Introduction 24 15 11 21 3 9 12 extrahepatic 16 8 31 26 Materials and methods 28 Immunohistochemistry Immunohistochemistry was performed with an automated staining device (Dako Autostainer, Glostrup, Denmark). Cyclooxygenase-2 In this study, a monoclonal rabbit anti-human COX-2 antibody (DCS, Hamburg, Germany) was used. Immunohistochemistry was performed on 5-μm-thick paraffin, and antigen retrieval was carried out with 0.01-M citrate buffer at pH 6.1 for 20 min in a hot water bath (95°C). The primary antibody was incubated for 30 min at 1:250 dilution. Antibody demonstration was achieved using the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation, Carpenteria, CA, USA) The replacement of the primary antibodies by mouse immunoglobin served as negative controls. Positive controls (colorectal carcinoma) were included in each staining series. In ICC, COX-2 was scored according to the amount of positive stained tumour cells. One complete tumour slide was examined for specific cytoplasmic COX-2 immunostaining. If none or less than 10% of the tumour cells showed specific COX-2 immunostaining regardless of staining intensity, the case was classified as negative. The cases with 11–50% of the positively stained tumour cells were classified as moderately positive and tumours with more than 50% stained tumour cells as strongly positive. Ki67 immunostaining and TdT-mediated dUTP nick-end labelling Ki67 immunohistochemistry was performed on 5-μm-thick paraffin sections. Dewaxed and rehydrated sections were incubated with hydrogen peroxide to block endogenous peroxidase. After the antigen retrieval in a hot water bath, the prediluted monoclonal anti-Ki67 antibody (Biogenex, San Ramon, USA) was incubated for 30 min; antibody demonstration was performed with the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation). The replacement of the primary antibodies by mouse immunoglobin served as negative controls. The growth fraction was defined as the percentage of Ki67-positive, randomly chosen nuclei per 600 tumour cells. In situ DNA fragmentation was established using the terminal desoxyribonucleotide transferase TdT-mediated dUTP nick-end-labelling technique (TUNEL) in paraffin-embedded sections. We used ApoTag™ plus peroxidase in situ apoptosis detection kit (Intergen). The staining procedures were performed according the manufacturer’s recommendations. The percentage of the stained apoptotic tumour cells per 600 randomly chosen tumour cells was calculated. To avoid miscounting of the necrotic cells, corresponding H&E sections were examined. Statistical analysis COX-2 immunostaining was assessed by two of the authors (K.J.S., H.R.) in a blind-trial fashion without knowledge of the clinical outcome. In case of disagreement, slides were re-evaluated by both investigators until agreement was reached. All data were converted to a PC and statistically analysed using SPSS version 12 for Windows Statistical Package for Social Sciences, Chicago, IL, USA. 19 χ 2 Results COX-2 immunohistochemistry 1 2 1 Fig. 1 asterisk left side asterisk right side arrow  Fig. 2 left right Inset  Table 1 COX-2 expression, demographic characteristics, and clinicopathological characteristics in 62 patients of the resected ICC   All COX-2 negative COX-2 moderate COX-2 strong P  n 62 12 (19.4) 25 (40.3) 25 (40.3)  Gender (male/female)  7/5 9/16 10/15 0.422 y 58.8 ± 11.5 52 ± 12.1 60.4 ± 9.8  60.6 ± 11.8 0.067 Grading     0.612 n 2 (3.2) 0 (0) 2 (100) 0 (0) n 41 (66.1) 7 (17.1) 15 (36.6) 19 (46.3) n 19 (30.6) 5 (26.3) 8 (42.1) 6 (31.6) UICC stage grouping     0.277  I 16 6 (37.5) 3 (18.8) 7 (43.8)  II 3 0 (0) 1 (33.3) 2 (66.7)  IIIA,B,C 38 5 (13.2) 19 (50) 14 (36.8)  IV 5 1 (20) 2 (40) 2 (40) Staging     0.390  T1 (n,%) 21 7 (33.3) 5 (23.8) 9 (42.9)  T2 (n,%) 7 0 (0) 4 (57.1) 3 (42.9)  T3 (n,%) 28 4 (14.3) 13 (46.4) 11 (39.3)  T4 (n,%) 6  1 (16.7) 3 (50) 2 (33.3) Lymph vessel infiltration     0.121   L0 47 7 (14.7) 18 (38.3) 22 (46.8)   L1 15 5 (33.3) 7 (46.7) 3 (20) Blood vessel infiltration     0.916  V0 39 8 (20.5) 15 (38.5) 16 (41)  V1 23 4 (17.4) 10 (43.5) 9 (39.1) a     0.281  0 37 10 (27) 15 (40.5) 12 (32.4)  1 21 2 (9.5) 10 (47.6) 9 (42.9) Distant metastasis     0.999  No 57 11 (19.3) 23 (40.4) 23 (40.4)  Yes 5 1 (20) 2 (40) 2 (40) b 32 7 (21.9) 11 (34.4) 14 (43.8) 0.545 Multifocal tumour 29 5 (17.2) 14 (48.3) 10 (34.5) Tumour size  8.1 8.75 ± 4.3  8.3 ± 2.9 7.4 ± 3.6 0.506 Resection status     0.627  R0 39 9 (32.1) 15 (38.5) 15 (38.5) c 23 3 (13) 10 (43.5) 10 (43.5) P a b c Clinical outcome by COX-2 expression n p 3 n p Fig. 3 p  p p p p 2 Table 2 Mean overall survival in relation to different patient subgroups Subgroups Mean overall survival in all ICC patients (months) All patients 16.5 R0-resected patients 19.7 R1-resected patients 11.4 R2-resected patients 9.3 COX-negative patients 22.8 Moderate COX-2 expression 18.7 Strong COX-2 expression 11.4 3 Table 3 Multivariate COX regression analysis for overall survival in 62 patients with resected intrahepatic cholangiocarcinoma   HR (95% CI)  P pTstage I–II/III–IV 1.63 (0.44–6.02) 0.465 UICC I–II, III–IV 1.20 (0.62–2.35) 0.589 Multifocality, solitary/multifocal 1.26 (0.41–3.89) 0.689 a 3.08 (1.27–7.44) 0.012 Vascular invasion, V0/V1 1.98 (0.88–4.41) 0.095 COX-2, negative/moderate/strong 1.07 (1.07–3.49) 0.028 a COX-2 and apoptosis/proliferation 4 Table 4 Apoptosis (TUNEL) and proliferative activity (Ki67) in relation to COX-2 expression   All (mean value/SD) COX-2 negative COX-2 moderate/strong P Apoptosis (percentage of positive tumour cells) 2.17 ± 2.40 3.84 ± 4.04 1.76 ± 1.73 0.028 Growth fraction (percentage of Ki67 positive tumour cells) 9.74 ± 7.37 8.55 ± 8.41 9.99 ± 7.34 0.486 P Discussion n extrahepatic 16 RAS TP53 13 14 16 20 29 1 4 7 12 12 16 5 16 10 30 9 22 2 23 3 17 6 18 27 32 33 8 31 25 26 In conclusion, this study demonstrates the independent prognostic value of immunohistochemical COX-2 protein expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors and facilitating an optimised therapeutic strategy. Our results suggest that COX-2 overexpression causes a shift towards increased tumour cell proliferation and decreased apoptosis contributing to the unfavourable clinical course.