Extrahepatic CCA Research Articles

Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma.

Most patients with cholangiocarcinoma (CCA) develop extrahepatic malignant biliary obstructions, which require palliative drainage to normalize bilirubin levels and to improve the patients' overall survival. Here, we report that the infusion of methotrexate-containing plasma-membrane microvesicles derived from apoptotic human tumour cells into the bile-duct lumen of patients with extrahepatic CCA mobilized and activated neutrophils and relieved biliary obstruction in 25% of the patients. Neutrophil recruitment by the microvesicles was associated with an increase in uridine diphosphate glucose and complement C5, and led to the degradation of the stromal barrier of CCA. The microvesicles induced pyroptosis of CCA cells through a gasdermin E-dependent pathway, and their intracellular contents released upon CCA-cell death activated patient-derived macrophages into producing proinflammatory cytokines, which attracted a secondary wave of neutrophils to the tumour site. Our findings suggest a possible treatment for the alleviation of obstructive extrahepatic CCA with few adverse effects, and highlight the potential of tumour-cell-derived microvesicles as drug carriers for antitumour therapies.

Usefulness of bile as a biomarker via ferroptosis and cysteine prenylation in cholangiocarcinoma; role of diagnosis and differentiation from benign biliary disease

BackgroundCholangiocarcinoma (CCA) is a malignant cancer of the biliary tract with a poor prognosis. Herein, we investigated possible mechanism of extrahepatic CCA (eCCA) by dysregulated iron metabolism and post-translational modifications (PTMs). Further, we evaluated potential biomarkers in the bile fluid for diagnosis of eCCA and differentiation between eCCA and benign biliary disease. MethodsFrom August 2018 to April 2019, we obtained bile fluids from 46 patients; 28 patients with eCCA (eCCA group) and 18 patients with common bile duct stone (Control group) via percutaneous transhepatic biliary drainage. We examined the levels of reduced glutathione (GSH), peroxide, ferrous iron [Fe+2], glutathione peroxidase (GPX) and farnesyl transferase/geranylgeranyl transferase type-1 subunit alpha (FNTA) concentration in bile fluids to clarify the mechanism of ferroptosis and prenylation. ResultsThe remarkable difference of PTMs was that FNTA which means prenylated cysteine as regulator was significantly decreased in eCCA than that of control. In addition, level of GSH, peroxide, GPX and ferrous iron [Fe+2] were significantly depleted in eCCA than control. These results demonstrate that PTM, dysregulated iron metabolism and GPX-regulated ferroptosis with GSH depletion through cysteine modification in bile are possible mechanisms of eCCA. Liquid Chromatography (LC)-Mass Spectrometry (MS) analysis, several oncogenic pathways including MYC target, apoptosis, fatty acid metabolism, P53 and mTORC1 were enriched in eCCA. ConclusionsIn conclusion, redox-dependent modification of cysteine and ferroptosis in bile fluids are possible mechanisms of eCCA. Several protein and oncogenic pathways related to PTM which are seen in eCCA tissues were also enriched in bile fluids. It suggests that bile fluid represents the oncogenic characteristics of eCCA tissues. Therefore, bile fluids have a role of a biomarker for diagnosis in eCCA, especially, differentiation of eCCA from benign biliary stricture.

Wnt/β-catenin signaling as an emerging potential key pharmacological target in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a fatal malignant tumor of biliary epithelial cells involving intra- or extra-hepatic bile ducts. The prognosis of CCA is generally poor due to its diagnosis at the late stages. The currently employed chemotherapeutic agents do not increase the survival rate in patients with unresectable CCA. Accordingly, there is a need to identify new therapeutic agents for the effective management of intra- and extra-hepatic CCA. Clinical as well as preclinical studies have suggested the key role of the activation of Wnt/β-catenin signaling pathway in the induction and progression of CCA. There is an up-regulation of different Wnt ligands including Wnt2, Wnt3, Wnt5, Wnt7 and Wnt10 along with redistribution of β-catenin (more expression in the nucleus and lesser on the cell surface due to nuclear translocation of β-catenin) in different types of malignant biliary tumors. Apart from the role of this pathway in the induction and progression of CCA, this pathway is also involved in inducing multidrug resistance by inducing the expression of P-glycoprotein efflux pump on the cancer cells. These deleterious effects of Wnt/β-catenin signaling are mediated in association with other signaling pathways involving microRNAs (miRNAs), PI3K/AKT/PTEN/GSK-3β, retinoic acid receptors (RARs), dickkopf-1 (DKK1), protein kinase A regulatory subunit 1 α (PRKAR1A/PKAI), (SLAP), liver kinase B1 (LKB1) and CXCR4. The selective inhibitors of Wnt/β-catenin signaling may be potentially employed to overcome multidrug-resistant, fatal CCA. The present review discusses the role of Wnt/β-catenin along with its relation with other signaling pathways in the induction and progression of CCA.

