Cholangiocarcinoma is a lethal tumour of the bile ducts. Cholangiocarcinoma fibroblast growth factor receptor gene fusions forms can be targeted by Pemigatinib (PGT). PGT a recently approved kinase inhibitor by the US-FDA, has its approval accelerated due to the disease viciousness. Development of a sensitive yet available and economical analytical platform to quantify PGT in human plasma is genuinely needed. Enabling monitoring of the therapeutic plan, hence, ensuring the drug efficacy and safety through pharmacokinetic studies. High-performance liquid chromatography with fluorescence detector (HPLC-FD) method is proposed using the native fluorescence of PGT. PGT and seliciclib (internal standard) chromatographic conditions optimisation, revealed favourable use of isocratic mobile phase consisting of methanol:ammonium acetate buffer (70:30v/v, pH5.0) pumped into C18-column (150 mm length × 4.6 mm internal diameter, 5 μm particle size), at 1 mL min-1 flow rate. PGT and seliciclib fluorescence excitation and emission were measured at 280 and 360 nm, respectively. Validation of the HPLC-FD method was processed based on the International Council for Harmonization guidelines. The method linearity range was 5-300 ng mL-1. The limits of detection and quantification were 2.8 and 8.5 ng mL-1, respectively. High precision and accuracy indicated by relative standard deviation ≤ 5.2, and recovery values of 95.4-102.2 %. Evident of the method greenness was verified by green analytical chemistry metric tools. HPLC-FD method was successfully applied to study PGT pharmacokinetics in rats. In conclusion, this study introduces a reliable analytical method of PGT in plasma for routine use in therapeutic drug monitoring for quality assurance and clinical follow-up.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) shares various features with small duct type intrahepatic cholangiocarcinoma (SmD-iCCA) and sometimes histological diagnosis may be difficult.

We examined genetic alterations such as hTERT promoter (hTERT), p53, and fibroblast growth factor receptor 2 (FGFR2) in 103 PLCs diagnosed as cHCC-CCA or SmD-iCCA. A cluster analysis was performed on the R software for re-classification of PLCs including cHCC-CCA and SmD-iCCA.

The primary liver carcinomas (PLCs) were divided into 5 clusters; 19 tumors (18 %) in Cluster-1 (with alterations in hTERT and/or p53), 24 (23 %) in Cluster-2 (FGFR2 and/or p53), 13 (13 %) in Cluster-3 (IDH2 or null), 19 (18 %) in Cluster-4 (MTAP and/or FGFR2), 28 (27 %) in Cluster-5 (ARID1A and/or PBRM1), being based on genetic alterations. Cluster-1 and Clusters-2 to- 5 formed distinct 2 groups. Cluster-1 was characterized by significantly bigger size, rich and higher histological grade of HCC component, significantly less cholangiolocellular carcinoma (CLC)-component, ductal plate malformation pattern and bile duct adenoma in the background livers. No SmD-iCCA was included in Cluster-1, whereas SmD-iCCA distributed evenly in Clusters 2-5. Cluster-4 was characterized by higher prevalence of hepatitis B and higher histological diversity scores.

PLCs diagnosed as cHCC-CCA or SmD-iCCAs could be divided into 5 clusters based on genetic alterations. Cluster-1 was HCC-like cluster characterized by hTERT alteration, rich and higher grade of HCC and bigger size. Clusters-2-5 may be iCCA-like clusters characterized by different genetic alterations. cHCC-CCA in Cluster-1 and Clusters-2-5 may be handled separately for further analysis and treatment.

Hepatobiliary cancers are heterogeneous and molecularly complex. Recent advances in next-generation sequencing (NGS) have enhanced the understanding of their molecular landscape and enabled deployment of biomarker-based gene- and immune-targeted therapies. This review examines the role of molecular testing and targeted therapies in these malignant neoplasms.

Patients with hepatobiliary cancers have poor outcomes. Precision oncology studies have shown that while many common molecular alterations are not currently targetable in hepatocellular carcinoma (HCC), a large number of actionable alterations characterize biliary tract cancers (BTCs), with several therapies now approved by the US Food and Drug Administration. Immunotherapy is increasingly adopted in clinical practice, either as monotherapy or combined with cytotoxic chemotherapy, for both HCC and BTCs. Moreover, multiple solid cancer tumor-agnostic therapies are approved (larotrectinib, entrectinib, and repotrectinib for NTRK fusions; selpercatinib for RET fusions; dabrafenib and trametinib combination for BRAF V600E mutations; dostarlimab or pembrolizumab for tumors with high microsatellite instability and pembrolizumab for tumor mutation burden ≥10 mutations/megabase), highlighting the need for NGS as well as ERBB2 (formerly HER2) immunohistochemistry (IHC) (with the recent approval of solid tissue-agnostic deruxtecan trastuzumab for ERBB2-positive [IHC 3+] cancer) across cancers. N-of-1 clinical trials using customized drug combinations matched to the tumor's molecular profile have yielded encouraging results and provide a promising framework for future clinical trial design.

Molecular testing and gene- and immune-targeted therapies are transforming hepatobiliary cancer treatment. Tumor-agnostic and N-of-1 clinical trials have challenged traditional clinical trial paradigms and provide the foundation for truly personalized oncology for patients with these aggressive cancers. Further work is needed to determine how to leverage these novel approaches into the management of operable disease.

The Japanese Society of Hepato-Biliary-Pancreatic Surgery guidelines propose a classification scheme that differs from the Union for International Cancer Control (UICC) system, in which the anatomic U-P point is the boundary between intrahepatic cholangiocarcinoma and perihilar cholangiocarcinoma (PCC).

To investigate whether this classification system improves clinicopathologic and genomic differentiation.

Fifty-eight PCC cases defined by the UICC system were collected and classified into intrahepatic PCC (IPCC) and extrahepatic PCC (EPCC) categories using U-P point division. They were analyzed by next-generation sequencing using a panel that targeted 425 cancer-related genes.

