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Background: A new circulating biomarker superior to carbohydrate antigen 19-9 (CA19-9) is needed for diagnosing pancreatobiliary cancer (PBca). The aim of this study was to identify serum microRNA (miRNA) signatures comprising reproducible and disease-related miRNAs., Methods: This multicenter study involved patients with treatment-naïve PBca and healthy participants. The optimized serum processing conditions were evaluated using t-distributed stochastic neighbor embedding (t-SNE) visualization. Serum miRNA candidates for disease association were selected using weighted gene coexpression network analysis (WGCNA). A miRNA signature combining multiple serum miRNAs was tested in exploratory, validation, and independent validation sets. The synthesis and secretion of diagnostic miRNAs were evaluated using human pancreatic cancer cells., Results: In total, 284 (150 healthy and 134 PBca) of 827 serum samples were processed within 2 h of blood collection before freezing, distributed in the same area as that in the t-SNE map, and assigned to an exploratory set. The 193 optimized samples were assigned to either the validation (50 healthy, 47 PBca) or independent validation (50 healthy, 46 PBca) set. Index-1, a combination of five serum miRNAs (hsa-miR-1343-5p, hsa-miR-4632-5p, hsa-miR-4665-5p, hsa-miR-665, and hsa-miR-6803-5p) with disease association in WGCNA, showed a sensitivity and specificity of > 80% and an AUC outperforming that of CA19-9 in the exploratory, validation, and independent validation sets. The AUC of Index-1 was superior to that of CA19-9 (0.856 vs. 0.649, p = 0.038) for detecting T1 tumors. miR-665, a component of Index-1, was expressed in human pancreatic cancer cells, and its transfection inhibited cell growth., Conclusions: The serum miRNA signature Index-1 is useful for detecting PBca and could facilitate the early diagnosis of PBca. These findings can help improve clinical PBca detection by providing an optimized biomarker that overcomes the limitations of the current standard., Competing Interests: Declarations. Ethics approval and consent to participate: This prospective observational study was approved by the ethics review committee of the National Cancer Center Hospital East and National Cancer Center Hospital (approval no. 2016–049, 2020–449) and the institutional review boards of collaborating institutions. The study for time-course effects was approved by the Human Tissue Samples Ethics Committee for R&D of Toray Industries, Inc. (HC2017-10, HC2021-3, and HC2021-18). All the studies complied with the principles of the Declaration of Helsinki and the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects. Written informed consent was obtained from all participants. Consent for publication: Not applicable. Competing interests: S.M. received honoraria and grants from Toray Industries, Inc., and grants from the Japan Agency for Medical Research and Development (AMED) under Grant Numbers 15ck0106027h0002 and 16ck106027h0003 during the submitted work, as well as research funding from Ajinomoto Co., Inc., PFDeNA Co., Ltd., and Mitsui Chemicals Inc. outside the submitted work. M.I. received research funding from Astellas Pharma Inc., Nihon Servier Co., Ltd., and Novartis Pharma K.K. and received honoraria from Guardant Health Japan Corp., Nihon Servier Co., Ltd., Taiho Pharmaceutical Co., Ltd., M.S.D. K.K., NIPPON KAYAKU Co., Ltd., Yakult Honsha Co., Ltd., Boehringer Ingelheim GmbH, D.F.P. (Delta Fly Pharma) Inc., Merus N.V., Invitae Corp., and Novartis Pharma K.K. outside the submitted work. M.U. received grants from Toray Industries, Inc., during the submitted work, as well as research funding from Taiho Pharmaceutical Co., Ltd., AstraZeneca K.K., M.S.D. K.K., Nihon Servier Co., Ltd., Ono Pharmaceutical Co., Ltd., Incyte Biosciences Japan G.K., Chugai Pharmaceutical Co., Ltd., Boehringer Ingelheim GmbH, Eisai Co., Ltd., Novartis Pharma K.K., Astellas Pharma Inc., J-pharma Co., Ltd., DFP (Delta Fly Pharma) Inc., Novocure GmbH, and Chiome Bioscience Inc. and honoraria from Taiho Pharmaceutical Co., Ltd., AstraZeneca K.K., Yakult Honsha Co., Ltd., M.S.D. K.K., Nihon Servier Co., Ltd., Ono Pharmaceutical Co., Ltd., Incyte Biosciences Japan G.K., Chugai Pharmaceutical Co., Ltd., Boehringer Ingelheim GmbH, J-pharma Co., Ltd., Daiichi Sankyo Co., Ltd., Eisai Co., Ltd., Takeda Pharmaceutical Co., Ltd., and Novartis Pharma K.K. outside the submitted work. K.H2. received honoraria and research funding from Toray Industries, Inc., during the submitted work. K.T. received research funding from Toray Industries, Inc., during the submitted work, received research funding from Hitachi High-Tech Corporation and H.U. Group Research Institute G.K., and holds a patent with H.U. Group Research Institute G.K. outside the submitted work. M.T., I.M., T.K., H.Y., H.I1, H.I2, E.H., K.N., M.N., and A.O. received research funding from Toray Industries, Inc., during the submitted work. H.S. is an employee of Toray Industries, Inc., and received grants from AMED under grant numbers 15ck0106027h0002 and 16ck106027h0003 during the submitted work. S.T. is an employee of Toray Industries, Inc., and received grants from AMED under grant numbers 15ck0106027h0002 and 16ck106027h0003 during the submitted work. The other authors declare that they have no competing interests., (© 2025. The Author(s).)