Comprehensive genomic profile of cholangiocarcinomas in China.

Cholangiocarcinoma (CCA) is a primary malignancy, which is often diagnosed as locally advanced or metastatic. Previous studies have revealed genomic characteristics of CCA in Western patients, however comprehensive genomic features of CCA in Chinese patients have not been well understood. To explore the specific genomic characteristics of Chinese patients with CCA, a total of 66 patients with CCA, including 44 intrahepatic CCA (iCCA) and 22 extrahepatic CCA (exCCA) cases, were studied. The most commonly altered genes in CCAs were TP53 (62.12%, 41/66), KRAS (36.36%, 24/66), SMAD4 (24.24%, 16/66), TERT (21.21%, 14/66), ARID1A (19.70%, 13/66), CDKN2A (19.70%, 13/66), KMT2C (9.09%, 6/66) and RBM10 (9.09%, 6/66), ERBB2 (7.58%, 5/66) and BRAF (7.58%, 5/66). Many gene mutations, including STK11, CCND1 and FGF19, were only found in iCCA. RBM10 mutations were found to be significantly higher in exCCA. The gene mutations of neurofibromin 1, STK11, CCND1 and FBXW7 specifically occurred in males, whereas gene mutations of ERBB2, AXIN2 and CREBBP specifically occurred in females. ERBB2 mutations were significantly associated with the sex of patients with CCA. Mutations in PIK3CA, FGFR2 and ZNF750 were significantly associated with the age of patients with CCA and TERT mutations were significantly associated with tumor differentiation. Alterations in KMT2C, PBRM1, AXIN2, MAGI2, BRCA2 and SPTA1 were associated with tumor mutational burden. The findings of the present study suggest that targeted sequencing, using next-generation sequencing technology, provides comprehensive and accurate information on genomic alterations, which will provide novel potential biomarkers for the diagnosis of CCA and may guide precise therapeutic strategies for Chinese patients with CCA.

Genomic Features and Clinical Characteristics of Adolescents and Young Adults With Cholangiocarcinoma.

Background: Adolescents and young adults (AYAs) diagnosed with cancer between ages 15 and 45 years may exhibit unique biologic and genomic characteristics as well as clinical features, resulting in differences in clinical characters and drug resistance. However, compared to other solid cancers, relatively few studies have been conducted in this age group in cholangiocarcinoma (CCA). This study is performed to investigate the clinical and molecular features of AYAs with CCA.Methods: Three cohorts, including the external dataset (TCGA and MSKCC) and the perihilar CCA databank of Chinese tertiary hospitals, were contained in this study. Pathway and process enrichment analysis had been carried out with the following ontology sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, and CORUM. Metascape and GEPIA datasets were used for bioinformatic analysis. P < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism (version 7.0; GraphPad Software, La Jolla, California) and R studio (version 3.6.1; R studio, Boston, Massachusetts).Results: Compared to older adults, AYAs with CCA presented with worse overall survival, although the difference was not significant. Specific to patients with stage IV CCAs who underwent chemotherapy, AYAs were associated with significantly poorer overall survival (OS) (p = 0.03, hazards ratio (HR) 3.01, 95% confidence interval (CI) 1.14-4.91). From the anatomical perspective, more extrahepatic CCA was detected in the AYA group. Microsatellite instability (MSI) occurred in 3% of older patients in the present study. Nevertheless, none of the AYAs had MSI status. In this study, AYAs gained an enhanced frequency of additional sex combs like 1 (ASXL1) (p = 0.02) and KMT2C (p = 0.02) mutation than their older counterparts. Besides ASXL1 and KMT2C, the genes enriched in AYAs with CCA were analyzed by pathway and process enrichment analysis. And those genes were found to be associated with poorer differentiation, deubiquitination, and WNT signal pathway. Moreover, AYAs were relevant to poor differentiation and advanced tumor stage.Conclusion: This study offered a preliminary landscape of the clinical and molecular features of early-onset biliary cancers. Further studies including more samples are essential to investigate whether ASXL1 and KMT2C could be considered as potentially targetable genomic signatures for young patients.

Quantitative proteomic analysis of bile in extrahepatic cholangiocarcinoma patients.