The IPCC group exhibited a significantly larger tumor size compared with the EPCC group (4.67 ± 2.44 cm versus 2.50 ± 0.91 cm, P = .002). The mutation frequency of the KRAS proto-oncogene, GTPase (KRAS) Q61 was also significantly higher in the IPCC group than in the EPCC group (16.7% versus 0.0%, P = .03). There were no statistically significant differences in other pathologic features or genomic characteristics, including tumor mutation burden and microsatellite instability. Significant differences in gene mutation rates, such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; 0.0% versus 15.8%, P = .01) and tumor protein p53 (TP53; 34.5% versus 63.2%, P = .04), were observed between PCC and adjacent biliary tract cancers.

This study offers valuable insight into the clinicopathologic and genomic features of PCC. It is proposed that the U-P point division may have limited potential to refine the characterization of PCC regarding these features, and that the UICC classification system can readily demonstrate the molecular specificity of PCC.

Recent years have witnessed significant advances in the imaging, molecular profiling, and systemic treatment of cholangiocarcinoma (CCA). Despite this progress, the early detection, precise classification, and effective management of CCA remain challenging. Owing to recent developments and the significant differences in CCA subtypes, EASL commissioned a panel of experts to draft evidence-based recommendations on the management of extrahepatic CCA, comprising distal and perihilar CCA. Particular attention is given to the need for accurate classification systems, the integration of emerging molecular insights, and practical strategies for diagnosis and treatment that reflect real-world clinical scenarios.

In the HERIZON BTC 01 trial for patients with HER2-positive biliary tract cancer (BTC) previously treated with systemic therapy, zanidatamab improved the objective response rate, disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). However, real-world data are needed to assess its efficacy and safety outside clinical trials.

We conducted an investigator initiated national multicenter retrospective study of most patients with BTC treated with zanidatamab in France as part of a compassionate access. The primary endpoint was PFS.

Our study included 20 patients with metastatic BTC enrolled between September 2022 and November 2024. The median age at diagnosis was 61.5 (interquartile range: 55-69) years and the majority of patients had gallbladder cancer (n = 12, 60 %). After a median follow-up of 8.5 (95 % confidence interval [CI]: 3.3-11.8) months, the median PFS was 6.7 (95 % CI 1.3-11.8) months, with an estimated OS at 1 year of 79.1 % (95 % CI: 53.2-91.6 %). The DCR was 65 %, with 40 % confirmed partial responses and a median duration of response of 7.3 (95 % CI: 2.06-16) months. Patients with immunohistochemistry (IHC) 3 + HER2 scores had a better PFS [8 (95 % CI: 1.5-18.4) months] than those with 2 + HER2 scores obtained by IHC followed by fluorescence in situ hybridization amplification or next-generation sequencing [1.4 (95 % CI: 1.1-6.8) months] (P = 0.02). No statistical difference in 1-year estimated OS rates was observed (P = 0.39). There were no grade 3 or 4 treatment-related adverse events or cardiac toxicities.

The benefits of in patients with HER2-positive BTC were confirmed. Zanidatamab should be considered for patients with this condition.

Anti-claudin 18.2 (anti-CLDN18.2) therapy has been approved for patients with CLDN18-positive gastric and gastroesophageal junction adenocarcinomas. The current study aims at evaluating the expression of CLDN18 in a large cohort of pathologically characterized biliary tract cancers (BTCs).

A series of 237 BTCs were collected and reviewed under the BITCOIN protocol. All samples were assessed for CLDN18 status using immunohistochemistry (clone 43-14A). Tumor positivity for CLDN18 was determined if ≥75% of tumor cells exhibited moderate-to-strong membranous staining.

CLDN18 expression was found in 29.5% of BTCs (70/237), with the highest rates in gallbladder carcinoma (GBC; 62.5%; 20/32) and extrahepatic cholangiocarcinoma (eCCA; 53.4%; 31/58), compared with intrahepatic cholangiocarcinoma (iCCA; 12.9%; 19/147) (P < 0.0001). CLDN18 positivity was detected in 5.5% of cases (13/237), most common in GBC (15.6%; 5/32), followed by eCCAs (8.6%; 5/58) and iCCAs (2.0%; 3/147) (P = 0.0045). Most CLDN18-positive samples (10/13) exhibited a heterogenous staining pattern. In iCCAs, large duct subtypes had higher CLDN18 expression [33.3% (10/30) versus 7.7% (9/117), P = 0.0002] and positivity [6.7% (2/30) versus 0.9% (1/117), P = 0.106] than small duct iCCAs. No significant differences were observed across GBC and eCCA histotypes, and CLDN18 was not associated with IDH1 or FGFR2 status in iCCAs.

This study demonstrates that CLDN18 expression is present in a subset of BTCs, with significantly higher positivity rates in GBCs and eCCAs compared with iCCAs. In iCCAs, CLDN18 expression was more frequent in the large duct subtype but was not associated with IDH1 or FGFR2 status. These findings suggest that CLDN18 could be a potential therapeutic target in BTCs, warranting further prospective studies to evaluate its clinical significance and impact on patient outcomes.

Patients with intrahepatic cholangiocarcinoma (iCCA) harboring FGFR2 fusion/rearrangement benefit from targeted therapies, highlighting the need for reliable testing strategies to identify FGFR2 alterations. We assessed 226 iCCA cases using RNA-based NGS, DNA-based NGS, and break-apart FISH to evaluate the effectiveness of these methods in detecting FGFR2 fusion/rearrangement. The detection rates for FGFR2 fusion/rearrangement were 9.7% (22/226) for RNA-based NGS, 7.1% (16/226) for DNA-based NGS, and 10.2% (23/226) for FISH. Among the 26 FGFR2 fusion/rearrangement-positive cases identified by any method, only 15 (57.7%) were positive by all three techniques, yielding a concordance rate of 95.1% (215/226). RNA-based NGS confirmed oncogenic FGFR2 fusion in 81% (21/26) of positive cases and identified five novel oncogenic fusions. Thirty-five percent (6/17) of the partner genes were located on chromosome 10, with BICC1 being the most common fusion partner, while the rest were distributed across the other 9 chromosomes. FISH demonstrated a sensitivity of 95.2% and specificity of 98.5%, compared to oncogenic FGFR2 fusions confirmed by RNA-based NGS, while DNA-based NGS exhibited a sensitivity of 71.4% and specificity of 99.5%, identifying FGFR2 mutations in 4 cases. FGFR2-FISH positive cases displayed no significant heterogeneity in positive cell distribution. Oncogenic FGFR2 fusion/rearrangement was associated with small duct type iCCA, especially in cases with positive serum HBsAg and absent cholangiolocarcinoma components and peripheral liver steatosis. This study provides a comprehensive comparison of three assays for detecting FGFR2 fusion/rearrangement, along with clinicopathologic characterization of oncogenic FGFR2 fusion in iCCA.

