Liver Fluke-associated Cholangiocarcinoma Research Articles

Incidence of liver fluke-associated cholangiocarcinoma in the area with high incidence of hemoglobin E disorder.

Incidence of liver fluke-associated cholangiocarcinoma in the area with high incidence of hemoglobin E disorder.

Abstract 6588: Systems biology-based drug repositioning and biomarker discovery for Thai liver fluke-associated cholangiocarcinoma

Abstract Our group has recently reported the successful use of comprehensive drug response and pan-omic profiling for identifying repurposable drug shortlists in Asian cholangiocarcinoma. We also developed the CCA45 gene-expression signature that could accurately predict the prognosis of CCA patients, especially those with Asian ethnic backgrounds. To further explore therapeutic candidates and potential resistance mechanisms, we have established CCA1 cell lines resistant to their subgroup-specific drug candidates: MEK and SRC inhibitors. Reverse-phase protein arrays (RPPA)-based proteomic profiling revealed upregulation of cell cycle, apoptosis, MAPK, and mTOR-related proteins in both MEKi- and SRCi-resistant cells. Src pathway activity was found to be more significantly reduced in SRCi-resistant cells. We identified CDK4/6 inhibitors as the second-line therapeutic candidates for MEK inhibitor-resistant cells by systematic drug screening. SRCi-resistant cells unexpectedly developed cross-resistance to MEK inhibitors and showed sensitivity to only a few CDK4/6 inhibitors. These data are beneficial for the clinical translation of our proposed drug candidates for Asian CCA, preparing the alternate regimens after drug resistance. We also adopted the DSP-based spatial transcriptomic platform to compare the tumor microenvironment between chemotherapy-sensitive and chemotherapy-resistant CCA patients. While the CTA panel showed consistent patterns of normalized gene expression to those of the WTA panel, the CTA panel can only measure 17 of the 45 genes required for our CCA45 signature. Out of these 17 genes, ITGB4, FGFR3, VEGFC, NOTCH1, and RRAD showed significant gene expression changes with reducing tumor percentage. The microenvironment of gemcitabine/cisplatin responder (R) non-responder (NR) exhibited negative regulation of ERK1/2 and MAPK, high WNT pathway activity, and high tumor-infiltrating lymphocyte (TIL). In contrast, the microenvironment of the non-responder (NR) subgroup showed high neutrophil-related activity, high NFκB, high Treg, and the exhausted immune phenotype. Overall, our study contributes toward realizing the precision medicine concept for CCA patients. Citation Format: Supawan Jamnongsong, Patipark Kueanjinda, Piyathida Tawornparcha, Kulthida Vaeteewoottacharn, Seiji Okada, Siwanon Jirawatnotai, Ananya Pongpaibul, Krittiya Korphaisarn, Somponnat Sampattavanich. Systems biology-based drug repositioning and biomarker discovery for Thai liver fluke-associated cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6588.

Multiple actions of NMS-P715, the monopolar spindle 1 (MPS1) mitotic checkpoint inhibitor in liver fluke-associated cholangiocarcinoma cells

AimNMS-P715 is a potent inhibitor of monopolar spindle 1 (MPS1) mitotic checkpoint kinase. Overexpression of MPS1 is associated with short survival times in patients with cholangiocarcinoma (CCA). This study investigated the anti-cancer effects of NMS-P715 in human CCA cell lines. Main methodsKKU-100 and KKU-213A CCA cell lines were treated with NMS-P715 and cell viability was determined using MTT and colony formation assays. Inhibitory effects of NMS-P715 on cell cycle and apoptosis were evaluated using flow cytometry. Expression of underlying mechanism-related proteins was examined by Western blotting. Mitotic catastrophe was assessed by counting abnormal nuclei. Transwell assays were used to examine cell migration and invasion. Key findingsMolecular docking showed that the NMS-P715/MPS1 complex was driven by an induced-fit mechanism. We provide new evidence that NMS-P715 potently inhibited cell proliferation and colony formation in both CCA cell lines. This was accompanied by induction of G2/M arrest and the consequent induction of mitotic catastrophe, a process that occurs during defective mitosis. The recent study showed that NMS-P715 activated caspase-dependent apoptosis and autophagosome formation with an increase of LC3 A/B-II protein expression in CCA cell lines. NMS-P715 also greatly impeded cell migration and invasion in CCA cell lines. The combination of NMS-P715 and gemcitabine or cisplatin showed synergistic effects on CCA cell proliferation. SignificanceThis study revealed for the first time that NMS-P715 is a promising candidate for combating CCA owing via multiple actions and may be suitable for further development in a clinical study.

