Background/Objectives: Biliary tract cancer (BTC) is a rare but aggressive malignancy that requires surgical treatment. However, postoperative recurrence rates are high, and reliable predictors of recurrence are limited. This study aimed to investigate the effectiveness of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) in predicting early recurrence after curative surgery and complete adjuvant therapy in patients with BTC. Methods: Twenty-four patients who underwent R0 and R1 resections and completed adjuvant therapy for BTC between September 2019 and March 2022 were followed up until March 2024. Patients were categorized into early recurrence (ER) and non-ER groups, using one year as the cutoff for recurrence. Results: The combination score derived from ultrashort fragments of cfDNA, vimentin-positive CTCs, and carbohydrate antigen (CA) 19-9 levels showed a statistically significant difference between the ER and non-ER groups (p-value < 0.001). The receiver operating characteristic curve from the combination score and CA 19-9 levels yielded areas under the curve of 0.891 and 0.750, respectively. Conclusions: Although further research is required, these findings suggest that cfDNA and CTCs may increase the accuracy of predicting postoperative recurrence in patients with BTC.

Bile contains many bacteria that can contribute to various diseases. Therefore, identifying bile microbiome differences between benign and malignant conditions is essential. In this study, bile samples were collected aseptically from 141 patients with biliary tract cancer (BTC) or benign biliary diseases (BBDs) who underwent endoscopic retrograde cholangiopancreatography or biliary tract surgery. Quality control PCR was performed to amplify the V3–V4 region of the bacterial 16S rRNA gene. Metagenomic sequencing of bile was successfully performed in 35 of 56 samples collected from patients with BTC and 24 of 85 samples from patients with BBD. The mean alpha diversity values comprised 2.788 ± 2.833 and 2.319 ± 1.355 in the BBD and BTC groups, respectively (p = 0.399). The bacterial species (4.7%) were shared between groups, whereas 12.3% and 83% were indicated to patients with BTC and BBD, respectively. Bacteroides coprocola, Prevotella copri, and Bacteroides plebeius were more frequently identified in the bile of patients with BTC, whereas Bacteroides vulgatus and Bacteroides uniformis were more abundant in the bile of patients with BBD. Distinct patterns of microorganism abundance between the two groups of patients suggest association of bile microbiome with disease status, so its diagnostic potential should be validated in further studies.

Background and Aim: Biliary tract cancers (BTCs) have a poor prognosis. One of the reasons for the prognosis is the difficulty of early diagnosis. The absence of a sufficient biomarker to screen for BTCs in high-risk populations makes the patient to be recognized in advanced status. Therefore, it is urgent to discover a biomarker for early detection of BTCs. This study analyzed extracellular vesicle (EV) peptides to find a new serum biomarker of BTCs. Methods: Plasma samples were obtained from 5 healthy people as control, 10 from patients with benign biliary diseases such as choledocholithiasis, sclerosing cholangitis, and gallbladder polyp, and 29 from BTCS patients. The EV peptides have been obtained from 2.5ml of patient plasma. The EV peptide samples were analyzed by liquid chromatography-data-dependent acquisition-tandem mass spectrometry (LC-DDA-MS/MS). The acquired peptides and proteins were investigated by searching in Fragpipe-MSFragger. Characterization was performed using the EVs database of previous studies. After that, Differential Expression Protein (DEP) analysis and Pathway/GO analysis are performed to know the biological process. Results: Total 25,382 peptides and 3,313 proteins were defined (PSM, peptide-level FDR 1%). Most of the gene ontology biological processes were related to vesicle-mediated transport. On DEP analysis, total 72 proteins were up-regulated, and 29 proteins were down-regulated. Compared to cancer samples, 9 proteins were up-regulated to both healthy and benign samples and 2 were down-regulated. According to biological process/pathway analysis, up-regulated 9 proteins were mostly related to cytoskeleton/ECM and Hemostasis. Down-regulated 2 proteins were related to endocytosis and immunity. Conclusions: By analyzing EV peptides, a total of 11 proteins have been identified. With validation experiments, we expect to finalize biomarker candidates. Citation Format: Dong Uk Kim, Sunghee Park, Sung Yong Han, Daye Jeon, Hyeji Lee, Jonghyun Lee, Hyeong Seok Nam, Dong Wook Lee, Chang-Min Cho, Min Kyu Jung, Jun Heo, Un-Beom Kang, Seong-Jun Park, Sunghyun Huh, Yourae Shin. Proteomic profiling of extracellular vesicles in patients with biliary tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3327.

Background: The role of normal tissues adjacent to tumors (NATs) in biliary tract cancer (BTC) remains unclear, despite their potential contributions to field cancerization. Methods: Targeted genomic profiling of tumor tissues, patient-matched NATs, and peripheral blood leukocytes from 13 patients with BTCs was performed. Clinicopathological data, including inflammatory conditions and precursor lesions (biliary intraepithelial neoplasia [BilIN] and intraductal papillary neoplasm of the bile duct), were integrated with genomic findings. Results: Tumor tissues exhibited recurrent alterations in genes regulating DNA damage response, cell cycle control, and oncogenic signaling. Importantly, rather than being genetically silent, NATs harbor early somatic variants distinct from those in both tumor and germline DNA. These alterations were not directly associated with cancer-related pathways, but rather with extracellular matrix-receptor interactions, suggesting that NATs may represent an intermediate step in carcinogenesis. All patients with extrahepatic cholangiocarcinoma presented with BilIN in adjacent tissues, providing histological evidence of field cancerization linked to chronic inflammation. Conclusions: This systematic comparison of tumors, NATs, and germline DNAs in BTCs revealed that NATs contain biologically relevant somatic mutations. The concordance between the inflammatory background, precursor lesions, and genomic alterations supports a multistep carcinogenic model and highlights opportunities for early BTC detection and risk stratification.