Background and Aims: Extrahepatic cholangiocarcinoma (CCA) without liver-fluke is increasing. Multifactorial carcinogenesis makes it hard to find biomarkers related to CCA. Although there are a few studies of bile proteomics, these showed different protein profiles because of having heterogeneous groups of patients and different sampling methods. Our aim was to identify the specific bile proteins of extrahepatic CCA patients.Methods: We collected bile from 23 patients undergoing endoscopic nasobiliary drainage in Korea University Guro Hospital from May 2018 to January 2019. The CCA group included 18 patients diagnosed with extrahepatic CCA, and the control group included 5 patients with benign biliary conditions. We analyzed bile proteome using liquid chromatography mass spectrometry. We compared the relative abundance of various proteins in the CCA and control groups.Results: In all, we identified a total of 245 proteins in the bile of CCA and control patients. Increased top 14 proteins in CCA patients were immunoglobulin kappa light chain, apolipoprotein B, inter-alpha-trypsin inhibitor heavy chain H4, apolipoprotein E, Mucin 5B, inter-alpha-trypsin inhibitor heavy chain H1, apolipoprotein A-IV, intercellular adhesion molecule 1, complement C7, complement C5, apolipoprotein C-III, albumin, antithrombin-III, and apolipoprotein A-II. However, the significantly increased proteins in bile of CCA patients comparing with control patients were immunoglobulin kappa light chain, apolipoprotein E, albumin, apolipoprotein A-I, antithrombin-III, α1-antitrypsin, serotransferrin, immunoglobulin heavy constant mu, immunoglobulin J chain, complement C4-A, and complement C3 (p<0.05).Conclusions: In this study, we identified several proteins that were significantly increased in the bile of extrahepatic CCA. Further study is needed to validate them as potential tumor-associated proteins that may be potential biomarkers for CCA.

96P - LRP2, a potential new biomarker for Chinese younger aged intrahepatic cholangiocarcinoma patients

BackgroundCholangiocarcinoma (CCA), which includes extrahepatic cholangiocarcinoma (exCCA) and intrahepatic cholangiocarcinoma (iCCA), is a primary malignancy which is often diagnosed as advanced stage and inoperable. The poor sensitivity of clinical diagnosis and lack of effective biomarkers to guide treatment are the main problems to be tackled. Identification of new and effective biomarkers is necessary to assist early diagnosis and prognosis of CCA. MethodsDeep sequencing targeting 450 cancer genes was performed on FFPE and matching blood samples were collected from 63 CCA patients. Genomic alterations (GAs) including single nucleotide variations, short and long insertions and deletions, copy number variations, and gene rearrangements were analyzed. Tumor mutational burden (TMB) was measured by an algorithm developed in-house. The correlation analysis was performed by One-way ANOVA. ResultsA total of 63 (35 male and 28 female) Chinese CCA patients, including 40 intrahepatic CCA (iCCA) and 23 extrahepatic CCA (exCCA), were analyzed in this study. The most commonly altered genes included TP53 (41.27%, 26/63), KRAS (31.75%, 20/63), ARIK1A and IDH1 (15.87%, 10/63, for both), SMAD4 (14.29%, 9/63), FGFR2 and BAP1 (12.70%, 8/63, for both) and CDKN2A (11.11%, 7/63). The mutation of LRP2 specifically occurred with low frequency (7.5%, 3/40) in iCCA patients. There were 24 patients aged 50 to 70 years, 6 patients aged more than 70 years and 10 patients aged less than 50 years (younger group). Notably, the mutations of LRP2 were detected only in the younger group, and statistical analysis showed the significant correlation between LRP2 and younger iCCA patients (P=0.02). Also, 2 of the 3 patients with LRP2 mutations harbored high TMB (TMB-H, more than 10 Muts/Mb). Statistical analysis showed a significant association of LRP2 and TMB-H. ConclusionsThe mutation of LRP2 is associated with younger Chinese iCCA patients. The correlation of LRP2 and TMB-H represents a better mutational burden and probably a better prognosis of younger iCCA patients with LRP2 mutations. Our results support that LRP2 may be a potential biomarker for the diagnosis and prognosis of younger Chinese iCCA patients. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Risk factors for intrahepatic and extrahepatic cholangiocarcinoma: A systematic review and meta-analysis

Cholangiocarcinoma (CCA) carries a poor prognosis, is increasing in incidence and its causes are poorly understood. Although some risk factors are known, they vary globally and collectively account for a minority of cases. The aim of this study was to perform a comprehensive meta-analysis of risk factors for intrahepatic (iCCA) and extrahepatic cholangiocarcinoma (eCCA), from Eastern and Western world studies. A literature search of case-control studies was performed to identify potential risk factors for iCCA and eCCA. Pooled odds ratios (ORs) with 95% CIs and heterogeneity were calculated. Funnel plots were used to assess publication bias, and meta-regression was used to select risk factors for comparison between Eastern and Western studies. A total of 13 risk factors were selected from 25 case-control studies in 7 geographically diverse countries. The strongest risk factors for both iCCA and eCCA were biliary cysts and stones, cirrhosis, hepatitis B and hepatitis C. Choledochal cysts conferred the greatest risk of both iCCA and eCCA with pooled ORs of 26.71 (95% CI 15.80-45.16) and 34.94 (24.36-50.12), respectively. No significant associations were found between hypertension and obesity for either iCCA or eCCA. Comparing Eastern and Western populations, there was a difference for the association of hepatitis B with iCCA (coefficient = -0.15195; 95% CI -0.278 to -0.025; p = 0.022). This is the most comprehensive meta-analysis of CCA risk factors to date. Some risk factors, such as diabetes, although less strong, are increasing globally and may be contributing to rising rates of this cancer. Cholangiocarcinoma (CCA) is a cancer arising in the bile ducts inside (intrahepatic CCA) and connected to the liver (extrahepatic CCA). It is a very aggressive cancer: 95% of patients die within 5 years. CCA rates are increasing globally, but the causes of CCA are poorly understood. The few risk factors that are known account for only a minority of cases. In this study, we found that the strongest risk factors for both intrahepatic and extrahepatic CCA are cysts and stones in the bile ducts, cirrhosis, and hepatitis B and C viruses. Some risk factors for CCA, such as diabetes, although less strong, are increasing globally and may be contributing to rising rates of CCA.