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited methods for early detection, necessitating the development of reliable biomarkers for diagnosis and management. However, conventional tumor markers, such as CA19-9 and CEA, exhibit insufficient diagnostic accuracy. Recent advancements in molecular genetics have identified several actionable mutations in CCA, enabling molecularly targeted therapies that improve survival in patients harboring these genetic alterations. Cancer panels, which facilitate multiplex genetic profiling, are critical for identifying these mutations. Studies indicate that several actionable mutations are detected in CCA cases, with patients receiving mutation-guided therapies achieving markedly better outcomes. Liquid biopsies, including cell-free DNA and circulating tumor DNA, offer real-time, non-invasive approaches to monitoring tumor dynamics, heterogeneity, and treatment responses. Furthermore, numerous studies have identified non-coding RNAs in serum and bile as promising biomarkers for the diagnosis and management of CCA. On the other hand, immunotherapy, particularly immune checkpoint inhibitors, has shown efficacy in subsets of CCA patients. However, the success of these therapies is often affected by the status of the tumor immune microenvironment (TME), underscoring the need for comprehensive TME analysis to predict responses to immune checkpoint inhibitors. Despite these advances, no single biomarker currently demonstrates sufficient sensitivity or specificity for clinical application. The integration of multi-omics approaches with cutting-edge technologies holds promise for enhancing diagnostic accuracy, optimizing treatment stratification, and advancing precision medicine in CCA. These developments highlight the transformative potential of biomarkers to improve early detection, prognostic assessment, and personalized therapeutic interventions for CCA.

Biliary tract cancer (BTC) remains among the most challenging malignancies with a poor prognosis and limited treatment options, particularly in pretreated patients. As most patients experience disease progression after first-line treatment, effective second-line and subsequent treatments are required. Although the addition of modified FOLFOX (fluorouracil, leucovorin, and oxaliplatin) to active symptom control improved the overall survival of patients with progressing advanced BTC despite gemcitabine plus cisplatin treatment, its efficacy was modest. Moreover, most clinical trials demonstrated modest efficacy of molecular-targeted agents for molecularly unselected pretreated advanced BTC. Patients with advanced BTC carry a relatively high druggable genetic alteration rate and have shown promising responses to molecular-matched therapies targeting gene alterations such as FGFR2 fusions/rearrangements, IDH1 mutation, and HER2 overexpression/amplification. Additionally, tumor-agnostic approaches, including BRAF V600E, NTRK fusion, and RET fusion, have expanded the treatment options for some patients. Immune checkpoint inhibitors have shown limited efficacy as mono- or combination therapy in patients with pretreated advanced BTC. Therefore, developmental efforts have shifted to immune checkpoint inhibitor and other combinations such as vascular endothelial growth factor receptor inhibitors or radiation. In addition to refining combination strategies to enhance the therapeutic potential of immune checkpoint inhibitor, future research should focus on elucidating the tumor microenvironment. This review delineates the evolution of systemic therapies in patients with pretreated advanced BTC. By examining past developments and recent advances through prospective trials, it highlights novel approaches that may improve outcomes in this challenging disease.

The purpose of this review is to summarize the current knowledge of cholangiocarcinoma molecular biology and to suggest a framework for implementation of next-generation sequencing in all stages of liver transplantation. This is timely as recent guidelines recommend increased use of these technologies with promising results.

The main themes covered here address germline and somatic genetic alterations recently discovered in cholangiocarcinoma, particularly those associated with prognosis and treatment responses, and nascent efforts to translate these into contemporary practice in the peri-liver transplantation period.

Early efforts to translate molecular profiling to cholangiocarcinoma care demonstrate a growing number of potentially actionable alterations. Still lacking is a consensus on what biomarkers and technologies to adopt, at what scale and cost, and how to integrate them most effectively into care with the ambition of increasing the number of patients eligible for liver transplantation and improving their long-term outcomes.

Fibrolamellar carcinoma (FLC) is a unique primary carcinoma of the liver that is characterized by distinct morphologic findings and a recurrent DNAJB1::PRKACA gene fusion. It typically presents in young individuals without underlying liver dysfunction. FLC is difficult to diagnose when based only on morphology, and misdiagnosis is common. Frequent differential diagnoses include conventional hepatocellular carcinoma and intrahepatic cholangiocarcinoma, both of which can show similar morphologic and immunohistochemical features. If based only on molecular analysis, other differential diagnoses have recently emerged, as the DNAJB1::PRKACA fusion has now been reported in cases of intraductal oncocytic papillary neoplasm and intraductal papillary mucinous neoplasm. In this article, we review our diagnostic approach to FLC, which relies on both morphologic and immunohistochemical features, as well as molecular analysis.

www.clinicaltrials.gov identifier is NCT06607302.

Biliary tract cancers (BTCs) constitute a heterogeneous group of malignancies with rising incidence and limited therapeutic options in advanced stages, leading to increased overall mortality. Extensive genomic profiling has identified key oncogenic drivers in BTCs that represent promising therapeutic targets and could change the treatment paradigm. Evidence suggests improved survival outcomes for patients with actionable molecular alterations who received matched targeted therapies. Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase and proto-oncogene that has been extensively studied as a prognostic biomarker and a therapeutic target in multiple solid organ malignancies. Recent clinical trials on the combination of trastuzumab with tucatinib, FOLFOX, or pertuzumab for previously treated, HER2-positive, advanced BTCs have shown improved outcomes compared to current second-line therapies. Early evidence from observational studies on trastuzumab-containing regimens as first-line suggests promising efficacy. Furthermore, the recent tumor-agnostic approval of trastuzumab deruxtecan for HER2-positive solid tumors has formally introduced HER2-directed agents in the BTC therapeutic arsenal. This review aims to summarize the rapidly evolving landscape of HER2-directed agents for BTCs, highlighting current evidence of survival benefit. Beginning with a concise presentation of the structural and functional aspects of HER2, we detail the frequency and prognostic significance of HER2 alterations in BTCs and discuss all available preclinical and clinical data on anti-HER2 agents tested for BTCs.