Background: Plasma cell-free DNA (cfDNA) fragmentomics has demonstrated significant differentiation power between cancer patients and healthy individuals, but little is known in pancreatic and biliary tract cancers. The aim of this study is to characterize the cfDNA fragmentomics in biliopancreatic cancers and develop an accurate method for cancer detection., Methods: One hundred forty-seven patients with biliopancreatic cancers and 71 non-cancer volunteers were enrolled, including 55 patients with cholangiocarcinoma, 30 with gallbladder cancer, and 62 with pancreatic cancer. Low-coverage whole-genome sequencing (median coverage: 2.9 ×) was performed on plasma cfDNA. Three cfDNA fragmentomic features, including fragment size, end motif and nucleosome footprint, were subjected to construct a stacked machine learning model for cancer detection. Integration of carbohydrate antigen 19-9 (CA19-9) was explored to improve model performance., Results: The stacked model presented robust performance for cancer detection (area under curve (AUC) of 0.978 in the training cohort, and AUC of 0.941 in the validation cohort), and remained consistent even when using extremely low-coverage sequencing depth of 0.5 × (AUC: 0.905). Besides, our method could also help differentiate biliopancreatic cancer subtypes. By integrating the stacked model and CA19-9 to generate the final detection model, a high accuracy in distinguishing biliopancreatic cancers from non-cancer samples with an AUC of 0.995 was achieved., Conclusions: Our model demonstrated ultrasensitivity of plasma cfDNA fragementomics in detecting biliopancreatic cancers, fulfilling the unmet accuracy of widely-used serum biomarker CA19-9, and provided an affordable way for accurate noninvasive biliopancreatic cancer screening in clinical practice., (© 2024. The Author(s).)

Background: Biliary tract cancer (BTC) is a relatively rare but aggressive gastrointestinal cancer with a high mortality rate. Cancer stem cell (CSC) populations play crucial roles in tumor biology and are responsible for the low response to anti-cancer treatment and the high recurrence rate. This study investigated the role of Transgelin-2 (TAGLN2), overexpressed in CSC in BTC cells, and analyzed its expression in patient tissues and serum to identify potential new targets for BTC., Methods: TAGLN2 expression was suppressed by small-interfering or short hairpin RNAs, and its effects on tumor biology were assessed in several BTC cell lines. Furthermore, the effects of TAGLN2 silencing on gemcitabine-resistant BTC cells, differentially expressed genes, proteins, and sensitivity to therapeutics or radiation were assessed. TAGLN2 expression was also assessed using western blotting and immunohistochemistry in samples obtained from patients with BTC to validate its clinical application., Results: Suppression of TAGLN2 in BTC cell lines decreased cell proliferation, migration, invasion, and tumor size, in addition to a reduction in CSC features, including clonogenicity, radioresistance, and chemoresistance. TAGLN2 was highly expressed in BTC tissues, especially in cancer-associated fibroblasts in the stroma. Patients with a low stromal immunohistochemical index had prolonged disease-free survival compared to those with a high stromal immunohistochemical index (11.5 vs. 7.4 months, P = 0.013). TAGLN2 expression was higher in the plasma of patients with BTC than that in those with benign diseases. TAGLN2 had a higher area under the curve (0.901) than CA19-9, a validated tumor biomarker (0.799; P < 0.001)., Conclusion: TAGLN2 plays a critical role in promoting BTC cell growth and motility and is involved in regulating BTC stemness. Silencing TAGLN2 expression enhanced cell sensitivity to radiation and chemotherapeutic drugs. The expression of TAGLN2 in patient tissue and plasma suggests its potential to serve as a secretory biomarker for BTC. Overall, targeting TAGLN2 could be an appropriate therapeutic strategy against advanced cancer following chemotherapy failure., (© 2024. The Author(s).)