The expression of gastrointestinal differentiation markers in extrahepatic cholangiocarcinoma: clinicopathological significance based on tumor location

The expression of gastrointestinal differentiation markers is associated with the tumorigenesis and prognosis of digestive cancers. However, little is known about the significance of gastrointestinal differentiation marker profiles in patients with extrahepatic cholangiocarcinoma (CCA), which is classified as perihilar and distal CCA. The purpose of this study was to clarify the role of gastrointestinal differentiation marker expression in extrahepatic CCA based on tumor location. We examined the expression of gastrointestinal differentiation markers in resected perihilar (n = 30) and distal (n = 54) CCAs based on the immunohistochemical expression of the following markers: MUC2, MUC5AC, MUC6, CD10, CDX-2, and cytokeratin 20. Expression scores were determined semiquantitatively based on the rate of positively stained cells. Furthermore, we performed hierarchical clustering of the CCAs based on the immunohistochemical expression scores to evaluate differences in the expression patterns of the 6 gastrointestinal differentiation markers. Consequently, perihilar and distal CCAs were stratified into 2 subgroups each. Among the perihilar CCAs, subgroup 1 was characterized by lower expression of MUC5AC and MUC6, a larger median tumor size, and a significantly worse prognosis compared with subgroup 2. Furthermore, the immunohistological subgroup (subgroup 1 versus 2) and TNM stage (stage III versus II) were independent predictors of patient survival. Among the distal CCAs, subgroup 1 was characterized by lower expression of MUC5AC compared with subgroup 2. We suggest that gastrointestinal differentiation marker profiles are useful for stratifying perihilar and distal CCAs. In addition, gastrointestinal differentiation markers play a crucial role in tumor development, particularly in perihilar CCA.

Abstract 1390: Molecular characterization of circulating tumor cells in cholangiocarcinoma patients: A new tool for treatment management

Abstract BACKGROUND: Cholangiocarcinoma (CCA) is a highly fatal disease mainly treated with standard chemotherapy, albeit with limited efficacy. New therapeutic options are greatly needed, but the use of targeted treatments is often prevented by the impossibility to obtain tissue biopsies for molecular characterization. Here, we propose the use of circulating tumor cells (CTCs) as an alternative source of tumor material to perform molecular characterization for the identification of novel therapeutic targets. MATERIALS AND METHODS: Blood samples (10 ml) from patients with advanced CCA were processed for CTC isolation as follows: -CTC enrichment with Parsortix -identification and single-cell recovery of epithelial CTCs (expressing epithelial markers) and non-conventional CTCs (lacking epithelial and leukocyte markers) using the DEPArray -whole genome amplification and quality check using Ampli1 kit and Ampli1 QC kit -mutational profiling using Ion AmpliSeq Cancer HotSpot Panel v2 and AmpliSeq somatic pipeline for variant calling -copy number alteration (CNA) analysis using Ampli1 LowPass kit, plus unsupervised clustering and frequency alteration analyses. RESULTS: We analyzed 88 single CTCs isolated from 38 blood samples longitudinally collected from 23 patients (12 with intrahepatic, 9 with extrahepatic CCA and 2 with gallbladder cancer). CNA profiles showed a high level of both inter- and intra-patient heterogeneity, with each CTC displaying a unique profile. Intra-patient heterogeneity was further confirmed by clustering analysis as, in most cases, CTCs from the same patient clustered independently. CTC clustering was also not affected by sampling time (before/during chemotherapy), nor by the anatomical location of primary tumor. Conversely, we observed an enrichment of CTCs derived from patients non-responding to therapy (showing a PD according to RECIST criteria) in 2 of the 4 identified clusters (p=0.00041). By pairwise comparison of CNAs among clusters, we identified 2 regions more frequently altered in one cluster enriched for CTCs from non-responders: 10q22.2 and 3p11.1. The latter encodes, among others, for EPHA3, a targetable gene whose involvement in chemoresistance will be investigated by in vitro studies. Mutational profiling of 19 CTCs (from 6 patients) also confirmed the high intra-patient heterogeneity with most mutations being present in only 1 CTC. This limits the applicability of this approach in patients with few CTCs. Nonetheless, in 1 patient presenting 9 CTCs, we identified 1 mutation in KIT shared by 7/9 CTCs, indicating it as a possible treatment target for this patient. CONCLUSIONS: Our results support the possibility of using CTC molecular characterization to identify both resistance mechanisms and patient-specific targets, thus opening the way for a shift in treatment management of CCA towards an innovative and personalized therapy. Citation Format: Carolina Reduzzi, Marta Vismara, Marco Silvestri, Monica Niger, Rosita Motta, Giorgia Peverelli, Patrizia Miodini, Luigi Celio, Filippo De Braud, Maria G. Daidone, Vera Cappelletti. Molecular characterization of circulating tumor cells in cholangiocarcinoma patients: A new tool for treatment management [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1390.

