Molecular Targets and Emerging Therapies for Advanced Gallbladder Cancer

Biliary tract cancers (BTCs), for their low incidence, have been often considered together. Gallbladder cancer (GBC) is the most common biliary tract malignancy, characterized by late diagnosis and poor prognosis, and although it is considered a rare tumor in western countries, other areas of the world show considerable incidence rates. In 2010, results from the large phase III ABC-02 clinical trial on GBC identified the gemcitabine and cisplatin combination as the most effective first-line regimen for both GBC and other BTCs. Since then, various systemic therapies have proven active in BTCs in both first- and second-line settings. Molecular profiling has highlighted important genetic differences between GBC and other BTCs, opening new ways for targeted therapy in advanced disease where standard chemotherapies show marginal benefit. Genome-wide data analysis have shown that GBC molecular landscape offer possible strategies for precision medicine approaches, and a better molecular understanding of the GBC is needed to better stratify patients for treatment. In this review, we discuss the molecular targetable agents for GBC, including the results that emerged by clinical trials exploring new treatment strategies.

Biliary Strictures and Cholangiocarcinoma – Untangling a Diagnostic Conundrum

Cholangiocarcinoma is an uncommon and highly aggressive biliary tract malignancy with few manifestations until late disease stages. Diagnosis is currently achieved through a combination of clinical, biochemical, radiological and histological techniques. A number of reported cancer biomarkers have the potential to be incorporated into diagnostic pathways, but all lack sufficient sensitivity and specificity limiting their possible use in screening and early diagnosis. The limitations of standard serum markers such as CA19-9, CA125 and CEA have driven researchers to identify multiple novel biomarkers, yet their clinical translation has been slow with a general requirement for further validation in larger patient cohorts. We review recent advances in the diagnostic pathway for suspected CCA as well as emerging diagnostic biomarkers for early detection, with a particular focus on non-invasive approaches.

EGFR and HER2neu Expression in Gall Bladder Carcinoma and Its Association with Clinicopathological Parameters - An Institutional Experience from North India

Objective: Gallbladder cancer (GBC) is the most common biliary tract malignancy found worldwide with very high incidence in North India especially Delhi region. It is characterized by poor prognosis and ineffective treatment especially in advanced stage. The aim of this study was to evaluate EGFR and HER2/neu immunoexpression in cancer patients and to correlate it with the clinicopathological parameters so as to identify GBC patients who can benefit from targeted therapy.Methods: Present study was conducted in the Department of Pathology, Hamdard Institute of Medical Sciences & Research, New Delhi. A total of 40 cases of Gallbladder carcinoma (GBC) were evaluated for Immunohistochemical expression of EGFR and HER2/neu. Clinicopathological parameters of GBC were studied and correlated with immunoexpression of EGFR and HER2 /neu. Result: The mean age of the GBC patients was 55.9 years with 90% being females. On histopathology, 34(85%) cases were conventional adenocarcinoma. The EGFR expression was positive in 29/40 cases (72.5%). It was significantly more positive in poorly differentiated grade and advanced stages of gall bladder carcinoma (P<0.05). The expression of HER2/neu was positive in 13/40 cases (32.5%). It was significantly more positive in well differentiated gall bladder carcinoma (P<0.05). Immunoexpression of EGFR was inversely related with HER2/neu expression and this association was statistically significant.Conclusion: Among GBC patients, EGFR expression and HER2/neu expression was 72.5% and 32.5%, respectively. Significant EGFR expression was seen in poorly differentiated and advanced stage cancers while significant HER2/neu expression was seen in well differentiated gall bladder carcinomas. To conclude, these two markers HER2/neu and EGFR can be used as predictive and prognostic markers respectively, with rationale to further explore the use of anti-HER2 and anti- EGFR therapy in gall bladder cancer.

