In recent years, the role of EGFR in CC has been the focus of several studies. EGFR expression was examined in 20 surgically resected liver tissues with CC by IHC and 25% (5/20) were EGFR-positive suggesting that this growth hormone receptor is associated with CC . Nonomura et al. found EGFR in 32% of cases with intrahepatic CC by immunocytochemistry and reported some cases of co-expression with EGF, suggesting autocrine growth stimulation . Another group analyzed the role of EGFR in intrahepatic CC . EGFR expression was found to be correlated with frequency of lymph node metastases, aberrant p53 expression, proliferative activity and differentiation of the carcinoma. In a recent publication on EGFR expression in 236 cases of CC EGFR expression was a significant prognostic factor and also a risk factor for tumour recurrence in intrahepatic CC . These results confirm that EGFR expression is associated with tumour progression.
EGFR has been attributed an important role in carcinogenesis of several tumor types and EGFR inhibitors are currently used in treatment of some of them [24–28]. Yoon et al. reported that EGF stimulation increased cell growth in CC cells . The effect was significantly diminished by EGFR kinase inhibitors. A growth inhibitory effect of cetuximab has demonstrated in several cell lines of non-CC origin, e.g. in squamous cell , colon , head and neck , non-small cell lung , prostate , renal carcinoma  and glioblastoma . It has been reported that the monoclonal anti-EGFR-antibody cetuximab was active against various tumors including colorectal , head and neck , non-small cell lung , prostate  and renal cancer in vivo. Sprinzl et. al. described the treatment of a patient with a non-resectable CC in a case report . Combination of cytotoxic chemotherapy together with cetuximab showed promising efficacy. Blocking EGFR on CC cells could represent a therapeutic approach in respect to survival and quality of life . A recent publication assessed the efficacy of cetuximab in the palliative treatment of patients with intrahepatic CC unresponsive to first-line gemcitabine-oxaliplatin pretreatment. In tumor cells EGFR expression was found by IHC in 7 from 9 patients without gene amplification . Therapy with both cetuximab and gemcitabine-oxaliplatin was suggested as palliative treatment in patients with advanced intrahepatic CC.
So far, there are no reports on the effects of cetuximab on growth inhibition in CC cell lines. The human CC cell lines used have been developed from different histological types and different stages of CC. EGI-1 was established from a bile duct carcinoma with advanced stage malignancy . The initial tumor presented with seeded metastases and was histologically characterized as a large cell adenocarcinoma of low differentiation. HuH28 was established from liver bile duct carcinoma. OZ was established from ascitic effusion of a patient who suffered from obstructive jaundice due to the clogging of the common bile duct with mucinous substances produced by adenocarcinoma cells . TFK-1 was grown from a surgically resected tumor (extrahepatic bile duct carcinoma) specimen, which had parts of a papillary adenocarcinoma and a differentiated tubular adenocarcinoma on histology . EGFR expression was confirmed on all four CC cell lines by RT-PCR, Western blot and IHC. For EGFR this is consistent with data on other human CC cell lines: HuCCT1 express EGFR mRNA  and KMBC contain EGFR protein . As inappropriate Ras activation is known to promote tumor cell proliferation we examined the four cell lines for KRAS gene mutations. In EGI-1 a heterozygous mutation was found. HuH28, OZ and TFK-1 were confirmed to display the wild-type allele. Constitutively activated Ras is associated with continuous growth stimulation. The cell lines EGI-1 and TFK-1 were chosen for growth inhibition experiments to further compare the two different KRAS genotypes. Cetuximab did not significantly inhibit cell growth in EGI-1 cells, but had a dose-dependent effect on growth of TFK-1 cells. This evidence suggests that the outcome of growth factor inhibitor treatment could be affected from the K-ras genotype.
Yoon et al found that EGFR activation was sustained following EGF stimulation in cholangiocarcinoma cells as compared to hepatoma cells . They used KMBC and Witt cell lines. EGFR activation resulted in p42/44 MAPK activation. Cell growth was increased in cholangiocarcinoma following EGF stimulation and this was significantly attenuated by kinase inhibitors. To our knowledge, as yet, no study has compared the growth inhibitory effects of EGFR antibodies and kinase inhibitors. This group further found a defective receptor internalization in a CC cell line. However, they did not look into KRAS mutations. It would be of interest to study cells used by Yoon et al. with respect to their KRAS status.