Abstract 1072: Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a fatal primary malignancy with limited treatment options and a dismal 5year survival rate of &amp;lt;10%. Poor prognosis and early recurrence after surgical treatment warrants further molecular characterization of these tumors to evaluate therapeutic sensitivities and identification of biomarkers for CCA. Although several Next Generation Sequencing (NGS) studies have revealed some targetable genomic aberrations, the lack of preclinical and translational models has severely limited the identification of clinically relevant targeted therapies for CCA. So far, very few putative molecular targets/pathways have been identified using patient-derived xenograft (PDX) models, CCA cell derived xenograft models (CDX) and CCA cell lines, only 4 of which have progressed to preclinical testing for therapeutic value. In this study we describe development and comprehensive characterization of patient derived xenograft models of CCA from freshly resected patient tissue. Patient tumor cells from 8 patients were engrafted subcutaneously in highly immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wj) mice for PDX development. Two (CCA-1 and CCA-7) of 8 (25 %) engraftments yielded successful PDX model. The PDX avatar and matched patient tumor samples were genomically characterized at the whole exome and transcriptome level to check the fidelity of PDX models. Exome sequencing analysis of PDX tumors demonstrate higher genomic aberrations as compared to their patient counterparts. Preliminary analysis of transcriptomic profiles from CCA TCGA dataset, long term established intra- and extra-hepatic CCA cell lines (Mz-ChA-1, TFK-1, SG231, CCLP-1, HuCC-T1 and HuH-28), CCA CDX model from Mz-ChA-1 cells and CCA PDX models demonstrate that CCA PDX models more faithfully recapitulate patient tumors as compared to long term established cell lines. In summary, this proof of concept study demonstrates that PDX avatars of CCA could serve as a good model system for preclinical target discovery and validation for a deadly and neglected disease such as CCA. Citation Format: Sanhita Rath, Sen Peng, Lauren Hartman, George Reid, Harshil Dhruv, Shannon Glaser, Fanyin Meng, Gianfranco Alpini, Scott Celinski. Development and characterization of patient derived xenograft (PDX) avatars of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1072.

Efficacy and safety of pembrolizumab in patients with PD-L1 positive advanced biliary tract cancer (BTC): A prospective cohort study.

4082 Background: For patients with advanced BTC, standard chemotherapy has limited benefit and no molecular targeted agents have been approved. Pembrolizumab is an anti PD-1 immune checkpoint inhibitor which has shown modest activity for advanced BTC patients in prior single-arm phase I/II studies. Considering the heterogeneity of BTC, more data are needed to evaluate the clinical outcomes of pembrolizumab in unresectable or metastatic BTC. Methods: In this prospective cohort study, 39 patients with PD-L1 positive BTC who received pembrolizumab in Asan Medical Center, Seoul, Korea were included (ClinicalTrials.gov identifier, NCT03695952). PD-L1 expression was assessed using immunohistochemistry and PD-L1 positive tumors were defined as the expression of PD-L1 in ≥ 1% of tumor cells. Pembrolizumab was given at a fixed dose of 200 mg intravenously, every 3 weeks. Results: The median age was 61 years old (range, 41-76) and 22 (56.4%) patients were male. Intrahepatic cholangiocarcinoma (CCA) was the most common type (n = 18, 46.2%), followed by gallbladder cancer (n = 12, 30.8%) and extrahepatic CCA (n = 9, 23.1%). Most of the patients had distant metastasis (n = 37, 94.9%). Pembrolizumab was administered as 2nd-, 3rd- and 4th or greater line chemotherapy in 18 (46.2%), 16 (41.0%) and 5 (12.8%) patients, respectively, and median 2 cycles (range 1-10) of pembrolizumab were given. In 36 patients whose response was assessable, partial response (PR) and stable disease were achieved in 4 (11.1%) and 13 (36.1%), respectively. In 19 (52.8%) patients, progressive disease was the best response. In patients with PR, the median time to response was 2.1 months (95% confidence interval (CI), 0.4 – 3.9). With a median follow-up duration of 4.4 months (95% CI, 2.4 – 6.4), median progression-free survival and overall survival was 1.5 months (95% CI, 0.4 – 2.6) and 4.3 months (95% CI, 2.6 – 6.1), respectively. No grade 3/4 adverse events (AEs) were reported and grade 1/2 fatigue (n = 4, 10.3%) was the most common AE. Conclusions: In PD-L1 positive BTC, pembrolizumab showed modest efficacy with 11.1% of response rates although our patients were heavily pretreated. Considering the limited therapeutic options and poor survival for these patients, further evaluation of immunotherapy including biomarker analysis is needed.