Mining of RNA Methylation-Related Genes and Elucidation of Their Molecular Biology in Gallbladder Carcinoma

Gallbladder carcinoma (GBC), which has high invasion and metastasis risks, remains the most common biliary tract malignancy. Surgical resection for GBC is the only effective treatment, but most patients miss the opportunity for curative surgery because of a lack of timely diagnosis. The aim of this study was to identify and verify early candidate diagnostic and prognostic RNA methylation related genes for GBC via integrated transcriptome bioinformatics analysis. Lists of GBC-related genes and methylation-related genes were collected from public databases to screen differentially expressed genes (DEGs) by using the limma package and the RobustRankAggreg (RRA) package. The core genes were collected with batch effects corrected by the RRA algorithm through protein interaction network analysis, signaling pathway enrichment analysis and gene ranking. Four modules obtained from four public microarray datasets were found to be related to GBC, and FGA, F2, HAO1, CFH, PIPOX, ITIH4, GNMT, MAT1A, MTHFD1, HPX, CTH, EPHX2, HSD17B6, AKR1C4, CFHR3, ENNP1, and NAT2 were revealed to be potential hub genes involved in methylation-related pathways and bile metabolism-related pathways. Among these, FGA, CFH, F2, HPX, and PIPOX were predicted to be methylated genes in GBC, but POPIX had no modification sites for RNA methylation. Furthermore, survival analysis of TCGA (the Cancer Genome Atlas) database showed that six genes among the hub genes, FGA, CFH, ENPP1, CFHR3, ITIH4, and NAT2, were highly expressed and significantly correlated with worse prognosis. Gene correlation analysis revealed that the FGA was positively correlated with the ENPP1, NAT2, and CFHR3, while CFH was positively correlated with the NAT2, CFHR3, and FGA. In addition, the results of immunohistochemistry (IHC) showed that the expressions of FGA, F2, CFH, PIPOX, ITIH4, GNMT, MAT1A, MTHFD1, HPX, CFHR3, NAT2, and ENPP1 were higher in GBC tissues than that in control tissues. In conclusion, two genes, FGA and CFH, were identified as RNA methylation-related genes also involved in bile metabolism in GBC, which may be novel biomarkers to early diagnose and evaluate prognosis for GBC.

Choledochal Cysts Resected during Childhood Show No Mutations of KRAS and BRAF as Early Markers of Malignancy in Cholangiocytes

Introduction In patients with choledochal cysts (CDC), a hyperplasia-dysplasia-carcinoma sequence can lead to biliary tract malignancy. The limited data available suggest that the risk decreases considerably after excision in childhood. We analyzed samples of resected CDC from pediatric patients histologically and performed mutational analysis of the proto-oncogenes KRAS and BRAF as early markers of malignant alteration in cholangiocytes. Materials and Methods After institutional review board approval, patients undergoing resection for CDC in our center from 2011 to 2019 were retrospectively identified. Histopathological reports were searched for inflammation and endothelial alteration. Cases with sufficient tissue specimen were tested for KRAS codon 12/13 and BRAF codon 600 mutations by pyrosequencing. Results In total, 42 patients underwent resection for choledochal cyst in the study period. Median age at surgery was 2.4 years (range = 18 days–18 years). Histopathological analysis showed no malignancy, but various degrees of inflammation or fibrosis in approximately 50% of the patients and in all age groups. Sufficient tissue for mutation analysis was available for 22 cases, all of which tested negative for KRAS or BRAF mutation. Conclusion In our series, chronic inflammatory changes were frequently present in CDC of infants and children. However, the lack of KRAS and BRAF mutations suggests that no malignant changes have been initiated in this group of European patients undergoing early resection.

A retrospective analysis of post second-line chemotherapy treatment outcomes for patients with advanced or metastatic cholangiocarcinoma and FGFR2 fusions.

4591 Background: Cholangiocarcinoma (CCA) is the most common biliary tract malignancy with an estimated incidence of 8,000–10,000 patients/year in the US. Chemotherapy is the most common second-line treatment with reported outcomes in patients with CCA. Response rates of < 10% and median progression-free survival (PFS) times of ~3–4 months have been reported with second-line chemotherapy regimens, including FOLFOX in the ABC-06 trial. Fibroblast growth factor receptor 2 ( FGFR2) fusions occur in 13–17% of CCA and multiple targeted agents are in development for patients with FGFR2 fusions. To date, the outcome of patients with CCA and FGFR2 fusions receiving standard second-line chemotherapy is unknown. Methods: Patients with advanced CCA and FGFR2 fusions after prior treatment with gemcitabine-based chemotherapy were enrolled in a single-arm phase 2 study (NCT02150967) and received the FGFR1–3 selective TKI infigratinib (previously BGJ398) 125 mg orally qd on d1–21, cycles repeated q28 days until unacceptable toxicity, disease progression, investigator discretion, or withdrawal of consent. A retrospective analysis of a subset of patients who received infigratinib as third- or later-line treatment was performed. Investigator-assessed PFS and best overall response (BOR, per RECIST 1.1) following second-line chemotherapy (pre-infigratinib) and third-line or later-line infigratinib were calculated. Results: Of the 71 patients (44 women; median age 53 years) with FGFR2 fusions enrolled at the time of analysis (datacut 8 August 2018), 37 (52%) were included in this retrospective analysis. Median PFS with standard second-line chemotherapy was 4.63 months (95% CI 2.69–7.16) compared with 6.77 months (95% CI 3.94–7.79) for third- and later-line infigratinib. BOR for second-line chemotherapy was 5.4% (95% CI 0.7–18.2) compared with 21.6% for third- and later-line infigratinib (95% CI 9.8–38.2). Conclusions: Outcomes from second-line chemotherapy in patients with CCA and FGFR2 fusions were similar to those reported in the literature for all patients with CCA regardless of genomic status and remain dismal. Infigratinib administered as third- and later-line treatment resulted in a meaningful PFS and ORR benefit in patients with CCA and FGFR2 fusions. Clinical trial information: NCT02150967 .