Limited treatment options exist for patients with locally advanced or metastatic biliary tract cancers (BTCs). Recently, several clinical trials provided preliminary evidence for human epidermal growth factor receptor 2 (HER2) as a new target for patients with HER2-expressing BTC. We conducted a systematic review and pooled analysis of the safety and efficacy of anti-HER2 agents in patients with advanced BTCs.

A comprehensive search of PubMed/MEDLINE and EMBASE was performed to identify phase I, II, or III clinical trials published between January 2019 and March 2024 that evaluated anti-HER2 therapy in locally advanced or metastatic BTC. Participant data included in the analysis were from trials evaluating the efficacy and safety of various anti-HER2 agents. The primary end points included objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). The secondary end points included incidence of treatment-related adverse events (TRAEs), rate of treatment discontinuation, and death.

The analysis included 368 patients from eight publications diagnosed with advanced BTC. Patients were treated with several anti-HER2 agents including zanidatamab, pertuzumab plus trastuzumab, tucatinib plus trastuzumab, trastuzumab deruxtecan, trastuzumab plus chemotherapy, trastuzumab-pkrb plus chemotherapy, and neratinib. The pooled ORR and DCR were 34% (95% CI, 24 to 44) and 64% (95% CI, 51 to 77), respectively. The pooled weighted PFS and median overall survival were 4.8 and 9.4 months, respectively. The pooled duration of response for the reporting trials was 5.0 months. In the study cohort, 82.6% of patients experienced any adverse event and 32.1% experienced a grade 3-4 adverse event. Only 5.7% of the patients discontinued treatment secondary to TRAEs.

In patients with HER2-expressing BTCs, anti-HER2 therapies are viable options, particularly in the second-line setting.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of the biliary tree, leading to significant liver function impairment over time. There is a strong association with inflammatory bowel diseases (IBD), together representing a distinct and complex medical condition. Patients with PSC-IBD face a heightened risk of various cancers, particularly colorectal carcinoma (CRC) and cholangiocarcinoma (CCA) as the most common types. In this review, we aim to characterize the distinctive features of PSC-IBD-associated carcinomas. Cancer pathogenesis in PSC-IBD is shaped by various factors including dysregulated bile acid metabolism, gut dysbiosis, and unique immune responses. PSC-IBD-associated CRC is often right-sided and warrants vigilant monitoring due to its higher incidence and unique morphological features compared to CRC arising in the terrain of IBD alone. CCA shares substantial genetic similarities with extrahepatic CCA and poses diagnostic challenges since it is frequently detected at advanced stages due to symptom overlap with PSC. Besides, reliable predictive biomarkers for targeted therapy remain largely unexplored. The distinct molecular, genetic, and histopathological profiles of CRC and CCA in PSC-IBD underscore the complexity of these malignancies and highlight the need for continued research to develop precise therapeutic strategies.

Cholangiocarcinoma (CCA) is a serious public health issue due to its insidious onset and dismal prognosis. The past few years have witnessed and highlighted the development of understanding and management of CCA. The combination of gemcitabine and cisplatin (GP) chemotherapy regimen with immunotherapy using immune checkpoint inhibitors has been considered the new standard first-line treatment alternative for advanced CCA. Notably, the proportion of patients with advanced CCA with targetable genetic mutations is approximately 40 %, and these patients may be considered for molecularly targeted therapy in the second-line treatment. In this review, we highlight the advances and progress in targeted therapies for advanced CCA, with special attention to data from Asian populations, including Chinese. In addition, we present in detail the phosphatase tension homolog (PTEN), a novel biomarker for both of first-line chemotherapy and second-line targeted therapy in advanced CCA, and its ability to forecast prognosis in patients with CCA. The mechanisms of rapid resistance to targeted agents warrant further investigation and address in light of the development of new targeted therapies. Precision medicine is gradually playing an increasing role in achieving optimal therapeutic outcomes.

BackgroundCholangiocarcinoma-with a growing incidence rate and poor prognosis-is not an uncommon cancer. Molecular profiling can reveal actionable aberrations in at least a third of the tumors. This is especially so in the case of intrahepatic cholangiocarcinoma (ICC), where mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1/2) make up 15%-20% of these tumors. IDH1/2 mutant ICC is a rare disease that has not been adequately reported. To expand the spectrum of findings seen in these patients, we present a single institution case series.Methods and resultsWe descriptively characterize the clinical, radiological, and histopathological findings of 12 such patients. IDH1/2 mutant ICC was found in elderly women, with two-thirds of patients having additional co-mutations. Anecdotally, patients who did receive systemic and/or locoregional therapies had long-term durable outcomes.ConclusionOur findings indicate an increasing need to personalize an approach for these patients with specific molecular alterations. With the advent of the IDH1 inhibitor ivosidenib and other inhibitors in this space, IDH1/2 mutation have both prognostic and predictive value. Our series builds upon the patterns and findings seen in these patients.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most prevalent primary liver cancers and pose significant challenges in oncology. While their etiology and incidence vary globally, the molecular landscape of these tumors is increasingly understood, offering new opportunities for precision medicine. In this joint multinational review, we present a comprehensive analysis of the key molecular pathways involved in the pathogenesis of HCC and CCA, highlighting actionable targets for emerging therapies. Recent advances in molecular diagnostics have significantly influenced treatment paradigms for both cancers. In HCC, while genetic alterations have not yet led to established diagnostic or therapeutic applications, targeting vascular endothelial growth factor (VEGF), immune checkpoints, and tyrosine kinase pathways has demonstrated considerable therapeutic potential. In CCA, genetic profiling has uncovered actionable alterations, such as FGFR2 fusions and IDH1 mutations, driving the development of targeted therapies. The growing complexity of precision oncology underscores the need for standardized molecular testing and streamlined diagnostic workflows to ensure timely and effective treatment. This review also emphasizes the importance of collaborative efforts between clinicians, pathologists, and oncologists to optimize outcomes. By synthesizing the latest molecular insights and treatment trends, this review provides a valuable resource to guide the personalized management of HCC and CCA.