Aberrant GLUT1 Expression Is Associated With Carcinogenesis and Progression of Liver Fluke-associated Cholangiocarcinoma.

Glucose transporter 1 (GLUT1) has been demonstrated to be overexpressed in various cancer tissues and play a significant role on growth, metastasis, and apoptosis in cancer cells. This study aimed to reveal the clinical relevance of glucose transporter 1 (GLUT1) in carcinogenesis and progression on liver fluke-associated cholangiocarcinoma (CCA). Expression of GLUT1 in CCA tissues from patients, as well as from a liver fluke-induced CCA hamster model, was determined using immunohistochemistry. CCA cell lines were transfected with GLUT1 siRNA and the roles of GLUT1 on cell growth as well as migration and invasion were investigated by using a clonogenic assay and Boyden chamber assays, respectively. GLUT1 was aberrantly expressed in hyperplastic/dysplastic bile ducts and CCA, but not in the normal bile ducts. High GLUT1 expression was significantly associated with non-papillary type, large tumor size, and short survival of patients. GLUT1 was expressed during cholangio-carcinogenesis and gradually increased with progression of histopathologic bile ducts. Silencing of GLUT1 expression significantly suppressed growth, migration, and invasion of CCA cell lines. GLUT1 plays important roles in carcinogenesis and progression of liver fluke-associated CCA. Targeting GLUT1 may be a strategy for treatment of metastasis in liver fluke-associated CCA.

Clinicopathologic features, tumor immune microenvironment and genomic landscape of Epstein-Barr virus-associated intrahepatic cholangiocarcinoma

Little is known about Epstein-Barr virus (EBV)-associated intrahepatic cholangiocarcinoma (EBVaICC) because of its rarity. We aimed to comprehensively investigate the clinicopathology, tumor immune microenvironment (TIME) and genomic landscape of this entity in southern China. We evaluated 303 intrahepatic cholangiocarcinomas (ICCs) using in situ hybridization for EBV. We compared clinicopathological parameters between EBVaICC and nonEBVaICC, and we analyzed EBV infection status, tumor-infiltrating lymphocytes (TILs) and genomic features of EBVaICC by immunohistochemistry, double staining, nested PCR, multiplex immunofluorescence staining, fluorescence in situ hybridization and whole-exome sequencing. EBVaICC accounted for 6.6% of ICCs and was associated with EBV latency type I infection and clonal EBV isolates. Patients with EBVaICC were more often female and younger, with solitary tumors, higher HBV infection rates and less frequent cirrhosis; the lymphoepithelioma-like (LEL) subtype was more common in EBVaICC. EBVaICC was associated with a significantly larger TIME component than nonEBVaICC. The LEL subtype of EBVaICC - associated with a significantly increased density and proportion of CD20+ B cells and CD8+ T cells - was associated with significantly higher 2-year survival rates than conventional EBVaICC and nonEBVaICC. Both PD-1 and PD-L1 in TILs, and PD-L1 in tumor cells, were overexpressed in EBVaICC. High PD-L1 expression in tumor cells and high CD8+ TIL densities were significantly more common in EBVaICC than in nonEBVaICC. Seven genes (MUC4, DNAH1, GLI2, LIPE, MYH7, RP11-766F14.2 and WDR36) were mutated in at least 3 patients. EBVaICC had a different mutational pattern to liver fluke-associated cholangiocarcinoma and HBV-associated ICC. EBVaICC, as a subset of ICC, has unique etiological, clinicopathological and genetic characteristics, with a significantly larger TIME component. Paradoxically, patients with EBVaICC could be candidates for immune checkpoint therapy. Epstein-Barr virus (EBV) is associated with a subtype of intrahepatic cholangiocarcinoma, with unique clinicopathological and genetic characteristics. The tumor immune microenvironment is also different in this tumor subtype and patients with EBV-associated intrahepatic cholangiocarcinoma may respond well to immune checkpoint inhibitors.

