The increasing of global incidence, poor prognosis and lack of effective therapy make the management of BTCs further emphasize the need of effective therapeutic agents.
Gene status and protein expression of EGFR and HER2 and their pathways may be potential biomarkers for predicting the response to EGFR/HER2 inhibitors.
We observed the presence of 100% EGFR expression in ICCs, 52.6% in ECCs and 38.5% in GBCs.
Mutations in the EGFR TK domain were present in 15% of cases . Furthermore, the incidence of K-RAS mutation was particularly low (6.1%). Interestingly, changes involving codon 12, frequently mutated in other tumor types, were not found in our series. Previous studies of K-RAS mutations in cholangiocarcinoma revealed divergent results [31–35]. A higher occurrence of K-RAS mutations was found in Japan and Germany (ranging from 39% to 54%) relative to other areas such as Thailand (from 0% to 8%) in which this tumor occurred with high frequency. Geographical differences in etiology or carcinogenesis of BTCs might explain this variability.
We observed a lower incidence of B-RAF mutations compared to that reported by Tannapfel and coworkers (8% vs. 21% respectively) .
We identified PI3K mutations in 4 cases (8.2%) and PTEN mutations in 2 cases (4.1%).
Multiple mutations of EGFR transducers were observed in some samples. Namely, a total of 14 mutations were found in 8 tumor samples and only 3 samples had a single point mutation. Consistent with previous reports [37, 38], the K-RAS and EGFR mutations were not present in the same sample but, in contrast with another report , K-RAS and B-RAF mutations were simultaneously present in one case. However, due to the genetic heterogeneity of tumor subclones, we cannot exclude that these mutations would be present in two distinct cell populations.
We observed a rare frequency of PTEN mutations and we did not find any loss of PTEN protein expression in comparison with normal cholangiocytes; rather a stronger labeling intensity and a high percentage of labeled cells were significantly present in tumor cells compared to normal counterparts. In particular, samples displaying EGFR pathway activation due to transducer mutations had the highest percentage of PTEN-labeled cells suggesting that a preserved PTEN function might counteract the EGFR downstream pathway activation. HER2 was overexpressed only in a small group of patients, in accordance with the results obtained by others  and no mutations on the TK domain were observed.
The inhibition of EGFR/HER2 pathways in BTCs cell lines demonstrated a broad range of response with EGFR TKIs being more efficient on ECC cell lines. In K-RAS-mutated EGI-1 cells the Dm of these drugs was twice the Dm on K-RAS WT TFK1. Furthermore, the presence of PI3K mutation and PTEN deletion in the HuH28 and TGBC1-TKB cells respectively could probably explain the resistance to these treatments [18, 20, 21]. Sorafenib was more effective in K-RAS-mutated ECC cell line in which the MAPK pathway had a high level of activation . Lapatinib was more effective in ICC cell line which had high levels of HER2 expression.
On the other hand, lapatinib was less effective on ECC cell lines. Furthermore, GBC cell line was considered resistant (Dm >10 μM). In fact, TFK-1 and EGI-1 cell lines expressed low level of HER2 and GBC cell line was negative. These results are consistent with data obtained in breast cancer and pancreatic preclinical models [41–44]. In addition, a phase II study on breast cancer patients revealed that all the responders showed high level of HER2 expression while the HER2 negative patients were non responders . In our study, the dual inhibition of EGFR and HER2 induced by lapatinib was less effective than the treatment with erlotinib and gefitinib in ECC cell lines. Indeed, in a study by Wiedmann et al., the treatment with NVP-AEE788, an EGFR/HER2/VEGFR-2, was more effective than erlotinib and gefitinib . The direct inhibition of VEGFR-2 could be the gain of function of this drug compared to EGFR and HER2 inhibition. In fact, VEGFR-2 was expressed in ECC cell lines . Moreover, VEGF was overexpressed in ICC and ECC samples from patients and regulated metastasis development . The inhibition of VEGFR and EGFR/HER2 signaling with NVP-AEE788 or vandetanib (ZD6474) might be another interesting alternative approach for the management of BTCs.
Everolimus was effective in all tested cell lines but not in HuH28. Nonetheless, everolimus inhibited the phosphorylation of mTOR in all cell lines. It seems reasonable that in HuH28 a mechanism of resistance that overcomes mTOR inhibition may be active.
Moreover, EGFR/HER2 pathway inhibitors had synergistic effect with gemcitabine treatment. The mTOR inhibition gave rise to the strong synergistic effect in combination with gemcitabine in extrahepatic cell lines. Chung et al demonstrated that more than 80% of extrahepatic BTC displayed mTOR activation  that correlated with poor prognosis. Interestingly, EGFR inhibitor erlotinib was able to overcome the resistance to gemcitabine in the intrahepatic cell line HuH28; in fact, intrahepatic specimens showed the highest EGFR expression. Surprisingly, this result was not obtained with gefitinib.
Deepening this study, by gene expression profiling of the cell lines will contribute to the comprehension of the different mechanisms involved in drug response.