As it was reported that cetuximab can induce apoptosis in tumor cells in vitro and in vivo [26, 42, 43], we tested the effect of cetuximab on cell survival. As we wanted to compare cell lines with activating KRAS mutation and without with respect to their response to cetuximab the cell lines EGI-1 (with activating KRAS mutations) and TFK-1 (without KRAS mutations) were chosen for growth inhibition experiments to further compare the two different KRAS genotypes. Cetuximab did not significantly inhibit cell growth in EGI-1 cells (containing the activating KRAS mutation), but had a dose-dependent effect on growth of TFK-1 cells. This suggests that the outcome of cetuximab treatment of CCC cell lines could be affected by the K-ras genotype. Therefore other modes of action might be involved in the effects observed on the growth of TFK-1 at least at lower cetuximab concentrations. The amount of EGFR present on the cell surface was not measured in our experiments. In colorectal cancer, there is no direct correlation between the amount of EGFR on the cell surface and the effects of EGFR blockade in vivo. And EGFR-negative colorectal cancer patients have been reported to respond to cetuximab treatment. From our data, it could be speculated that similar effects might be seen in CC. Further research is needed to demonstrate whether histology can predict a response to EGFR blockade in CC. Growth inhibition of cetuximab in TFK-1 cells was dose-dependent. This may imply that higher concentrations in vivo have more pronounced therapeutic effects. In contrast to cholangiocarcinoma in colorectal cancer, dose-escalating studies are on its way and their results are awaited.
More limitations apply to our study: we studied cell lines and it is inherent that the results may not be readily transferable to the in vivo situation. Moreover, although we used four different cell lines for the mRNA and protein expression experiments and two for the studies on the effect of cetuximab, these numbers are still low compared to the myriad of differences which should be found in different tumors in vivo. Nevertheless, the results of this study help to generate relevant questions for research in the treatment of cholangiocarcinoma. One of them is that KRAS mutations should be examined in the clinical trials on the effect of EGFR blockade in this tumor type.
In our experiments HGFR was detected in all four CC cell lines by RT-PCR, Western Blot and IHC. Others found HGFR mRNA in rat cell line CC-62, which was derived from a combined hepatocellular and cholangiocellular carcinoma . HGFR expression is high in well-differentiated tumors and relatively low in poorly differentiated tumors . Antagonizing the binding of HGF to HGFR also inhibited invasion in HuCC-T1, a human CC cell line, in vitro and in vivo  suggesting that blockade of HGFR might be a therapeutic strategy which should be the focus of further studies.
IGF1R expression was confirmed in the four CC cell lines tested by RT-PCR and IHC. Western blot showed protein expression in EGI-1 and TFK-1. Alvaro et. al. reported that IGF1R is expressed in the CC cell lines HuH-28, TFK-1 and Mz-ChA-1  which supports our finding. IGF1R antagonists can inhibit proliferation of CC cell lines after serum deprivation and re-administration. In addition, IGF1R antisense oligonucleotides diminished cell growth in HuH-28 cells . Increased expression of IGF1R promotes ligand-dependent malignant transformation in various cell systems . To our knowledge, there have been no reports on the expression on IGF2R in CC cell lines or CC. In our experiments IGF2R mRNA and protein expression was found in EGI-1, HuH28, OZ and TFK-1 with all methods applied.
VEGFR1 expression was found by RT-PCR and IHC in the four CC cell lines tested but not with Western blot. To date, there is poor knowledge about the expression of VEGFR in CC. Benckert et. al. confirmed VEGFR1 in 15 of 19 tumor samples in human CC biopsies by IHC and in in situ hybridization . It was suggested that a malignant phenotype is associated with increased VEGFR1 expression.
This study has several limitations. First, only CC cell lines were used. These cells lines may have gained additional mutations during the many passages under in vitro conditions. This may be a reason for the detection of VEGFR3 mRNA in the absence of detectable protein. Further, we only investigated the blockade of EGFR. Other growth factor receptors may be crucial for the growth of CC cells. In addition we regarded TFK-1 cells as an example of CCC cell lines with WT KRAS and did not study HuH28 and OZ cells. This all limits the impact of this study.