Background: Associations of High-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, total cholesterol (CHL), and triglyceride (TRG) concentrations with risk of biliary tract cancer (BtC) were conflicting in observational studies. We aim to investigate the causal link between circulating lipids and BtC using genetic information., Methods: Single nucleotide polymorphisms of the four circulating lipids (n = 34,421) and BtC (418 cases and 159,201 controls) were retrieved from two independent GWAS studies performed in East Asian populations. Two-sample univariate and multivariate Mendelian Randomization (MR) analyses were conducted to determine the causal link between circulating lipids and BtC., Results: No significant horizontal pleiotropy was detected for all circulating lipids according to the MR-PRESSO global test (P = 0.458, 0.368, 0.522, and 0.587 for HDL, LDL, CHL, and TRG, respectively). No significant evidence of heterogeneity and directional pleiotropy was detected by the Cochran's Q test and MR-Egger regression. Univariate MR estimates from inverse variance weighting method suggested that one standard deviation (1-SD) increase of inverse-normal transformed HDL (OR = 1.38, 95% CI 0.98-1.94), LDL (OR = 1.46, 95% CI 0.96-2.23), and CHL (OR = 1.34, 95% CI 0.83-2.16) were not significantly associated with BtC risk. Whereas 1-SD increase of inverse-normal transformed TRG showed a significantly negative association with BtC risk (OR = 0.48, 95% CI 0.31-0.74). In multivariate MR analyses including all the four lipid traits, we found that 1-SD increase of LDL and TRG was significantly associated with elevated (OR = 1.32, 95% CI 1.04-2.01) and decreased (OR = 0.54, 95% CI 0.42-0.68) risk of BtC, respectively., Conclusion: Circulating lipids, particularly LDL and TRG, may have roles in the development of BtC. However, the results of this study should be replicated in MR with larger GWAS sample sizes for BtC., (© 2022. The Author(s).)

Background: DNA methylation-associated studies on biliary tract cancer (BTC), including cholangiocarcinoma (CCA) and gallbladder cancer (GBC), may improve the BTC classification scheme. We proposed to identify the shared methylation changes of BTCs and investigate their associations with genomic aberrations, immune characteristics, and survival outcomes., Methods: Multi-dimensional data concerning mutation, DNA methylation, immune-related features, and clinical data of 57 CCAs and 48 GBCs from Eastern Hepatobiliary Surgery Hospital (EHSH) and 36 CCAs in the TCGA-CHOL cohort were analyzed., Results: In our cohort including 24 intrahepatic CCAs (iCCAs), 20 perihilar CCAs (pCCAs), 13 distal CCAs (dCCAs), and 48 GBCs, 3369 common differentially methylated regions (DMRs) were identified by comparing tumor and non-tumor samples. A lower level of methylation changes of these common DMRs was associated with fewer copy number variations, fewer mutational burden, and remarkably longer overall survival (OS, hazard ratio [HR] = 0.07, 95% confidence interval [CI] 0.01-0.65, P = 0.017). Additionally, a 12-marker model was developed and validated for prognostication after curative surgery (HR = 0.21, 95% CI 0.10-0.43, P < 0.001), which exhibited undifferentiated prognostic effects in subgroups defined by anatomic location (iCCAs, d/pCCAs, GBCs), TNM stage, and tumor purity. Its prognostic utility remained significant in multivariable analysis (HR = 0.26, 95% CI 0.11-0.59, P = 0.001). Moreover, the BTCs with minimal methylation changes exhibited higher immune-related signatures, infiltration of CD8 + lymphocytes, and programmed death-ligand 1 (PD-L1) expression, indicating an inflamed tumor immune microenvironment (TIME) with PD-L1 expression elicited by immune attack, potentially suggesting better immunotherapy efficacy., Conclusions: In BTCs, DNA methylation is a powerful tool for molecular classification, serving as a robust indicator of genomic aberrations, survival outcomes, and tumor immune microenvironment. Our integrative analysis provides insights into the prognostication after curative surgery and patient selection for immunotherapy., (© 2022. The Author(s).)