A novel mouse model of orthotopic extrahepatic cholangiocarcinoma confirmed with molecular imaging.

BackgroundCholangiocarcinoma (CCA) is the second most common primary hepatic malignancy in humans. Although early-stage CCA can be managed with surgery, CCA is considered incurable at advanced stages and results in poor quality of life and overall survival. A good orthotopic CCA animal model is essential to perform basic studies investigating CCA in order to understand the molecular pathways that underlie cancer development, and to develop new therapies for the prevention and treatment of CCA. However, to the best of our knowledge, orthotopic extrahepatic CCA animal models have not yet been reported in the literature.MethodsIn this study, we established an orthotopic extrahepatic CCA model in mice using a two-step surgical procedure.ResultsThe characteristics of this model were confirmed using molecular imaging and histological analysis.ConclusionsWe believe that this CCA animal model, which can be established quickly, easily, and with good reproducibility, will help guide research on novel diagnostic and treatment strategies for extrahepatic CCA.

The postoperative peak number of leukocytes after hepatectomy is a significant prognostic factor for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a lethal disease. A new predictive factor to identify patients suitable for adjuvant chemotherapy is needed. The relationship between the long-term prognosis and the perioperative immune responses in patients with CCA remains unclear. We therefore investigated the clinical impact of perioperative immune responses on the long-term prognosis in patients receiving hepatectomy for CCA. We investigated 81 patients who underwent hepatectomy between February 2000 and October 2012: 57 intra-hepatic CCA (iCCA) patients and 24 extra-hepatic CCA (eCCA) patients. We checked the postoperative level of C-reactive protein and the numbers of leukocytes. A multivariate analysis of the clinicopathological factors identified 2 significant risk factors for the overall survival: The postoperative maximum number of leukocytes (PNL) among patient factors (P=0.0406) and the TNM-stage among tumor factors (P=0.0059). On evaluating the distribution of each kind of leukocyte with a multivariate analysis, both the postoperative maximum number of neutrophils (PNN) and the postoperative maximum number of eosinophils (PNE) were detected as significant factors among leukocytes (PNN/PNE, P=0.0367/0.0083). In conclusion, the PNL after hepatectomy was significantly associated with the long-term prognosis in patients with CCA. Changes in the numbers of leukocytes after hepatectomy may be a marker on treatment for CCA.

Pre-operative biliary drainage is associated with shortened survival time in patients with cholangiocarcinoma.

Background and objectiveAlthough pre-operative biliary drainage (PBD) is frequently performed in patients with cholangiocarcinoma (CCA), its impact on patient survival is unclear. Our aim was to evaluate the impact of PBD on overall survival of patients with extra-hepatic CCA.MethodsThis was a retrospective study using the Surveillance, Epidemiology, and End Results (SEER)-Medicare data. Patients who underwent biliary drainage within 3 months prior to and/or after diagnosis of CCA were included in the PBD cohort. Patients who did not receive biliary drainage were included in the non-PBD cohort. Cox proportional hazard regression was used to determine independent predictors of survival.ResultsOf 3862 patients with extra-hepatic CCA, 433 (11.2%) underwent curative surgical resection, with a median survival of 14 months (95% confidence interval [95% CI], 10–21 months) in the PBD cohort (n = 126) vs 31 months (95% CI, 26–39 months) in the non-PBD cohort (n = 307) (P < 0.001), during the median follow-up duration for the surgical cohort of 26 months (range, 1–60 months). Among the 433 patients, 126 (29.1%) underwent PBD and had significantly higher Charlson comorbidity index and advanced SEER stage than those without PBD before surgery. On multivariable analysis in patients who underwent curative surgical resection, after adjusting patient demographics, tumor characteristics, Charlson comorbidity index, radiotherapy and chemotherapy, PBD was significantly associated with shortened survival time (hazard ratio, 2.35; 95% CI, 1.34–4.10; P = 0.003).ConclusionsPBD appears negative impact on long-term survival in patients with potentially resectable CCA and should be avoided if possible.

Frequency and feasibility of detecting FGFR mRNA expression in archival samples of patients with cholangiocarcinoma (CCA).

