Circulating tumor DNA in biliary tract cancers: A review of current applications

Table 1 Studies evaluating circulating tumor DNA for early detection

Ref.	Title	Clinical relevance
Phallen et al[46], 2017	Direct detection of early-stage cancers using circulating tumor DNA	Investigates ctDNA levels in patients with early-stage solid tumors; higher levels are associated with recurrence and worse overall survival
Liu et al[37], 2024	Cell-free DNA in plasma reveals genomic similarity between biliary tract inflammatory lesion and biliary tract cancer	Compares molecular profiles of biliary tract cancer and inflammatory bile diseases to aid differential diagnosis
Mencel et al[40], 2022	Liquid biopsy for diagnosis in patients with suspected pancreatic and biliary tract cancers: PREVAIL ctDNA pilot trial	Evaluates the utility of ctDNA in confirming cancer diagnosis in patients with radiologically suspected pancreatic or biliary malignancies
Yang et al[45], 2023	Multimodal integration of liquid biopsy and radiology for the noninvasive diagnosis of gallbladder cancer and benign disorders	Develops a ctDNA-based tool for the preoperative diagnosis of gallbladder cancer, supporting surgical decision-making
He et al[49], 2025	Bile-derived cfDNA of syncytin-1 and SLC7A11 as a potential molecular marker for early diagnosis of cholangiocarcinoma	Explores the role of syncytin-1 and SLC7A11 expression in cholangiocarcinoma pathogenesis
Konczalla et al[39], 2020	Clinical significance of circulating tumor cells in gastrointestinal carcinomas	Reviews the potential of CTCs for cancer screening and patient stratification
Mishra et al[24], 2024	Diagnostic utility of next-generation sequencing in circulating free DNA and a comparison with matched tissue in gallbladder carcinoma	Analyzes genomic alterations in cfDNA from gallbladder cancer to improve diagnostic precision and inform therapeutic strategies

cfDNA: Cell free DNA; CTC: Circulating tumor cells; ctDNA: Circulating tumor DNA; SLC7A11: Solute carrier family 7 member 11.

Table 2 Studies evaluating circulating tumor DNA for minimal residual disease

Ref.	Title	Clinical relevance
Alcaide et al[73], 2020	Evaluating the quantity, quality and size distribution of cell-free DNA by multiplex droplet digital PCR	Presents a novel droplet digital PCR assay to identify suboptimal samples and aberrant cfDNA size distributions, the latter typically associated with high ctDNA levels
King et al[76], 2023	Prospectivelongitudinal tumor-informed ctDNA in resectable biliary tract cancers	Assesses the utility of ctDNA levels in evaluating response in the absence of radiographically visible disease. ctDNA showed a higher detection rate than CA 19-9 prior to resection
Park et al[77], 2024	Ultrashort cell-free DNA fragments and vimentin-positive circulating tumor cells for predicting early recurrence in patients with biliary tract cancer	Investigates the effectiveness of cell-free DNA and circulating tumor cells in predicting early recurrence after curative surgery and adjuvant therapy in patients with BTC
Yoo et al[78], 2024	Circulating tumor DNA status and dynamics predict recurrence in patients with resected extrahepatic cholangiocarcinoma	Evaluates superiority of ctDNA over conventional biomarkers in predicting recurrence and informing adjuvant chemotherapy in resected extrahepatic cholangiocarcinoma
Yu et al[79], 2025	Detecting early recurrence with circulating tumor DNA in stage I-III biliary tract cancer after curative resection	Evaluates serial ctDNA testing for surveillance after curative resection in early-stage BTC. Identified recurrence in 93.8% of cases, with a median lead time of 3.7 months

BTC: Biliary tract cancer; cfDNA: Cell free DNA; ctDNA: Circulating tumor DNA.