Antitumor Effect of Shikonin, a PKM2 Inhibitor, in Cholangiocarcinoma Cell Lines.

Pyruvate kinase M2 (PKM2) is an enzyme that is predominantly overexpressed in various types of cancer. The role of PKM2 in liver fluke-associated cholangiocarcinoma (CCA) remains unclear. This study aimed to investigate the antitumor activity of shikonin, a PKM2 inhibitor, in CCA cells. Immunohistochemistry and immunoblotting were used to determine PKM2 expression in CCA tissues and cells. Antiproliferative effects of shikonin were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation and trypan blue exclusion assays. The anti-metastatic activity of shikonin was determined using the Boyden chamber assay. Mechanisms by which shikonin inhibited CCA progression were determined. PKM2 was overexpressed in CCA compared to normal bile duct epithelial cells. Shikonin significantly inhibited growth, and migration of CCA cells while inducing their death. A mechanistic study revealed that antitumor effects of shikonin in CCA cells depended on increased production of reactive oxygen species. Shikonin may be a novel therapeutic agent for patients with CCA.

Characterisation of the Urinary Metabolic Profile of Liver Fluke-Associated Cholangiocarcinoma

BackgroundHuman infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis.MethodsGlobal molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand.ResultsMultivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury.ConclusionsSeveral metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

Modeling liver fluke transmission in northeast Thailand: Impacts of development, hydrology, and control

Human infection with the Southeast Asian liver fluke Opisthorchis viverrini and liver fluke-associated cholangiocarcinoma cause significant disease burden in Southeast Asia. While there has been considerable work to understand liver fluke pathology and to reduce infection prevalence, there remains a limited understanding of the environmental determinants of parasite transmission dynamics to inform treatment and control programs. A particular setting where targeted control efforts have taken place is the Lawa Lake complex in northeast Thailand. Here, we describe the recent history of host infections, as well as the hydrologic characteristics of this floodplain ecosystem that influence the extent of snail habitat and fish mobility and the transport of human waste and parasite cercariae. Using mathematical modeling, we outline a framework for reconstructing environmental transmission of O. viverrini over the course of the Lawa Project control program from its inception in 2008 until 2016, using locally acquired but fragmentary longitudinal infection data for both humans and environmental hosts. The role of water flow in facilitating movement between snail, fish, human, and reservoir hosts is a particular focus with respect to its relevant scales and its impact on success of interventions. In this setting, we argue that an understanding of the key environmental drivers of disease transmission processes is central to the effectiveness of any environmental intervention.

Dysregulated Expression of MITF in Subsets of Hepatocellular Carcinoma and Cholangiocarcinoma.

Cholangiocarcinoma represents the second most common primary liver tumor after hepatocellular carcinoma. Mahanine, a carbazole alkaloid derived from Murraya koenigii (Linn.) Spreng, has been used as folk medicine in Thailand, where the liver fluke-associated cholangiocarcinoma is common. The expression of microphthalmia-associated transcription factor (MITF) is maintained at immunohistochemically undetectable levels in hepatocytes and cholangiocytes. To explore the regulation of MITF expression in the liver, we immunohistochemically analyzed the MITF expression using hepatocellular carcinoma and cholangiocarcinoma specimens of the human liver cancer tissue array. MITF immunoreactivity was detected in subsets of hepatocellular carcinoma (6 out of 38 specimens; 16%) and cholangiocarcinoma (2/7 specimens; 29%). Moreover, immunoreactivity for glioma-associated oncogene 1 (GLI1), a transcription factor of the Hedgehog signaling pathway, was detected in 55% of hepatocellular carcinoma (21/38 specimens) and 86% of cholangiocarcinoma (6/7 specimens). Importantly, MITF was detectable only in the GLI1-positive hepatocellular carcinoma and cholangiocarcinoma, and MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. Subsequently, the effect of mahanine was analyzed in HepG2 human hepatocellular carcinoma and HuCCT1 and KKU-100 human cholangiocarcinoma cells. Mahanine (25 µM) showed the potent cytotoxicity in these hepatic cancer cell lines, which was associated with increased expression levels of MITF, as judged by Western blot analysis. MITF is over-expressed in subsets of hepatocellular carcinoma and cholangiocarcinoma, and detectable MITF immunoreactivity is associated with poor prognosis in patients with hepatocellular carcinoma. MITF expression levels may be determined in hepatic cancer cells by the balance between the Hedgehog signaling and the cellular stress.

