants to institution from Alentis Therapeteutics, Arvinas, H3 Biomedicines, Tango Therapeutics, RayzeBio, Medinlink, Revolution Medicines, Incyte, Seagen, Pfizer, Nuvectis, Cogent, Astra Zeneca, Compass Therapeutics, Biond, Sanofi, Elevar, Elevation, Agios, Relay Therapeutics and Basilea; received consulting fees from Amplia, Servier, Revolution Medicines, George Clinical, Guardant and Imugene; received stock from ADC Therapeutics, Abeona, Assertio, and Homology; reports participation on a data safety monitoring board or advisory board on Accession Therapeutics, Khora, Breakthru Bio, Reignite Therapeutics, Karkinos, Vionix, RayzeBio, Moderna, Revolution Medicines, Servier, Cogent, Sirtex, Processa, Jazz Pharmaceuticals, Elevar, Merck, Eisai, Compass Therapeutics, Kalivir Immunotherapeutics, Zielbio, Tempus, Cardinal Health, Exelixis, Kriya Therapeutics, Zymeworks, Orum Therapeutics, IMVAX, Cedilla, Senti Biosciences, and Diffusion Pharmaceuticals. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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