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

Abstract

Background: Advanced 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.

Methods: Immunohistochemistry, 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.

Results: EGFR 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.

Conclusion: These 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.

Figure 1

Pattern of EGFR expression in the biliary tumors. EGFR expression as typical cell membrane immunostaining: weak (1+ score; A); moderate (2+ score, B) and strong (3+ score, C). Magnification X400.

Figure 2

Pattern of ErbB2 expression by HercepTest in the biliary tumors. Scoring from 0 to 3+ (A, B, negative; C 2+ and D 3+). Magnification X200.

Figure 3

Western blot analysis of EGFR/HER2 expression and phosphorylation of related signal transducers in BTC cell lines.

Figure 4

Dose effect curves of gemcitabine (gem) combined with EGFR/HER2 pathway targeted therapies on TFK1 (A); EGI-1 (B); HuH28(C) and TGBC1-TKB (D). After 72 h of treatment with 1:5 scalar doses (10 μM, 2 μM, 400 nM, 80 nM, 16 nM) of gem alone or in combination with targeted therapy at a fixed 1:1 ratio, antiproliferative effect was evaluated using viability test and Chou-Talalay equation by CalcuSyn software as described in the Methods section.