Genomic Profiling of Biliary Tract Cancers and Implications for Clinical Practice.

Biliary tract cancers are relatively uncommon, have an aggressive disease course and a dismal clinical outcome. Until recently, there have been very few clinical advances in the management of these patients and gemcitabine-based chemotherapy has been the only widely accepted systemic therapy. The advent of next generation sequencing technologies can potentially change the treatment paradigm of this disease. Targeted therapy directed against actionable mutations and identification of molecular subsets with distinct prognostic significance is now feasible in clinical practice. Mutation profiling has highlighted the genomic differences between the intra, extrahepatic cholangiocarcinoma, and gallbladder cancer. The mutational spectrum of intrahepatic cholangiocarcinoma differs according to geographic location and ethnicity. There is a higher incidence of chromatin modulating gene mutations in Western patients as compared with Asian patients with liver fluke-associated cholangiocarcinoma. KRAS and p53 mutations are associated with an aggressive disease prognosis while FGFR mutations may signify a relatively indolent disease course of intrahepatic cholangiocarcinoma. FGFR and IDH mutations have promising agents in clinical trials at this time. An estimated 15% of gallbladder cancers have Her2/neu amplification and can be targeted by trastuzumab. On the other hand, an estimated 10-15% of cholangiocarcinomas have DNA repair mutations and maybe candidates for immune therapies with checkpoint inhibitors. The promise of targeted therapies for biliary tract cancers can be fulfilled with well-designed, prospective, and multi-center clinical trials.

Serial analysis of gene expression reveals promising therapeutic targets for liver fluke-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) continues to be a serious health problem and is the most common fatal cancer in northeastern Thailand. Comprehensive gene expression analysis was here used to identify possible therapeutic targets for CCA treatment. We assessed liver fluke-associated CCA tissue using serial analysis of gene expression (SAGE) and compared the data to normal liver tissue as a part of the Cancer Genome Anatomy Project (CGAP). The analysis identified 509 differentially expressed genes. Of 142 up-regulated examples, we selected candidates including TMSB10, GAL3, VDR, CYPA and CD147 for further validation in CCA tissues by immunohistochemistry. VDR, CYPA and CD147 were confirmed to be consistently overexpressed in the samples tested. The therapeutic and diagnostic potential of these genes warrants further investigation.

