Won et al. BMC Cancer 2010, 10:564 http://www.biomedcentral.com/1471-2407/10/564 chemotherapy remain poorly understood. Biliary tract cancers can be divided into bile duct cancer and gall- bladder (GB) cancer. GB and bile duct cancer have been considered similar, and hence have been treated with the same treatment protocols or clinical trials. However, they are currently considered different disease entities, because they have been shown to be distinct in clinical behavior and response to anti-cancer treatments. 5-fluorouracil (5-FU) is the most evaluated drug for GB and bile duct malignancies as a single agent or in combination. Recently, gemcitabine and cisplatin have shown improved survival of patients with biliary tract cancer compared with gemcitabine alone [2]. Thymidine phosphorylase (TP) is an enzyme that converts 5’-deoxy-5-fluorouridine (5’-DFUR) to 5-FU. The expression of TP might be correlated with the efficacy of 5-FU-based chemotherapy [3]. Overexpression of the excision repair cross-complementation group 1 (ERCC1) gene, which is crucial in the repair of cisplatin-induced DNA adducts, has been reported to negatively influence to effectiveness of cisplatin-based chemotherapy [4]. This study aims to compare the clinico-pathological characteristics and prognostic factors of GB and bile duct cancers, and also investigate the expression and clinical significance of TP and ERCC1, including several cell-cycle regulatory proteins in biliary tract cancers. Methods Patients One hundred and sixty-one patients who underwent curative or palliative surgery for extrahepatic cholangio- carinoma, Klatskin tumor or GB cancer in Kangnam St. Mary’s Hospital between October 1994 and December 2003 were evaluated. We excluded patients with intrahe- patic cholangiocarinoma, because this type of cancer has been known to have different clinico-pathological char- acteristics from the other two types of bile duct cancers. Among 161 patients, 65 (40.4%) patients had been diag- nosed with GB cancer, and 96 (59.6%) patients with extrahepatic and Klatskin tumor (BDC). Clinical records and pathological reports were reviewed retrospectively. The following clinical data were collected: patient age, gender, objectives of operation, adjuvant therapy, tumor staging, recurrence, and survival. Ethics committee approval was obtained from the Institutional Review Board of The Catholic University of Korea, Seoul St. Mary’s Hospital, Seoul, Korea. Informed consent was provided according to the Declaration of Helsinki. Tissue microarray and Immunohistochemical staining To construct the tissue microarray block, small core biopsies (3.0 mm diameter) were taken from non-necro- tic, morphologically representative areas of paraffin- embedded tumor tissues. One hundred and sixty-one Page 2 of 8 tissue cores from each specimen were assembled on a recipient paraffin block with a precision instrument (Micro Digital Co. Korea). After construction, 5 μm sec- tions were cut and hematoxylin-eosin staining was per- formed on the initial slide for histological verification. Immunohistochemical staining was performed on the 5 μm sections of the tissue microarray blocks. Paraffin sec- tions were deparaffinized in xylene and rehydrated in serial graded ethanol, and then subjected to microwave antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. Sec- tions were incubated for 1 hour at room temperature or at 4 ℃ overnight with the following primary antibodies at the specified dilutions: TP (Zymed Laboratories, South San Francisco, CA, USA) diluted 1:100, ERCC1 (Neo- Markers, Fremont, CA, USA) diluted 1:200, survivin (NeoMarkers, Fremont, CA, USA) diluted 1:50, p53 (DAKO Corporation, Carpinteria, CA, USA) diluted 1:100, and cyclin D1 (DAKO Corporation, Carpinteria, CA, USA) diluted 1:50. Immunohistochemical staining was performed using the rabbit or mouse DAKO Chem- Mate TM EnVision TM system and the Peroxidase/DAB kit (DAKO). Sections were then counterstained with Mayer hematoxylin and dehydrated, cleared and mounted. The results were interpreted by two indepen- dent pathologists who were blinded to the specific diag- nosis and prognosis for each case. The staining intensity was scored on a three-tiered scale: score 0 = less than 10% of cells positive; 1 = 10-49% positive; and 2 = more than 50% of cells positive. The criterion for positive stain- ing was more than 1 + of tumor cells that showed dis- tinct nuclear or cytoplasmic staining (Figure 1) [5-8]. Statistical analysis Overall survival (OS) was defined as the time from the date of diagnosis to the date of death or last follow-up. Time to recurrence (TTR) was defined as the time from the date of diagnosis to the date of recurrence. Continu- ous and categorical variables were compared using the Student’s t-test and the chi-square test. Univariate survi- val analysis and survival curves were estimated using the Kaplan-Meier method, and the log-rank test was applied to assess statistical differences. Multivariate analysis was performed with the Cox hazards regression model. All statistical analyses were performed using the SPSS pro- gram (version 15.0) and p < 0.05 was considered statisti- cally significant. Results Patient characteristics The clinico-pathological characteristics of the 161 patients included in our study are summarized in Table 1. The median age of the patients was 62 (range 35-87) years. GB cancer was more frequent in women, and