The study examines Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA), a serious malignancy common in Southeast Asia. Through multi-regional whole-exome sequencing of 52 tumor samples and 13 adjacent tissues from 13 patients, significant intratumoral heterogeneity (ITH) and inter-patient heterogeneity are shown. Chronic liver fluke infection induces a distinct mutational landscape, with 48-90% of mutations concentrated in each region of the tumor. The average mutation burden is 95 non-synonymous mutations per area, exceeding previous CCA investigations. Critical driver mutations in TP53, SMAD4, and other genes underscore their significance in pathogenesis. Mutational markers elucidate mechanisms including spontaneous deamination and impaired DNA repair. Unique mutation patterns distinguish OV-associated CCA from other variants. Chromosomal instability in patient K110 signifies aggressive tumor behavior and unfavorable prognosis. Targetable mutations such as ERBB2 underscore the possibility for personalized therapeutics. These findings underscore the necessity for personalized strategies for treatment that target both trunk and branch mutations in endemic areas.

Liposomal irinotecan (nal-IRI) combined with fluorouracil (5-FU)/leucovorin (LV) as a second-line treatment for biliary tract cancer (BTC) following progression on gemcitabine-based therapy yielded conflicting outcomes in the Korean NIFTY and German NALIRICC trials. This necessitated a comprehensive pooled analysis to evaluate its efficacy and safety.

Individual patient data were pooled from the intention-to-treat populations of the NIFTY and NALIRICC trials. The primary endpoint was progression-free survival (PFS).

A total of 278 patients were included: 137 in the nal-IRI plus 5-FU/LV group and 141 in the 5-FU/LV group. The nal-IRI plus 5-FU/LV group showed significantly longer median PFS (3.6 months [95% CI 2.7-4.4] vs. 1.8 months [95% CI 1.5-2.6]; hazard ratio 0.65, p <0.001). Median overall survival was 8.1 months (95% CI 6.0-8.9) and 6.1 months (95% CI 5.3-7.5), respectively (hazard ratio 0.77, p = 0.051). Objective response rates were also higher in the nal-IRI plus 5-FU/LV group than in the 5-FU/LV group (17.5% vs. 2.8%; p <0.001). Post-study irinotecan-containing therapy was administered in 4 (2.9%) and 21 (15.3%) patients in the nal-IRI plus 5-FU/LV group and 5-FU/LV group, respectively. Adverse events varied by ethnicity, with gastrointestinal toxicities more common in Germans and neutropenia more prevalent in Koreans; treatment discontinuation without disease progression occurred in 31.3% vs. 8.0%, respectively.

The addition of nal-IRI to 5-FU/LV significantly improved PFS and objective response rates, supporting its potential as subsequent-line therapy. Differences in safety profiles underscore the relevance of ethnicity for nal-IRI in patients with BTC.

Current standard of care for second-line therapy in patients with advanced biliary tract cancer (BTC) is FOLFOX. This study provides robust evidence supporting the potential role of adding liposomal irinotecan (nal-IRI) to fluorouracil and leucovorin (5-FU/LV) as a subsequent therapy for patients with BTC who have progressed on gemcitabine-based regimens. The findings demonstrate significant improvements in progression-free survival and objective response rates in a patient population for whom treatment options are limited. Furthermore, the study underscores the necessity of considering ethnic differences in adverse event profiles to optimize treatment administration and patient outcomes.

NCT03524508 and NCT03043547.

Due to the limited efficacy of chemotherapy alone in the treatment of unresectable biliary tract cancer, we performed conversion surgery in patients with unresectable biliary tract cancer who responded to down-staging chemotherapy.

Patients with unresectable biliary tract cancer who initiated chemotherapy between 2007 and 2018 were included in this study. We evaluated the short- and long-term outcomes of patients with initially unresectable biliary tract cancer who underwent conversion surgery.

A total of 101 patients with unresectable biliary tract cancers treated with chemotherapy were eligible for the present study. A total of 20 patients eventually underwent conversion surgery; these patients had locally advanced disease in 6 cases, liver metastasis in 6 cases, para-aortic lymph node metastasis in 5 cases, and peritoneal dissemination in 3 cases. The mean operative time was 823 min, and the mean intraoperative blood loss was 1902 mL. Histological R0 resections were performed in 17 patients. Postoperative complications of Clavien-Dindo grade IIIa or higher occurred in 10 patients, with no surgery-associated deaths. The 5-year survival rate was significantly higher in patients who underwent conversion surgery (65.0%) than in those who did not (4.3%, p < 0.001).

Conversion surgery for initially unresectable biliary tract cancer resulted in favorable overall survival and was safely performed despite its high surgical invasiveness. Conversion surgery for an initially unresectable biliary tract cancer is worth considering.

Liver cancer poses a global health challenge with limited therapeutic options. Notably, the limited success of current therapies in patients with primary liver cancers (PLCs) may be attributed to the high heterogeneity of both hepatocellular carcinoma (HCCs) and intrahepatic cholangiocarcinoma (iCCAs). This heterogeneity evolves over time as tumor-initiating stem cells, or cancer stem cells (CSCs), undergo (epi)genetic alterations or encounter microenvironmental changes within the tumor microenvironment. These modifications enable CSCs to exhibit plasticity, differentiating into various resistant tumor cell types. Addressing this challenge requires urgent efforts to develop personalized treatments guided by biomarkers, with a specific focus on targeting CSCs. The lack of effective precision treatments for PLCs is partly due to the scarcity of ex vivo preclinical models that accurately capture the complexity of CSC-related tumors and can predict therapeutic responses. Fortunately, recent advancements in the establishment of patient-derived liver cancer cell lines and organoids have opened new avenues for precision medicine research. Notably, patient-derived organoid (PDO) cultures have demonstrated self-assembly and self-renewal capabilities, retaining essential characteristics of their respective in vivo tissues, including both inter- and intratumoral heterogeneities. The emergence of PDOs derived from PLCs serves as patient avatars, enabling preclinical investigations for patient stratification, screening of anticancer drugs, efficacy testing, and thereby advancing the field of precision medicine. This review offers a comprehensive summary of the advancements in constructing PLC-derived PDO models. Emphasis is placed on the role of CSCs, which not only contribute significantly to the establishment of PDO cultures but also faithfully capture tumor heterogeneity and the ensuing development of therapy resistance. The exploration of PDOs' benefits in personalized medicine research is undertaken, including a discussion of their limitations, particularly in terms of culture conditions, reproducibility, and scalability.