Abstract IA14: Targeting the Wnt signaling pathway in cancer

Abstract Mutations in the adenomatous polyposis coli (APC) gene, resulting in aberrant Wnt pathway activation, were first identified in hereditary and sporadic colorectal cancers in 1991. Today, 24 years later, there are still no approved Wnt pathway inhibitors on the market. In contrast, oncogenic BRAF mutations were discovered in various solid tumors in 2002. Within 9 years the first RAF inhibitor, vemurafenib, received FDA approval for the treatment of BRAF-mutant metastatic melanoma. Why has it been so difficult to develop Wnt pathway inhibitors? The first and most significant challenge has been to identify protein targets within the Wnt pathway that are amenable to pharmacologic inhibition. Other prominent signaling pathways dysregulated in cancer (for example, the MAPK pathway) consist of a series of activating phosphorylation events carried out by kinases, many of which can be inhibited by small molecules. However, the critical regulatory event in the Wnt pathway is the degradation of the transcriptional co-activator β-catenin by the β-catenin destruction complex, composed of APC, Axin, casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). Instead of activating the pathway, the kinases CK1 and GSK3 serve an inhibitory role, sequentially phosphorylating the amino terminus of β-catenin, triggering its ubiquitination and degradation. Confident that the Wnt pathway was important for tumorigenesis, albeit difficult to drug, Novartis initiated a high-throughput screen to identify small molecule inhibitors of Wnt secretion without a priori knowledge of their protein targets. This effort yielded WNT974, a potent and selective inhibitor of Porcupine, the O-acyltransferase that palmitoylates Wnt ligands, enabling them to be secreted. With the first synthesis of WNT974 in 2009, and efforts by Novartis and others to target additional upstream Wnt pathway components (including Frizzled, LRP6 and R-spondin), a second challenge had to be faced. Upstream Wnt pathway inhibitors are unlikely to be efficacious in tumors harboring downstream mutations in APC or β-catenin. Which, if any, human cancers are Wnt ligand-dependent, and therefore, likely to be sensitive to upstream Wnt pathway inhibitors? Recent insights into how the R-spondin/Lgr5/Rnf43 module regulates surface levels of Wnt receptors provided a critical first step towards a patient selection strategy. We now know that inactivating mutations in the Wnt antagonist RNF43 occur frequently in microsatellite-unstable colorectal, gastric, and endometrial cancers (80%, 55%, and 51%, respectively), pancreatic ductal adenocarcinoma (10%), and liver fluke-associated cholangiocarcinoma (9%). Its homolog ZNRF3 is commonly mutated in adrenocortical carcinoma (21%), and fusions that increase the expression of the Wnt agonists RSPO2 or RSPO3 are reported in 10% of colorectal cancers. Our preclinical studies in cell line and patient-derived xenograft models have confirmed that inactivating mutations of RNF43 or R-spondin fusions confer Wnt ligand dependency. Currently, a Phase 1 clinical trial with WNT974 is underway in patients with advanced cancer. Preliminary clinical data suggest WNT974 has a manageable safety profile, with dysguesia (49%) as the most common adverse event suspected to be drug related. Based on pharmacokinetic, pharmacodynamic and tolerability data, a recommended dose for expansion of 10 mg QD has been selected. In response to the emerging scientific evidence implicating the R-spondin/Lgr5/Rnf43 module in a wide variety of cancers, we adapted the clinical trial design, restricting the expansion part to patients harboring upstream Wnt pathway mutations. While there was no evidence of WNT974 anti-tumor activity in the dose escalation, one patient in the dose expansion with an RNF43-mutant appendiceal cancer had stable disease with tumor shrinkage of -27%, raising the possibility that WNT974 may have anti-tumor activity in a molecularly selected population. The intense efforts to understand the basic biology of the Wnt pathway and Wnt-driven cancers have made progress toward development of Wnt pathway inhibitors possible. Citation Format: Margaret McLaughlin. Targeting the Wnt signaling pathway in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA14.

Suppression of trophoblast cell surface antigen 2 enhances proliferation and migration in liver fluke-associated cholangiocarcinoma

Background and aim. Trophoblast cell surface antigen 2 (TROP2) or tumor-associated calcium signal transducer 2 (TACSTD2) is a 36-kDa type I transmembrane glycoprotein and exerts dual functions as an oncogene and tumor suppressor in cancer cells. In this study, we investigated the expression and functions of TROP2 in liver fluke-associated cholangiocarcinoma (CCA).Material and methods. TROP2 expression in 85 CCA tissues was detected by using immunohistochemistry. The methylation status of TROP2 promoter was studied in 15 matched pairs of normal and CCA formalin fixed paraffin embedded (FFPE) tissues using the bisulfite genomic sequencing (BGS) method. The functions of TROP2 on cancer cell behavior were investigated using siRNA in CCA cell lines. Proliferation, migration and invasion assays were performed. A PCR array was used to evaluate the impact of TROP2 knockdown on the gene expression profiles.Results. TROP2 was highly expressed in all normal bile duct epithelia, but significantly down-regulated in CCA cells. Sixty percent of CCA revealed promoter hypermethylation compared to the corresponding adjacent normal tissues. TROP2 knockdown significantly enhanced the proliferation and migration in CCA cell lines, and altered the expressions of MARCK, EMP1and FILIP1L.Conclusion. We provide new evidence that TROP2 is epigenetically down-regulated and operates as a negative regulator of cell proliferation and migration in liver fluke-associated CCA.

