Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma

Background: Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC. Patients and methods: One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Methigh and c-Metlow groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines. Results: The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Methigh expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Methigh expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Methigh expression was an independent predictor of poor overall and disease-free survival in patients with IHCC. Conclusions: c-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC.


Cholangiocarcinoma (CC) is a highly malignant invasive carcinoma arising through malignant transformation of cholangiocytes.
Epidemiologic studies have demonstrated that the incidence and mortality rates of this disease, especially those of intrahepatic CC (IHCC), are increasing worldwide (Mouzas et al, 2002;Okuda et al, 2002;Blechacz and Gores, 2008;Hezel and Zhu, 2008;Yachimski and Pratt, 2008;Aljiffry et al, 2009).
It is difficult to diagnose CC at an early stage because of the lack of clinical symptoms at this point, and most patients have unresectable disease at clinical presentation. Surgical resection is the only curative therapy, but among those patients who receive it, recurrence rates are high (Hezel and Zhu, 2008). To date, no randomised study has demonstrated any clear survival benefit of a specific chemotherapeutic regimen for cases of unresectable and recurrent CC (Aljiffry et al, 2009). Existing phase II data and a more recent meta-analysis suggest that gemcitabine and gemcitabine-based platinum regimens offer a slight advantage over other regimens (Hezel and Zhu, 2008).
Recently, a new treatment strategy for CC has been proposed, in the light of better understanding of the molecular mechanisms of carcinogenesis: it has been proposed that receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), vascular epithelial growth factor (VEGF) and c-Met, are promising targets for treatment of CC (Socoteanu et al, 2008;Yoshikawa et al, 2008). In a previous report, we have indicated that EGFR and VEGF could be promising molecules for targeted therapy of CC (Yoshikawa et al, 2008(Yoshikawa et al, , 2009. c-Met, also known as scatter factor, is a high-affinity receptor for hepatocyte growth factor (HGF). Activation of HGF-c-Met signalling initiates cell invasiveness and triggers metastasis through direct involvement of tumour angiogenesis (Zhang et al, 2003). Upon ligand binding, c-Met activates multiple downstream signal transduction pathways, including the Grb2-Ras-mitogen-activated protein kinase (MAPK) cascade, the phosphatidylinositol-3 kinase (PI3K) pathway, and the signal transducer and activator of transcription (STAT) pathway (Weidner et al, 1993;Furge et al, 2000). c-Met partners include the integrin a6b4, CD44, plexin B, Fas and other RTKs such as RON, EGFR and ErbB2 (Gentile et al, 2008).
c-Met and EGFR are considered to assemble oncogenic signalling networks. Amplified c-Met activates members of the EGFR family and, conversely, mutated or amplified EGFR activates c-Met in vitro (Guo et al, 2008). EGFR is frequently coexpressed with c-Met in cell lines of lung, head and neck, breast, colon, and brain tumours (Reznik et al, 2008).
To improve our understanding of the clinical significance of c-Met in CC, the primary aim of this study is to clarify the frequency of c-Met overexpression. Following with this analysis, the second aim of this study is to analyse its association with clinicopathological factors, along with molecular data (EGFR, HER2, and VEGF expression), in the largest cohort (111 cases of IHCC and 136 cases of extrahepatic CC (EHCC)) of surgical specimens of CC. We also examined the expression of c-Met and EGFR in CC cell lines.