Biliary tract cancer (BTC) originates from the biliary epithelium of the small ducts within the liver (intrahepatic cholangiocarcinoma, IHCC), the main ducts of the hilum (extrahepatic cholangiocarcinoma, EHCC), or in the gallbladder (gallbladder cancer, GC). Due to presentation with nonspecific symptoms as well as absence of screening, most patients present with advanced disease and unfavorable prognosis.

The ABC-02 trial established the current first-line chemotherapy with gemcitabine/platinum for advanced BTC in 2010. Since then, multiple therapies have become available exploring different targetable alterations, emphasizing the importance of molecular profiling in all patients with BTC as well as understanding the distinct toxicity profile associated with these therapies. Besides chemotherapy, immunotherapy as well as targeted therapies for FGFR2, IDH1, and HER2 will be discussed in this manuscript. We performed a non-systematic review, largely based on high-quality articles on the topic of interest with no predefined protocol.

The primary objective of this manuscript is to conduct a thorough review of diverse aspects related to the safety of systemic treatment in BTC. As the benefit of these therapies depends on compliance and/or tolerance, the authors aim to discuss different toxicity profiles and to provide insights into strategies for overcoming them.

Cholangiocarcinomas are a highly heterogeneous group of malignancies that, despite recent progress in the understanding of their molecular pathogenesis and clinical management, continue to pose a major challenge to public health. The traditional view posits that cholangiocarcinomas derive from the neoplastic transformation of cholangiocytes lining the biliary tree. However, increasing genetic and experimental evidence has recently pointed to a more complex, and nuanced, scenario for the potential cell of origin of cholangiocarcinomas. Hepatocytes as well as hepatic stem/progenitor cells are being considered as additional potential sources, depending on microenvironmental contexts, including liver injury. The hypothesis of potentially diverse cells of origin for cholangiocarcinoma, albeit controversial, is certainly not surprising given the plasticity of the cells populating the liver as well as the existence of liver cancer subtypes with mixed histologic and molecular features. This review carefully examines the current pathologic, genomic, and experimental evidence supporting the existence of multiple cells of origin of liver and biliary tract cancers, with particular focus on cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma.

Cholangiocarcinoma (CCA) has rising incidence and mortality rates. Outcomes from combination systemic, loco-regional therapy (LRT) and liver transplantation (LT) are improving, but more granular data are needed to inform evidence-based management, including patient selection and immunosuppression.

Patients with peri-hilar (PH) and intrahepatic (IH) CCA who underwent LT at a single center between January 2008 and February 2023 were reviewed retrospectively. Primary outcomes were overall survival (OS) and recurrence-free survival (RFS) with significance determined by Cox proportional hazards model.

During the study period, 53 patients underwent LT for either PH (n = 27), or IH (26). Cohort had mean age 58.5 years old (IQR, 47.0-63.0), body mass index (BMI) 25.9 (IQR, 22.9-30.0) kg/m2, and mean biologic MELD 9 (IQR, 7-17). Most frequent etiology was PSC (n = 12, 22.6%). Forty-nine patients (92.5%) received neoadjuvant therapy, including systemic (n = 48, 90.6%) and locoregional therapy (LRT) (n = 22, 41.5%), to which PH tumors were both most and least responsive (P = .03). On explant pathology, tumor were a median size of 3.5 cm and lympho-vascular invasion (LVI) was present in 13 (24.5%) cases. Median follow-up post-transplant was 910 days (IQR, 407-1509). Probabilities of OS and RFS at 3-years post-LT were 69.2% (95% CI, 56.9%-84.2%) and 57.4% (95% CI, 43.7%-75.4%). In multivariable analysis, OS was associated with tumor type and LVI, and RFS with age, BMI, PSC and LRT. After a median post-LT period of 38 days (IQR, 27-79.5), 39 (71.7%) patients started mTOR inhibition with lowered tacrolimus goal. Cox proportional hazard model showed significant association of OS with mTOR inhibition, though this was not validated by a time-dependent co-variate approach.

In this single center cohort of CCA, post-LT outcomes were significantly greater for patients with IH tumors and no LVI. Immunosuppression with mTOR inhibition was not consistently associated with outcomes.

Cholangiocarcinoma (CCA) is a highly fatal malignancy with an increasing prevalence, a high mortality rate, poor overall survival, and limited responsiveness to conventional chemoradiotherapy. Targeted therapies addressing specific gene mutations have expanded treatment options for some patient populations. The introduction of chimeric antigen receptor-modified T-cell (CAR-T) immunotherapy and personalized vaccines have opened up a new avenue for managing various cancers. Considerable efforts have been dedicated to preclinical research and ongoing clinical trials of immunotherapeutic approaches including CAR-T therapy, vaccines, and antibody-based therapies such as antibody drug conjugates. However, the potential of CAR-T therapy and vaccines in treating advanced unresectable/metastatic cholangiocarcinoma remains largely unexplored. This article offers an overview of the current landscape of antibody-based immunotherapy, particularly CAR-T therapy and vaccines in the context of cholangiocarcinoma treatment. It outlines a framework for selecting CAR-T and vaccine targets and delves into the biology of promising targetable antigens, as well as potential future therapeutic targets.

Due to malnutrition and tumor cachexia, body composition (BC) is frequently altered and known to adversely affect short- and long-term results in patients with cholangiocarcinoma (CCA). Here, we explored immune cell populations in the tumor and liver of CCA patients with respect to BC.