Abstract C45: Phase I study of WNT974, a first-in-class Porcupine inhibitor, in advanced solid tumors

Abstract Background: Aberrant Wnt pathway activation is critical for initiation and maintenance of multiple tumor types, yet therapeutic inhibition of Wnt/β-catenin signaling has been challenged by the lack of tractable targets. WNT974 is an oral investigational agent that potently and selectively inhibits Porcupine, a membrane-bound O-acyltransferase enzyme required for Wnt secretion. Based on preclinical studies, cancers that are Wnt ligand-dependent due to upstream aberrations in the Wnt pathway (e.g. mutations in RNF43 or its homolog ZNRF3, and fusions in R-spondin [RSPO]) are predicted to be sensitive to WNT974. RNF43 is frequently mutated in microsatellite unstable colorectal, gastric, and endometrial cancers (80%, 55%, and 51% respectively), pancreatic ductal adenocarcinoma (10%), and liver fluke-associated cholangiocarcinoma (9%). ZNRF3 is mutated in adrenocortical carcinoma (21%). RSPO2 and RSPO3 fusions occur in 10% of colorectal cancers. Methods: A Phase I clinical trial is being conducted in patients (pts) with advanced cancer to evaluate the safety and tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) properties, and antitumor activity of WNT974 (NCT01351103). Dose escalation was completed in a molecularly unselected population and guided by an adaptive Bayesian logistic regression model based on dose limiting toxicities and adverse events (AEs). Pre- and on-treatment specimens (blood, skin, and tumor) were collected for PK/PD evaluation. Expansion cohort enrollment is restricted to pts with tumors harboring molecular alterations that portend Wnt ligand dependence (e.g. RNF43 mutation, RSPO fusion). Results: As of data cut-off, March 2, 2015, 68 pts have enrolled. Sixty-six pts were treated in the dose escalation at dose ranges and schedules of 5-30 mg QD; 30-45 mg QD 4 days on, 3 days off; and 5 mg BID. WNT974 showed rapid absorption (median Tmax 1-3 h) and a mean elimination half-life of 5-8 h. Accumulation on Cycle 1 Day 15 was small (ratio <2). PK exposure was dose proportional and inter-patient variability was generally moderate. Reported AEs (>10%) suspected to be related to WNT974 included: dysgeusia (49%), decreased appetite (28%), nausea (27%), fatigue (19%), diarrhea (18%), vomiting (16%), hypercalcemia (13%), alopecia (10%), asthenia (10%), and hypomagnesemia (10%). Grade 3/4 related AEs (>2%) included: asthenia (4%), fatigue (4%), decreased appetite (3%), and enteritis (3%). Measurement of AXIN2, a ß-catenin target gene, in matched skin specimens, showed Wnt pathway inhibition at a wide range of doses. A recommended dose for expansion of 10 mg QD was chosen based on PK, PD, and tolerability data; maximum tolerated dose was not determined. There was no evidence of WNT974 antitumor activity in the dose escalation. As of data cut-off, two pts had initiated study treatment in the dose expansion and one, a pt with appendiceal cancer with an RNF43 mutation, had completed the first response assessment. This pt had stable disease with tumor shrinkage of -27%. Updated clinical data will be reported. Conclusion: WNT974 is a first-in-class Porcupine inhibitor. Preliminary clinical data suggest WNT974 has a manageable safety profile and potential for antitumor activity in a molecularly selected population. Studies to further evaluate the safety, tolerability, and antitumor activity of WNT974 as a single agent and in combination are ongoing. Citation Format: Filip Janku, Roisin Connolly, Patricia LoRusso, Maya de Jonge, Ulka Vaishampayan, Jordi Rodon, Guillem Argilés, Andrea Myers, Shu-Fang Hsu Schmitz, Yan Ji, Margaret McLaughlin, Michael R. Palmer, Jennifer Morawiak. Phase I study of WNT974, a first-in-class Porcupine inhibitor, in advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C45.