281 Background: Fibroblast growth factor receptor (FGFR) inhibitors show promising activity in patients with intrahepatic CCA harboring FGFR2 fusions, but these alterations have a modest frequency of 15-20%. FGFR mRNA expression has shown preliminary potential as a predictive biomarker for FGFR inhibition in advanced urothelial cancer and is currently being used for patient selection in clinical trials. To assess if we can increase the number of patients with CCA eligible for FGFR inhibitor trials, we evaluated the frequency and feasibility of detecting FGFR mRNA overexpression in archival CCA tissue. Methods: Patients with intrahepatic and extrahepatic CCA who received treatment at the MGH between 1/2004 and 12/2013 were included. Tissue microarrays of archival specimens were stained using the FGFR1-3 pooled, FGFR1, and FGFR2 RNA in situ hybridization probes (RNAscope 2.5 assay). Clinicopathologic data were obtained from the medical record. CCA cell lines were assessed for sensitivity to the FGFR selective inhibitor BGJ398 and also for FGFR expression by qPCR. Results: 90/111 (81%) TMA specimens were evaluable: median age 62.6 years, female 63%, median tumor size 5.1cm, intrahepatic/extrahepatic 71%/29%%. Among the 90 samples, 14% samples stained negative for the pooled FGFR1-3 probe, and 23%, 32%, and 30% stained 1+, 2+, and 3+ positive, respectively. Of note, majority of the samples stained 3+ for FGFR2. Examination of 11 ICC cell lines lacking FGFR gene abnormalities revealed that the single line with marked overexpression of FGFR1 exhibited hypersensitivity to BGJ398. Conclusions: Selection of patients with CCA for treatment with FGFR inhibitors based on mRNA expression levels is feasible on archival tissue with the FGFR RNA ISH probes, and this technique may identify a subpopulation of patients without FGFR genetic abnormalities who may benefit from FGFR inhibitors. [Table: see text]

Resection margin distance in extrahepatic cholangiocarcinoma: How much is enough?

455 Background: Surgical resection is required for curative treatment of patients with extra-hepatic cholangiocarcinoma (EH-CCA). The objective of this study was to determine if the distance of surgical margin was associated with outcome. Methods: Patients who underwent curative-intent resection for EH-CCA between 2000 and 2015 at 10 hepatobiliary centers across the U.S. were evaluated using prospectively collected data. Cox proportional hazard model was utilized to evaluate the influence of the extent of the margin on outcome. Results: 538 patients with EH-CCA who underwent curative-intent resection were included: 383 (71%) undergoing R0 resection, 153 (28%) undergoing R1 resection, and 2 with R2 resection. A negative surgical margin (R0) was associated with improved recurrence-free (RFS) and overall survival (OS) (RFS: 10.5% vs. 3.6% (R1) and OS: 25.8% vs. 9.3% (R1). Subsequently, further analysis on 161 patients with complete data on distance of resection margin, all undergoing R0 resection, was performed to assess the impact of extent of margin on outcome. On multi-variable analysis, the resection margin distance, analyzed as a continuous variable, was not associated with either improved RFS (RR 1.00, 95% CI 0.96-1.05; p 0.71) or OS (RR 0.99, 95% CI 0.96-1.01; p 0.49). Increasing age, increased tumor size, and LN metastasis were identified as independent predictors of OS; while RFS were mainly dependent on tumor size and LN metastasis (Table). Conclusions: Achieving R0 resection is acceptable for EH-CCA tumors, and obtaining additional margin does not confer a benefit on overall survival. Increasing age, tumor size, and LN metastasis are independent predictors of RFS and OS, but increased margin width is not associated with improvement in either. Multivariable analysis of factors affecting OS of patients with extra-hepatic CCA who underwent surgical resection, with significant factors noted in bold. [Table: see text]

Preoperative assessment of longitudinal extension margin of cholangiocarcinoma with per-oral video-cholangioscopy

Introduction: Preoperative assessment of longitudinal spread of cholangiocarcinoma (CCA) is crucial to select surgical method for performing R0 resection in patients with widespread extrahepatic CCA. Enhanced CT is essential but is not enough to detect longitudinal resection margin (LRM). Preoperative per-oral video-cholangioscopy (POCS) can be helpful but has not been necessarily indispensable. We examined whether POCS can supportive to diagnose LRM for CCA. Method: Between 2011 and 2018, there were 50 patients with distal extrahepatic or peri-hilar CCA who underwent preoperative PVCS followed by surgery. Diagnostic accuracy was examined by comparing results of preoperative PVCS and postoperative histopathologic LRM of surgical specimens. Result: The 50 patients underwent pancreaticoduodenectomy (PD), hemi-hepatectomy (HH), or PD-plus-HH. PVCS was performed since 2015, and 20 patients underwent PVCS. The incidence of positive LRM was 8/29 (27.6%) in patients without PVCS and 2/20 (10%) with PVCS. The overall incidence of positive LRM was not significant. However, the two patients with positive LRM, diagnosed an appropriate surgical method after PVCS was PD-plus-HH, but could not be performed due to patient side cause of advanced age. Therefore, the accuracy of preoperative PVCS was satisfactory for diagnosing LRM. Conclusion: Appropriate surgery cannot be always performed in CCA patients because of advanced age, insufficient hepatic function, or inadequate remnant liver volume. However, preoperative PVCS, undoubtedly in combination with MD-CT findings, can be helpful to detect LRM and select surgical method in patients with CCA.