A cohort of 96 patients who underwent surgery for CCA was investigated by multiplexed immunofluorescence (MIF) techniques with computer-based analysis on whole-tissue slide scans to quantify and characterize immune cells in normal liver and tumor regions. BC was characterized by obesity, sarcopenia, myosteatosis, visceral obesity and sarcopenic obesity. Associations between BC and immune cell populations were determined by univariate and multivariable binary logistic regressions.

BC was frequently altered in intrahepatic CCA (iCCA, n = 48), with 47.9% of the patients showing obesity, 70.8% sarcopenia, 18.8% sarcopenic obesity, 58.3% myosteatosis and 54.2% visceral obesity as well as in perihilar CCA (pCCA, n = 48) with 45.8% of the patients showing obesity, 54.0 sarcopenia, 14.6% sarcopenic obesity, 47.9% myosteatosis and 56.3% visceral obesity. From an immune cell perspective, independent associations within the tumor compartment were observed for iCCA (myosteatosis: TIM-3+CD8+cells; obesity: PD-1+TIM-3+CD4+cells) and for pCCA (myosteatosis: PD-L2+CD68-cells and CD4+cells). Further, independent associations were observed within the normal liver parenchyma for iCCA (visceral obesity: PD-1+PD-L1+PD-L2+CD68+cells) and for pCCA (sarcopenia: CD68+cells and TIM-3+CD8+cells; visceral obesity: ICOS+-TIGIT+CD8+cells and sarcopenic obesity: PD-1+PD-L1+PD-L2+CD8+cells).

This is the first systematic analysis of the association of BC and immune cells in cholangiocarcinoma showing a strong association between BC and distinct immune cell populations within the tumor itself as well as within the normal parenchyma.

Biliary tract cancer (BTC) is becoming more common worldwide, with geographic differences in incidence and risk factors. In Europe, BTC may be associated with primary sclerosing cholangitis, lithiasis, and liver cirrhosis, but is more frequently observed as a sporadic disease. BTC increasingly affects patients under 60 years, resulting in a significant social and economic burden. Early diagnosis remains challenging due to vague symptoms in 50% of patients with BTC, and lack of specific biomarkers, resulting in late presentation and poor prognosis. The identification of patients at increased risk and reliable biomarkers require collaborative efforts to make faster progress. This Series paper highlights the disparities in access to diagnostic tools and multidisciplinary care in Europe, particularly in economically disadvantaged regions, while identifying priority areas for improvement. Addressing these inequities requires harmonised guidelines, accelerated pathways to curative treatments, and improved awareness among healthcare professionals and the public. Multidisciplinary teams (MDTs) are crucial for the diagnosis of BTC and for improving patient outcomes, yet inconsistencies exist in their implementation not only between different countries, but also between different centres within a country. Collaboration and standardisation of diagnostic and treatment protocols across Europe are essential to effectively address the management of patients with BTC.

The biliary tract is a ductal network comprising the intrahepatic (IHBDs) and extrahepatic bile duct (EHBDs). Biliary duct disorders include cholangitis, neoplasms, and injury. However, the underlying mechanisms are not fully understood. With advancements in 3D culture technology, cholangiocyte organoids (COs) derived from primary tissues or induced pluripotent stem cells (iPSCs) can accurately replicate the structural and functional properties of biliary tissues. These organoids have become powerful tools for studying the pathogenesis of biliary diseases, such as cystic fibrosis and primary sclerosing cholangitis, and for developing new therapeutic strategies for cholangiocarcinoma. Additionally, COs have the potential to repair bile duct injuries and facilitate transplantation therapies. This review also discusses the use of organoids in genetically engineered mouse models to provide mechanistic insights into tumorigenesis and cancer progression. Continued innovation and standardization of organoid technology are crucial for advancing precision medicine for biliary diseases and cancer.

Metachronous biliary tract cancer (BTC) is a rare occurrence after curative resection of primary BTC. The genetic alterations and pathogenesis associated with metachronous BTC remain poorly understood.

We analyzed four patients with metachronous BTC who underwent resection at the Nagoya University Hospital between 2010 and 2024. Gene panel examination was performed on both primary and secondary tumors using next-generation sequencing.

The median interval between resection of the primary tumor and diagnosis of metachronous BTC was 24 months. Genetic alterations were observed in all paired primary and metachronous carcinomas. The number of genetic mutations was higher in metachronous lesions than in primary lesions. CDKN2A and SMAD4 were the most frequently mutated genes in all metachronous lesions. Common genetic mutations between primary and metachronous lesions were confirmed in all four cases, suggesting a common clonal origin.

This study demonstrated that characteristic genetic alterations and their accumulation play important roles in metachronous BTC. This suggests that the increasing burden of gene mutations may play a crucial role in the carcinogenesis of metachronous BTC. Further investigation is required to validate these findings and elucidate the underlying molecular mechanisms.

Biliary tract cancer (BTC) is a rare but highly aggressive malignancy that includes intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma, and gallbladder cancer (GBC). While BTC has a low global incidence, its regional variations are notable. Among nations, Korea has the second-highest incidence of BTC globally, with the highest mortality rate worldwide, underscoring the need for a deeper understanding of this cancer. Liver fluke infection and hepatitis B virus infection are key risk factors unique to Korea, contributing to regional differences in BTC incidence. Additionally, genomic alterations in Korean patients with BTC differ from those in other populations, including lower frequencies of IDH1 mutations and FGFR2 fusions in ICC and a higher prevalence of ERBB2 amplification in GBC. Recognizing the clinical significance of these alterations, ivosidenib and pemigatinib have been approved in Korea for BTC patients with IDH1 mutations and FGFR2 fusions, respectively. This review explores the epidemiology, risk factors, and molecular features of BTC, along with corresponding targeted therapies. Furthermore, we compare the unique characteristics of BTC in Korea with global data to inform future research and clinical practice.