Urinary microRNA-192 and microRNA-21 as potential indicators for liver fluke-associated cholangiocarcinoma risk group

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region.

Circulating miR-192 in liver fluke-associated cholangiocarcinoma patients: a prospective prognostic indicator.

This study aimed to investigate the miR-192 levels in patients' sera of liver fluke-associated cholangiocarcinoma (CCA) for a prospective prognostic indicator. MicroRNA polymerase chain reaction (PCR) array was performed using pooled serum samples from 11 CCA patients and nine healthy subjects. Selected miRNAs were verified for the differential levels in both sera and tumor tissues (of patients and Opisthorchis viverrini (Ov)-induced CCA model) using TaqMan miRNA expression assay. Our results demonstrated that miR-192 was significantly higher in the serum of CCA patients than that in healthy subjects giving a sensitivity of 74% and specificity of 72% (area under the curve [AUC] = 0.803; 95% confidence interval [CI], 0.708-0.897, P < 0.0001). Serum miR-192 examined in Ov infected subjects and subjects with periductal fibrosis were increased but not statistically significantly when compared with healthy subjects. High levels of serum miR-192 were significantly correlated with lymph node metastasis (P = 0.047) and shorter survival compared with individuals with low levels of serum miR-192 (hazard ratio [HR] 2.076, 95% CI 1.004-4.291, P = 0.049). We also found that the expression levels of miR-192 appeared to be elevated in both CCA tissues of patients and in Ov-induced CCA tissues of a hamster model. This finding indicates that elevated levels of miR-192 may be involved in CCA genesis and have a potential utility as a noninvasive prognostic indicator for CCA patients.

270: TPRG1L is a novel microRNA-21 target: a possible linkage between miR-21 and cellular senescence in liver fluke-associated cholangiocarcinoma

270: TPRG1L is a novel microRNA-21 target: a possible linkage between miR-21 and cellular senescence in liver fluke-associated cholangiocarcinoma

Risk biomarkers for assessment and chemoprevention of liver fluke-associated cholangiocarcinoma

Human liver fluke, Opisthorchis viverrini (Ov), is the major risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Our approach focuses on genetic progression and molecular changes in the carcinogenic pathway of liver fluke-associated CCA aimed at assessing patients at risk of CCA and using chemoprevention as the secondary cancer prevention to reduce the incidence of CCA. This review summarizes altered gene expressions, biomolecules and their modification, i.e. DNA adducts, oxidized proteins, oxysterols and fibrotic markers in hamster- and human-CCA. Potential risk biomarker(s) and chemopreventive agent(s) criteria and selection were based on results from experimental and epidemiological studies identifying hepatobiliary disease, including CCA. Laboratory results reveal that oxidative stress induced by Ov infection leads to bimolecular damage, tissue remodeling especially periductal fibrosis and alteration of gene expressions, which could be involved in all steps of CCA carcinogenesis. Some of these molecules are reported to change their levels in opisthorchiasis, periductal fibrosis diagnosed by ultrasonography and CCA. Chemoprevention in experimental CCA tumorigenesis is discussed. These multiple risk biomarkers could now be explored for screening including chemopreventive intervention of subjects living in endemic areas where the prevalence of opisthorchiasis remains high.