Table 3 Studies evaluating circulating tumor DNA in advanced settings

Ref.	Title	Clinical relevance
Zhang et al[108], 2020	Prognostic and predictive impact of circulating tumor DNA in patients with advanced cancers treated with immune checkpoint blockade	Analyzed ctDNA from multiple tumor types in durvalumab trials, showing that higher pretreatment VAF is associated with worse overall survival, supporting its role as a prognostic not predictive biomarker
Gouda et al[85], 2022.	Longitudinal monitoring of circulating tumor DNA to predict treatment outcomes in advanced cancers	Evaluation of longitudinal ctDNA to predict early dynamic changes with advanced solid tumors
Bratman et al[87], 2020.	Personalized circulating tumor DNA analysis as a predictive biomarker in solid tumor patients treated with pembrolizumab	Examines ctDNA in advanced solid tumor patients treated with pembrolizumab, showing that baseline ctDNA and its kinetics during treatment correlate with clinical outcomes
Aguado et al[89], 2020	IDH1 mutation detection in plasma ctDNA and association with clinical response in patients with advanced intrahepatic cholangiocarcinoma from the phase 3 ClarIDHy study	Extends analysis from the ClarIDHy trial, demonstrating 92% concordance between plasma and tissue for mIDH1-R132 detection in iCCA, supporting its use in liquid biopsy for patient selection when tumor tissue is limited
Andersen et al[97], 2022	The clinical impact of methylated homeobox A9 ctDNA in patients with non-resectable biliary tract cancer treated with erlotinib and bevacizumab	Demonstrates that rising plasma meth-HOXA9 Levels after one treatment cycle are significantly associated with worse survival in late-stage BTC
Uson Junior et al[98], 2022	Cell-free tumor DNA dominant clone allele frequency is associated with poor outcomes in advanced biliary cancers treated with platinum-based chemotherapy	Demonstrates that higher dominant clone allele frequency in pretreatment ctDNA is associated with significantly worse progression-free and overall survival in metastatic BTC patients receiving platinum-based chemotherapy
Meng et al[100], 2006	Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines	Identifies dysregulated miRNAs in cholangiocarcinoma and shows that targeting miR-21, miR-141, and miR-200b alters tumor growth and gemcitabine sensitivity
Berchuck et al[23], 2022	The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer	Demonstrates that ctDNA sequencing in cholangiocarcinoma shows high concordance with tumor tissue, reflects tumor burden, and can track mutational evolution during chemotherapy
Ettrich et al[27], 2019	Genotyping of circulating tumor DNA in cholangiocarcinoma reveals diagnostic and prognostic information	Demonstrates that ctDNA sequencing in CCA enables noninvasive monitoring of tumor mutations, with high concordance to tissue, correlation with tumor burden and PFS, and dynamic changes during chemotherapy
Okamura et al[109], 2021.	Comprehensive genomic landscape and precision therapeutic approach in biliary tract cancers	Shows that genomic profiling via ctDNA and/or tissue-DNA is feasible in BTC, with higher concordance between ctDNA and metastatic tissue, and that matched targeted therapies based on profiling improve PFS and disease control rates
Weinberg et al[52], 2019	Molecular profiling of biliary cancers reveals distinct molecular alterations and potential therapeutic targets	Comprehensively profiles BTCs, revealing distinct molecular alterations by subtype and supporting the use of site-specific molecular profiling to guide therapy and clinical trial design
Nakamura et al[110], 2015	Genomic spectra of biliary tract cancer	Molecularly characterizes BTCs, identifying subtype-specific genomic alterations and mutational signatures, with nearly 40% harboring actionable targets and a hypermutated subgroup potentially responsive to immunotherapy
Jusakul et al[51], 2017	Whole-genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma	Performs integrative genomic and epigenomic analysis of CCAs, revealing distinct molecular subtypes
Farshidfar et al[55], 2017	Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles	Integrative TCGA analysis of predominantly intrahepatic CCA identifies an IDH-mutant subtype with distinct epigenetic and metabolic features, revealing molecular heterogeneity with potential therapeutic relevance
Thongyoo et al[64], 2025	KRAS mutations in cholangiocarcinoma: Prevalence, prognostic value, and KRAS G12/G13 detection in cell-free DNA	Assesses KRAS G12/G13 mutations in cfDNA as non-invasive prognostic biomarkers in CCA; elevated MAF combined with CA19-9 Levels predicts poorer survival
Leone et al[111], 2006	Somatic mutations of epidermal growth factor receptor in bile duct and gallbladder carcinoma	Identifies somatic EGFR mutations and pathway activation in biliary tract cancers
Tannapfel et al[68], 2003	Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma	Identifies BRAF and KRAS mutations in cholangiocarcinoma; supports ctDNA-based profiling of MAPK pathway alterations to stratify patients for targeted therapies
Toyota et al[101], 2015	Mechanism of gemcitabine-induced suppression of human cholangiocellular carcinoma cell growth	Exploration of microRNAs and angiogenic molecules as biomarkers of sensitivity or resistance to gemcitabine in CCC; potential targets for overcoming chemoresistance
Carotenuto et al[102], 2020	Modulation of biliary cancer chemo‐resistance through microRNA‐mediated rewiring of the expansion of CD133+ cells	Targeting microRNA 1249 to overcome chemoresistance to gemcitabine–cisplatin in BTC
Gerlinger et al[93], 2012.	Intratumor heterogeneity and branched evolution revealed by multiregion sequencing	Identification of Highlights the limitations of single-site, reinforcing ctDNA as a non-invasive method to capture spatial and temporal tumor heterogeneity for molecular profiling and treatment monitoring
Deshpande et al[112], 2011	Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma	Highlights ctDNA for subtype differentiation and therapeutic decision-making by identification of subtype-specific mutations
Kawakami et al[66], 2021	Stepwise correlation of TP53 mutations from pancreaticobiliary maljunction to gallbladder carcinoma: A retrospective study	Supports the use of TP53 mutations as a biomarker for early detection and risk stratification in GBC patients, potentially detectable via ctDNA
Kim et al[67], 2015	Molecular subgroup analysis of clinical outcomes in a phase 3 study of gemcitabine and oxaliplatin with or without erlotinib in advanced biliary tract cancer	Highlights the potential utility of ctDNA for assessing KRAS and PIK3CA mutational status as predictive biomarkers to guide anti-EGFR therapy in BTC
Saetta et al[113], 2004	Mutational analysis of BRAF in gallbladder carcinomas in association with KRAS and p53 mutations and microsatellite instability	Supports the inclusion of BRAF exon 15 (V600-equivalent codon 599) in ctDNA panels for gallbladder cancer, enabling detection of mutually exclusive RAS/RAF alterations with potential diagnostic and therapeutic relevance

BTC: Biliary tract cancer; CCA: Cholangiocarcinoma; CCC: Cholangiocellular carcinoma; ctDNA: Circulating tumor DNA; EGFR: Epidermal growth factor receptor; GBC: Gallbladder carcinoma; HOXA9; Homeobox A9; iCCA: Intrahepatic cholangiocarcinoma; MAF: Mutant allele frequency; MAPK: Mitogen-activated protein kinase; mIDH1; Mutated form of the isocitrate dehydrogenase 1 enzyme; PIK3CA; Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PFS: Progression-free survival; TCGA: The Cancer Genome Atlas; TP53: Tumor protein p53; VAF: Variant allele frequency.