PATIENTS AND METHODS Patients
A total of 247 patients with CC were examined in the present study. The patients had undergone surgery and been diagnosed histologically as having adenocarcinoma of the bile duct, except for cancer of gallbladder and ampulla of Vater, at the National Cancer Center Hospital, Tokyo, between February 1990 and July 2005. Patients who had other malignancies or had died within four weeks after surgery were excluded. Clinical and pathological data were obtained from the medical records of the patients. To examine the correlations of c-Met with other RTKs (EGFR, HER2, or VEGF), qualified cases including previous data for overexpression of these molecules (Yoshikawa et al, 2008) were examined. The studied patients included 168 men and 79 women ranging in age from 33 to 82 years (median 65 years), who had been observed for periods ranging from 1.4 to 204.5 months (median 29.8 months). The cases were divided into two groups, IHCC and EHCC, in accordance with the TNM Classification of Malignant Tumours (Sobin and Wittekind, 2002) defined by the Union for International Cancer Control (UICC) and the World Health Organization Histological Classification of Tumours (Hamilton and Altonen, 2000). There were 111 cases of IHCC and 136 cases of EHCC. In this study, peri-hilar EHCC and distal EHCC are combined as EHCC because it is difficult to categorise EHCC based on the origin of the cystic duct. Tumour recurrence was defined as tumour growth in any site of the body after the operation, which was diagnosed clinically, radiologically, or pathologically, but mainly by computed tomography and ultrasonography. Only tumour death was used for analysis. The research protocol was approved by the Ethics Committee of the National Cancer Center, Tokyo, Japan. All patients gave written informed consent for inclusion in this study.

Immunohistochemistry
Immunohistochemistry (IHC) was performed on 247 formalinfixed, paraffin-embedded tissue sections. Immunohistochemical staining for c-Met was performed using a polymer-based method (Envision þ Dual link-system-HRP (Dako, Glostrup, Denmark)). Figure 3 Immunoblot analysis of c-Met, phosphorylated-Met pY1234/ 1235), EGFR, and phosphorylated EGFR (pY1173) in CC cell lines. MKN45 cell (a human gastric cancer cell) is a positive control of c-Met and phosphorylated-Met expression (Smolen et al, 2006). b-actin is a loading control. 3,3 0 -Diaminobenzidine tetrahydrochloride was used as the chromogen, and the tissue sections were counterstained with haematoxylin.

Met
Intensities of c-Met immunoreactivity were defined as: 0, complete absence of membrane staining or membrane staining in less than 30% of cancer cells; 1 þ , faint and partial membrane staining in at least 30% of cancer cells; 2 þ , strong and complete staining in at least 30% of cancer cells. The cases were divided into two groups, c-Met low (0 or 1 þ ) or c-Met high (2 þ ), for purposes of statistical analysis. The sections were evaluated by three observers, MM, HO, and TS, without knowledge of the clinical data. HO and TS are board-certified pathologists. IHC of EGFR and assessment of its expression were done as described previously (Yoshikawa et al, 2008).

Cell lines
NCC-CC1, NCC-CC3-1, NCC-CC3-2, and NCC-CC4 cells were established from human IHCC, and NCC-BD1and NCC-BD2 from human EHCC, at the National Cancer Center Research Institute (Ojima et al, 2010). TKKK, HuCCT1, OZ, TGBC24TKB, and MKN45 were purchased from RIKEN Bio Resource Center or from the Japanese Collection of Research Bioresources. TKKK, TGBC24TKB, and HuCCT1 were established from IHCC, and OZ was from EHCC. MKN45 was a gastric cancer cell line that was used as a positive control, because of its high expression of c-Met and phospho-Met (Smolen et al, 2006). All of the cell lines had been derived from Japanese patients. The originally established six CC cell lines, HuCCT1 and MKN45 were maintained in RPMI with 10% bovine serum. TGBC24TKB, TKKK, and OZ were maintained in Dulbecco's modified Eagle medium with 10% bovine serum.

Western blotting
Subconfluent cells were lysed at 41C for 30 min using lysis buffer containing 10 mM Tris-HCl (pH 7.5), 1% Triton X-100, and 150 mM NaCl with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphate inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein concentration was determined using a

RESULTS
Immunohistochemical analysis of c-Met in human CC specimens c-Met staining was localised in both the cell membrane and cytoplasm of CC cells (Figure 1) (Figure 2).

c-Met and EGFR expression in CC cell lines
Expression of c-Met, phospho-Met, EGFR, and phospho-EGFR in ten CC cells and one gastric cancer cells were estimated by  Western blotting (Figure 3). Expression of c-Met was observed in nine CC cells. Coexpression of c-Met and EGFR was detected in eight of them (except NCC-CC3-1). Prominent c-Met phosphorylation was detected in five cell lines (HuCCT1, OZ, NCC-BD2, TGBC24TKB, and NCC-BD1) and simultaneous activation of c-Met and EGFR was observed in seven cell lines including these five.