Gallbladder carcinoma (GBC) is an extremely aggressive tumor of the biliary tract with a bleak prognosis, and the evidence supporting the benefit of available systemic therapy for advanced GBC is scarce. Herein, this study intended to investigate the real-world outcome of chemotherapy combined with programmed death-1 (PD-1) inhibitor for the management of unresectable or recurrent GBC.

From January 2018 to December 2023, consecutive patients who were treated with systematic treatment, including chemotherapy or the combination of chemotherapy plus PD-1 inhibitor, for unresectable or recurrent GBC were retrospectively identified. Clinical data regarding baseline characteristics, therapeutic response, adverse events (AEs), and oncological outcomes were collected.

The eligible patients were allocated to combination therapy arm (n = 46) and mono-chemotherapy arm (n = 19). After propensity score matching (PSM), 16 patients were allocated in each arm. The overall survival (OS) and progression-free survival (PFS) of combination therapy were marginally superior to mono-chemotherapy both before and after PSM. The combination therapy exhibited advantage over mono-chemotherapy in regards to partial response (PR) (before PSM: P = 0.009; after PSM: P = 0.037) and objective response rate (ORR) (before PSM: P = 0.006; after PSM: P = 0.015). In combined therapy cohort, 1 patient achieve a complete response, and 13 patients were assessed as appropriate for surgical excision, among which 1 patient refused further surgical intervention.

In patients with unresectable or recurrent GBC, the combination of chemotherapy and PD-1 inhibitor as first-line therapy exhibited prolonged OS and PFS, and increased PR and ORR over those receiving chemotherapy alone, with an acceptable toxicity profile. The combination therapy may be a potential conversion therapy in unresectable GBC patients.

Biliary tract cancer (BTC), encompassing diseases such as intrahepatic (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer, is not only increasing but also poses a significant and urgent health threat due to its high malignancy. Genomic differences point to the possibility that these subtypes represent distinct diseases. Elucidation of the specific distribution of T-cell subsets, critical to cancer immunity, across these diseases could provide better insights into the unique biology of BTC subtypes and help identify potential precision medicine strategies. To address this, we conducted single-cell RNA sequencing and T-cell receptor sequencing on CD3+ T cells from 36 samples from 16 patients with BTC across all subtypes and analyzed 355 pathologic slides to examine the spatial distribution of T cells and tertiary lymphoid structures. Compared with ICC and gallbladder cancer, ECC possessed a unique immune profile characterized by T-cell exhaustion, elevated CXCL13 expression in CD4+ T helper-like and CD8+CXCL13+ exhausted T cells, more mature tertiary lymphoid structures, and fewer desert immunophenotypes. Conversely, ICC displayed an inflamed immunophenotype with an enrichment of IFN-related pathways and high expression of LGALS1 in activated regulatory T cells, associated with immunosuppression. Inhibition of LGALS1 reduced tumor growth and regulatory T-cell prevalence in ICC mouse models. Overall, this study unveils T-cell diversity across BTC subtypes at the single-cell and spatial level that could open paths for tailored immunotherapies. Significance: Single-cell and spatial analyses detailed the T-cell characteristics specific to anatomic subtypes of biliary tract cancer, identifying unique immunologic features that could potentially be harnessed to improve patient outcomes.

Biliary tract malignancies, including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer, represent a group of aggressive cancers with poor prognosis due to late-stage diagnosis, limited treatment options, and resistance to conventional therapies like chemotherapy and radiotherapy. These challenges emphasize the urgent need for innovative therapeutic approaches. In recent years, cell-based therapies have emerged as a promising avenue, offering potential solutions through immune modulation, genetic engineering, and targeted intervention in the tumor microenvironment. This Mini-review provides an overview of current advancements in cell-based therapies for biliary malignancies, encompassing immune cell-based strategies such as CAR-T cells, NK cells, dendritic cell vaccines, and tumor-infiltrating lymphocytes. We also examine strategies to overcome the immunosuppressive tumor microenvironment and discuss the integration of cell therapies into multimodal treatment regimens. By synthesizing preclinical and clinical findings, this review highlights key insights and future directions, aiming to assist researchers and clinicians in translating these approaches into effective treatments. The transformative potential of cell-based therapies discussed here makes this review a valuable resource for advancing biliary malignancy research and clinical applications.

Cholangiocarcinoma (CCA) is a diverse group of epithelial malignant tumors arising from the biliary tract, characterized by high molecular heterogeneity. It is classified into intrahepatic (iCCA) and extrahepatic CCA (eCCA) based on the location of the primary tumor. CCA accounts for approximately 15% of all primary liver cancers, with iCCA comprising 10-20% of all CCAs. iCCA is especially known for its characteristic aggressiveness and refractoriness, leading to poor prognosis. Despite the increasing global incidence and mortality rates, surgery remains the only available standard treatment approach for a subset (25%) of patients with early-stage, resectable iCCA. The paucity of effective systemic medical therapies restricts therapeutic options for patients with advanced or metastatic iCCA. In the past decade, advances in the understanding of the molecular complexity of these tumors have provided fruitful insights for the identification of promising new druggable targets and the development of feasible therapeutic strategies that may improve treatment outcomes for patients with iCCA. In this review, we aim to highlight critical up-to-date studies and medicinal chemistry aspects, focusing on novel targeted approaches utilizing promising candidates for molecular targeted therapy in iCCA. These candidates include aberrations in isocitrate dehydrogenase (IDH) 1/2, fibroblast growth factor receptor (FGFR), B-Raf proto-oncogene (BRAF), neurotrophic tyrosine receptor kinase (NTRK), human epidermal growth factor receptor 2 (HER2), and programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1). Furthermore, this review provides an overview of potential inhibitors aimed at overcoming acquired drug resistance in these actionable targets for iCCA.

We report the rare case of severe cognitive disorder and ataxia caused by entrectinib in a patient with NTRK1 fusion gene-positive intrahepatic cholangiocarcinoma. The case was a 56-year-old woman after nine courses of GCS therapy. The patient experienced grade 1 muscle weakness on day 4 and grade 3, cognitive disorder on day 7 after receiving entrectinib, which led to hospitalization. The symptoms were reversible and tended to improve after withdrawal of entrectinib. It is crucial to increase awareness of TRKi-specific adverse events and their proper management.

Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.