Honokiol and Magnolol Inhibit Growth, Metastasis and Induce Apoptosis in Human Cholangiocarcinoma

Cholangiocarcinoma (CCA) is biliary tract malignancy. Because no specific biomarkers are available, CCA patients frequently present with disseminated tumour that is too late for curative treatment, leading to a high mortality rate. Honokiol and magnolol are the hydroxylated biphenyl compounds isolated from Magnolia officinalis. Many studies have reported that honokiol and magnolol have antitumour effects on various types of cancer, but the evidence of the effects of these compounds on CCA cells has not yet been reported. This study therefore aims to evaluate the antitumour activities of honokiol and magnolol on CCA cell lines. The CCA cell lines were incubated with honokiol and magnolol before determining their responses. The results indicate that low concentrations of honokiol and magnolol suppressed CCA proliferation by induction of cell cycle arrest at G0/G1 and down-regulation of cyclin D1 protein. Moreover, these compounds exhibited an antimetastasis ability mediated by inhibiting migration, adhesion, and the MMP activities of CCA cells. In addition, at high concentrations of honokiol and magnolol activated CCA cell death associated with the apoptosis signalling pathway, along either an intrinsic or extrinsic pathway. Our data provides evidence that honokiol and magnolol have potential anticancer properties and are promising compounds for alternative CCA treatment.

Epidermal growth factor-like domain multiple 7 (EGFL7): Expression and possible effect on biliary epithelium growth in cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. The intrahepatic subtype comprises two forms: mucin-iCCA and mixed-iCCA. Epidermal growth factor-like domain multiple (EGFL7) is overexpressed in less differentiated liver tumors. The aim of this study was to assess the presence of EGFL7 due to its possible role in the growth of CCA. Hematoxylin and Eosin and periodic acid-Schiff staining were used to evaluate the morphological aspects and glycogen deposition. Immunohistochemistry and immunofluorescence were performed to identify the presence of EGFL7 both in tumor sections ex vivo and in appropriate cell lines in culture. We found that EGFL7 is expressed in malignant cholangiocytes of mixed-iCCA and absent in mucin-iCCA. In conclusion the expression of EGFL7 might be useful in the classification of CCA subtypes.

MYBL2 is a Potential Prognostic Marker that Promotes Cell Proliferation in Gallbladder Cancer

Background: Gallbladder cancer (GBC) is an aggressive and highly lethal biliary tract malignancy, with extremely poor prognosis. In the present study, we analyzed the potential involvement of MYBL2, a member of the Myb transcription factor family, in the carcinogenesis of human GBC. Methods: MYBL2 expression levels were measured in GBC and cholecystitis tissue specimens using quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) assays. The effects of MYBL2 on cell proliferation and DNA synthesis were evaluated using Cell Counting Kit-8 assay (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) retention assay, flow cytometry analysis, western blot, and a xenograft model of GBC cells in nude mice. Results: MYBL2 expression was increased in GBC tissues and associated with histological differentiation, tumour invasion, clinical stage and unfavourable overall survival in GBC patients. The downregulation of MYBL2 expression resulted in the inhibition of GBC cell proliferation, and DNA replication in vitro, and the growth of xenografted tumours in nude mice. Conversely, MYBL2 overexpression resulted in the opposite effects. Conclusions: MYBL2 overexpression promotes GBC cell proliferation through the regulation of the cell cycle at the S and G2/M phase transitions. Thus, MYBL2 could serve as a potential prognostic and therapeutic biomarker in GBC patients.