Correlations between c-Met and clinicopathological factors
The relationships between c-Met expression and clinicopathological factors of IHCC and EHCC were evaluated and are shown in Tables 1 and 2. Increased expression of c-Met was significantly correlated with overexpression of EGFR in IHCC (P ¼ 0.0063), and histopathological classification (P ¼ 0.0239) and overexpression of EGFR (P ¼ 0.0056) in EHCC. No other clinical factors were associated with c-Met expression.
In EHCC, the c-Met high group tended to have a poor 5-year survival rate, but not to a significant degree. Univariate analysis also showed that c-Met high was not a significant factor for survival. Therefore, multivariate analysis was not performed for EHCC.

DISCUSSION
In the present study, we have demonstrated the importance of c-Met overexpression in the prognosis and treatment of CC. We found that c-Met expression was correlated with EGFR overexpression in CC, and that it was also a significant prognostic factor in IHCC. In previous studies, the frequency of c-Met overexpression ranged from 21 to 58% in IHCC (Terada et al, 1998;Aishima et al, 2002;Nakazawa et al, 2005) and from 0 to 80% in EHCC (Hida et al, 1999;Nakazawa et al, 2005). This rather broad range is probably attributable to the small numbers of cases studied, or to differences in the definition of positivity. Moreover, no correlation between c-Met overexpression and clinical outcome of CC has been demonstrated previously. Here we showed that increased expression of c-Met was significantly associated with decreased overall and disease-free survival in patients with IHCC. The reason why c-Met expression was not a prognostic factor in EHCC may be partly explained by variables associated with their anatomic behaviour and methods of surgery. Simultaneous expression of c-Met and EGFR has been observed in clinical specimens of primary chordoma (Weinberger et al, 2005) and gastrinoma (Peghini et al, 2002). Accumulated evidence has suggested that cross-talk occurs between c-Met and EGFR in several cancer cell lines (Jo et al, 2000;Farazi et al, 2006;Guo et al, 2008). Here we showed that c-Met expression was correlated with EGFR expression in clinical specimen of CC. We found that both EGFR and c-Met are broadly activated in CC cell lines. Eight CC cells coexpressed both c-Met and EGFR and coactivation of both proteins was detected in seven CC cell lines. It has been proposed that amplified c-Met drives the activity of EGFR family members and that mutated and amplified EGFR can drive c-Met activity (Guo et al, 2008). Mutual or unidirectional interaction between EGFR and MET activation has been reported in several cell lines (Bergstrom et al, 2000;Jo et al, 2000;Reznik et al, 2008). It is thought that either c-Met or EGFR stands at the top of the hierarchy of the downstream signalling pathway governed by the two molecules in a subset of cancer.
Collectively, it seems reasonable that efficient molecular therapy for CC should target multiple kinases such as c-Met, EGFR, and VEGFR. c-Met activation is regarded as one of the molecular mechanisms involved in the acquisition of resistance to anti-EGFR therapy, as activation of the alternative RTK pathway would bypass the EGFR pathway (Dempke and Heinemann, 2009). Therefore, inhibition of c-Met, either alone or in combination with an EGFR inhibitor, may be clinically beneficial in the setting of EGFR inhibitor resistance (Eder et al, 2009). Several studies have focused on combination therapy with c-Met inhibitors and agents targeting EGFR family members (Toschi and Janne, 2008).
In conclusion, c-Met overexpression is significantly correlated with overexpression of EGFR in CC and with prognosis in IHCC. Further molecular investigation of the interaction between EGFR and c-Met in this fatal disease is urgently needed.