Background: Pancreatic and biliary tract cancer (PC and BTC, respectively) are difficult to diagnose because of their clinical characteristics; however, recent studies suggest that serum microRNAs (miRNAs) might be the key to developing more efficient diagnostic methods for these cancers., Methods: We analysed the genome-wide expression of serum miRNAs in PC and BTC patients to identify novel biomarker candidates using high-throughput sequencing and experimentally validated miRNAs on clinical samples., Results: Statistical and classification analysis of the serum miRNA-expression profiles of 55 patient samples showed distinguishable patterns between cancer patients and healthy controls; however, we were unable to distinguish the two cancers. We found that three of the highest performing miRNAs were capable of distinguishing cancer patients from controls, with an accuracy of 92.7%. Additionally, dysregulation of these three cancer-specific miRNAs was demonstrated in an independent sample group by quantitative reverse transcription polymerase chain reaction., Conclusions: These results suggested three candidate serum miRNAs (mir-744-5p, mir-409-3p, and mir-128-3p) as potential biomarkers for PC and BTC diagnosis.

Background: Biliary tract carcinoma (BTC) is a fatal malignancy which aggressiveness contrasts sharply with its relatively mild and late clinical presentation. Novel molecular markers for early diagnosis and precise treatment are urgently needed. The purpose of this study was to evaluate the diagnostic and prognostic value of promoter hypermethylation of the SHOX2 and SEPT9 gene loci in BTC., Methods: Relative DNA methylation of SHOX2 and SEPT9 was quantified in tumor specimens and matched normal adjacent tissue (NAT) from 71 BTC patients, as well as in plasma samples from an independent prospective cohort of 20 cholangiocarcinoma patients and 100 control patients. Receiver operating characteristic (ROC) curve analyses were performed to probe the diagnostic ability of both methylation markers. DNA methylation was correlated to clinicopathological data and to overall survival., Results: SHOX2 methylation was significantly higher in tumor tissue than in NAT irrespective of tumor localization ( p  < 0.001) and correctly identified 71% of BTC specimens with 100% specificity (AUC = 0.918; 95% CI 0.865-0.971). SEPT9 hypermethylation was significantly more frequent in gallbladder carcinomas compared to cholangiocarcinomas ( p  = 0.01) and was associated with large primary tumors ( p  = 0.01) as well as age ( p  = 0.03). Cox proportional hazard analysis confirmed microscopic residual tumor at the surgical margin (R1-resection) as an independent prognostic factor, while SHOX2 and SEPT9 methylation showed no correlation with overall survival. Elevated DNA methylation levels were also found in plasma derived from cholangiocarcinoma patients. SHOX2 and SEPT9 methylation as a marker panel achieved a sensitivity of 45% and a specificity of 99% in differentiating between samples from patients with and without cholangiocarcinoma (AUC = 0.752; 95% CI 0.631-0.873)., Conclusions: SHOX2 and SEPT9 are frequently methylated in biliary tract cancers. Promoter hypermethylation of SHOX2 and SEPT9 may therefore serve as a minimally invasive biomarker supporting diagnosis finding and therapy monitoring in clinical specimens.

Background: Biliary cancers are highly aggressive tumors that are often diagnosed an advanced disease stage and have a poor outcome with systemic therapy. Recent efforts towards molecular characterization have identified a subset of biliary patients that have HER2/neu amplification or mutation. HER2/neu amplification is associated with response to HER2/neu-directed therapy in breast and gastric cancers. However, the efficacy of HER2/neu-targeted therapy in biliary cancers is unknown., Patients and Methods: We retrospectively reviewed cases of advanced gallbladder cancer and cholangiocarcinoma with HER2/neu genetic aberrations or protein overexpression who received HER2/neu-directed therapy between 2007 and 2014. Clinical data were retrieved from medical records, and imaging studies were independently reviewed., Results: Nine patients with gallbladder cancer and five patients with cholangiocarcinoma had received HER2/neu-directed therapy (trastuzumab, lapatinib, or pertuzumab) during the study period. In the gallbladder cancer group, HER2/neu gene amplification or overexpression was detected in eight cases. These patients experienced disease stability (n = 3), partial response (n = 4), or complete response (n = 1) with HER2/neu-directed therapy. One patient had HER2/neu mutation and experienced a mixed response after lapatinib therapy. The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. One patient developed HER2/neu amplification as a secondary event after FGFR-directed therapy for FGF3-TACC3 gene fusion. The cholangiocarcinoma cases treated in this series had a higher proportion of HER2/neu mutations, and no radiological responses were seen in these patients despite HER2/neu-directed therapy., Conclusions: HER2/neu blockade is a promising treatment strategy for gallbladder cancer patients with gene amplification and deserves further exploration in a multi-center study.