A Novel Mouse Model of Orthotopic Extrahepatic Cholangiocarcinoma Confirmed with Molecular Imaging

Background: Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy in humans. Although early-stage CCA can be managed with surgery, CCA is considered incurable at advanced stages and results in poor quality of life and overall survival. A good orthotopic cholangiocarcinoma (CCA) animal model is essential to perform basic studies investigating CCA in order to understand the molecular pathways that underlie cancer development, and to develop new therapies for the prevention and treatment of CCA. However, to the best of our knowledge, orthotopic extrahepatic CCA animal models have not yet been reported in the literature. Methods: In this study, we established an orthotopic extrahepatic CCA model in mice using a two-step surgical procedure. Results: The characteristics of this model were confirmed using molecular imaging and histological analysis. Conclusions: We believe that this CCA animal model, which can be established quickly, easily, and with good reproducibility, will help guide research on novel diagnostic and treatment strategies for extrahepatic CCA. Funding Statement: The medical and health technology Project in Guangzhou 20171A010304. Declaration of Interests: The authors disclose no potential conflicts of interest. Ethics Approval Statement: All experimental protocols were approved by the Institutional Animal Care and Ethics Committee of Zhujiang Hospital of Southern Medical University, and all procedures were performed in accordance with the approved guidelines.

Patterns of Failure and Need for Biliary Intervention among Patients with Biliary Tract Cancers Treated with Definitive Surgery and Adjuvant Chemoradiation

Adjuvant chemoradiation therapy (CRT) after surgical resection of biliary tract cancers (BTC) has been shown to improve patient outcomes. However, there are limited data regarding patterns of failure after adjuvant chemotherapy and CRT with modern treatment regimens. In this study, we sought to characterize patterns of failure and need for biliary intervention after adjuvant CRT for gallbladder carcinoma (GBC) and cholangiocarcinoma (CCA). We present a retrospective analysis of 80 patients with GBC (n=29), extrahepatic CCA (n=30), perihilar CCA (n=13), or intrahepatic CCA (n=8). All patients underwent curative-intent resection with adjuvant CRT at a single institution from 2007–2017. Median CRT dose was 50.4 Gy (range: 36–65 Gy), which was administered with concurrent 5-FU-based chemotherapy. All but two patients received adjuvant chemotherapy. Tumor genomic mutation profiling was available for 30 patients (38%). Kaplan-Meier method was used to calculate rates of overall survival (OS) and locoregional failure (LRF), and log rank method was used to compare groups. Biliary interventions included ERCP with and without biliary stenting and percutaneous biliary tube placement. Study variables included age, performance status, site of primary disease, margin status, stage, receipt of oncologic resection, lymph node (LN) positivity, presence of lymphovascular invasion (LVI), tumor differentiation, and KRAS and TP53 mutational status. The median follow-up time was 30.5 months, and the median OS was 35.0 months. Among the entire cohort, 34% had T1/T2 disease, 40% were LN positive, and 53% underwent margin-negative (R0) resection. The overall rates of LRF and OS at 2 years were 21% and 62%, respectively. LRF was the initial site of failure in 16/80 patients (20%), and 19 patients (24%) had any LRF. On univariate analysis, only LN positivity (p=0.02) was positively associated with LRF. Site of primary disease (GBC vs. CCA), stage, LVI, and tumor mutation profile were not associated with LRF. Margin status was also not associated with LRF (2-year LRF: 18% vs. 29% in R0 vs. margin-positive (R1) resections, respectively, p=0.76). Among all patients, 23 (29%) required subsequent biliary intervention. Biliary intervention was required in 63% (12/19) of patients with LRF, compared to 18% (11/61) of patients without LRF. Of the 12 patients with LRF who required biliary intervention, 4 (33%) went on to die from biliary complications. Despite adjuvant CRT, there are high rates of LRF among patients with BTC, including GBC. Lymph node positivity may increase the risk of LRF. R1 resection was not associated with higher rates of LRF, suggesting that CRT may partially mitigate the risk of LRF in this high-risk group. LRF results in high rates of subsequent biliary interventions and can yield significant morbidity. Novel strategies should be considered to further improve upon rates of LRF.