Suppression of thymosin β10 increases cell migration and metastasis of cholangiocarcinoma

BackgroundThymosin β10 (Tβ10) expression is associated with malignant phenotypes in many cancers. However, the role and mechanisms of Tβ10 in liver fluke-associated cholangiocarcinoma (CCA) are not fully understood. In this study, we investigated the expression of Tβ10 in CCA tumor tissues and cell lines as well as molecular mechanisms of Tβ10 in tumor metastasis of CCA cell lines.MethodsTβ10 expression was determined by real time RT-PCR or immunocytochemistry. Tβ10 silence or overexpression in CCA cells was achieved using gene delivery techniques. Cell migration was assessed using modified Boyden chamber and wound healing assay. The effect of silencing Tβ10 on CCA tumor metastasis was determined in nude mice. Phosphorylation of ERK1/2 and the expression of EGR1, Snail and matrix metalloproteinases (MMPs) were studied.ResultsTen pairs of CCA tissues (primary and metastatic tumors) and 5 CCA cell lines were studied. With real time RT-PCR and immunostaining analysis, Tβ10 was highly expressed in primary tumors of CCA; while it was relatively low in the metastatic tumors. Five CCA cell lines showed differential expression levels of Tβ10. Silence of Tβ10 significantly increased cell migration, invasion and wound healing of CCA cells in vitro; reversely, overexpression of Tβ10 reduced cell migration compared with control cells (P<0.05). In addition, silence of Tβ10 in CCA cells increased liver metastasis in a nude mouse model of CCA implantation into the spleen. Furthermore, silence of Tβ10 activated ERK1/2 and increased the expression of Snail and MMPs in CCA cell lines. Ras-GTPase inhibitor, FPT inhibitor III, effectively blocked Tβ10 silence-associated ERK1/2 activation, Snail expression and cell migration.ConclusionsLow expression of Tβ10 is associated with metastatic phenotype of CCA in vitro and in vivo, which may be mediated by the activation of Ras, ERK1/2 and upregulation of Snail and MMPs. This study suggests a new molecular pathway of CCA pathogenesis and a novel strategy to treat or prevent CCA metastasis.

Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. Chronic inflammation such as parasite infection and primary sclerosing cholangitis can be an etiological factor of cholangiocarcinoma. Using a proteomic approach and double-fluorescent staining, we identified high expression and colocalization of albumin and cytokeratin-19 in liver fluke-associated cholangiocarcinoma tissues, compared with normal livers from cholangiocarcinoma patients and cadaveric donors, respectively. Albumin was detected not only in cells of hyperplastic bile ducts and cholangiocarcinoma, but also in liver stem/progenitor cell origin, such as canal of Hering, ductules, and ductular reactions, suggesting the involvement of stem/progenitor cells in cholangiocarcinoma development. To clarify the involvement of liver stem/progenitor cells in cholangiocarcinoma, we examined several stem/progenitor cell markers (CD133, CD44, OV6, and Oct3/4) in cholangiocarcinoma tissues analyzed by immunohistochemical staining, and measured 8-oxodG levels by using HPLC-ECD as an inflammation-related DNA lesion. In addition, a stem/progenitor cell factor Bmi1, 8-nitroguanine (formed during nitrative DNA damage), DNA damage response (DDR) proteins (phosphorylated ATM and γ-H2AX), and manganese-SOD (Mn-SOD) were analyzed by immunohistochemistry. Stem/progenitor cell markers (CD133, OV6, CD44, and Oct3/4) were positively stained in 56, 38, 47, and 56% of 34 cholangiocarcinoma cases, respectively. Quantitative analysis of 8-oxodG revealed significantly increased levels in CD133- and/or Oct3/4-positive tumor tissues compared to negative tumor tissues, as well as 8-nitroguanine formation detected by immunohistochemistry. In the cases of CD44- and/or OV6-positive tissue, no significant difference was observed. Cholangiocarcinoma patients with CD133- and/or Oct3/4-positive tumor tissues showed significantly lower expression of Mn-SOD and higher DDR protein, γ-H2AX. Moreover, CD133- and/or Oct3/4-positive cholangiocarcinoma patients had significant associations with tumor histology types, tumor stage, and poor prognoses. Our results suggest that CD133 and Oct3/4 in cholangiocarcinoma are associated with increased formation of DNA lesions and the DDR protein, which may be involved in genetic instability and lead to cholangiocarcinoma development with aggressive clinical features.