Background: Standard chemotherapy in unresectable biliary tract carcinoma (BTC) patients is based on gemcitabine combined with platinum derivatives. However, primary or acquired resistance is inevitable and no second-line chemotherapy is demonstrated to be effective. Thus, there is an urgent need to identify new alternative (chemo)therapy approaches., Methods: We evaluated the mechanism of action of ET-743 in preclinical models of BTC. Six BTC cell lines (TFK-1, EGI-1, TGBC1, WITT, KMCH, HuH28), two primary cell cultures derived from BTC patients, the EGI-1 and a new established BTC patient-derived xenografts, were used as preclinical models to investigate the anti-tumor activity of ET-743 in vitro and in vivo. Gene expression profiling was also analyzed upon ET-743 treatment in in vivo models., Results: We found that ET-743 inhibited cell growth of BTC cell lines and primary cultures (IC50 ranging from 0.37 to 3.08 nM) preferentially inducing apoptosis and activation of the complex DNA damage-repair proteins (p-ATM, p-p53 and p-Histone H2A.x) in vitro. In EGI-1 and patient-derived xenografts, ET-743 induced tumor growth delay and reduction of vasculogenesis. In vivo ET-743 induced a deregulation of genes involved in cell adhesion, stress-related response, and in pathways involved in cholangiocarcinogenesis, such as the IL-6, Sonic Hedgehog and Wnt signaling pathways., Conclusions: These results suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials in BTC patients resistant to standard chemotherapy.

Background: Genetic variants in inflammation-related genes have been associated with biliary stones and biliary tract cancers in previous studies., Methods: To follow-up on these findings, we examined 35 single nucleotide polymorphism (SNPs) in 5 genes related to inflammation (IL8, NFKBIL, RNASEL, TNF, and VEGFA) in 456 participants with incident biliary tract cancer cases (262 gallbladder, 141 extrahepatic bile duct, 53 ampulla of Vater), 982 participants with biliary stones, and 860 healthy controls in a population-based case-control study in Shanghai, China., Results: Suggestive associations were observed for SNPs in VEGFA with biliary stones, IL8 with gallbladder and ampulla of Vater cancers, and RNASEL with ampulla of Vater cancer (false discovery rate≤0.2)., Conclusion: These findings provide additional support for the role of inflammation in biliary stones and biliary tract cancer risk and need further validation.

Background: Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC., Methods: Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs., Results: EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis., Conclusion: These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

Background: The pathogenesis of biliary cancers is ill-defined. This study investigates changes in gene expression and copy number in biliary cancers and correlates these changes with anatomical site of origin, histopathology and outcome., Methods: We performed gene expression and CGH analysis on 34 biliary tract cancer specimens. Results were confirmed by RT-PCR. Clinical-pathologic correlation was made using functional over-representation analysis of the top 100 mutations associated with each variable., Results: There were 545 genes with altered expression in extrahepatic cholangiocarcinoma, 2,354 in intrahepatic cholangiocarcinoma, and 1,281 in gallbladder cancer. Unsupervised hierarchical clustering analysis indicated there was no difference in the global gene expression patterns between each biliary cancer subgroup. CGH analysis revealed that short segments of chromosomes 1p, 3p, 6q, 8p, 9p, and 14q were commonly deleted across all cancer subtypes. Commonly amplified regions included segments of 1q, 3q, 5p, 7p, 7q, 8q, and 20q. Over-representation analysis revealed an association between altered expression of functional gene groupings and pathologic features., Conclusion: This study defined regions of the genome associated with changes in DNA copy number and gene expression in specific subtypes of biliary cancers. The findings have implications for identification of therapeutic targets, screening, and prognostication.