Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas

Abstract

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

Significance of epithelial growth factor in the epithelial–mesenchymal transition of human gallbladder cancer cells

“…Human recombinant EGF (Peprotec EC, Rocky Hill, NJ, USA), human recombinant HGF (R&D Systems, Minneapolis, MN, USA), and Wnt inhibitor (IWP3; Miltenyi Biotec, Bergisch Gladbach, Germany) were purchased. The effect of various concentrations of EGF (1,5,10,25,50, 100 ng/mL) and HGF (1,10,30,60, 120 ng/mL) on morphology was examined in GBC cells. At 10 ng/mL and higher concentrations of EGF, and at 30 ng/mL and higher concentrations of HGF, there were clear effects on cell morphology (see Fig.…”

“…(7) Amplification and point mutations of the EGFR gene have been reported to be 1% and 15 -26.5%, respectively, in GBC. (8)(9)(10) The HGF receptor c-Met is involved in the early carcinogenesis of BTC. (11) c-Met is expressed in 74% of invasive GBC and is associated with invasive depth.…”

Biliary cancer: intrahepatic cholangiocarcinoma vs. extrahepatic cholangiocarcinoma vs. gallbladder cancers: classification and therapeutic implications

“…Its amplification or overexpression has been shown to play an important role in the development and progression of several solid tumor malignancies, and is an effective therapeutic target in breast and gastric cancer (41)(42)(43). Studies evaluating HER-2 overexpression in BC have identified HER-2 mutations in gallbladder (about 10%) and EHCCs (up to 25%), and have been associated with a more aggressive phenotype (27,44). While small case series demonstrated anti-tumor activity with anti HER-2 directed therapy in patients whose tumors expressed HER-2 overexpression or amplification, these findings did not translate in BC patients Novel genomic alterations identified in IHCC…”

“…ErbB receptors and the binding to its ligands are integral to biliary carcinogenesis. Anti EGFR and HER-2 therapies have demonstrated interesting anti-tumor activity in preclinical studies (27), resulting in their investigation in clinical trials. …”

Cholangiocarcinoma: from molecular biology to treatment

“…In in vivo studies, the authors found that oral administration of sorafenib significantly inhibited tumor growth of heterotopic xenografts [58]. Other in vitro study has shown the existence of synergy in the combined use of sorafenib with doxorubicin and gemcitabine in CC treatment [62].…”