Eppenberger et al. BMC Cancer 2010, 10:93 http://www.biomedcentral.com/1471-2407/10/93 Table 1 Characteristics of patients with mismatch repair- proficient colorectal cancer Clinico-pathological feature Gender (n = 381) Tumour location (n = 369) pT stage (n = 373) pN stage (n = 357) Tumour grade (n = 374) Vascular invasion (n = 374) Tumour border configuration (n = 374) Peritumoural lymphocytic infiltration (n = 374) Local recurrence (n = 377) Liver metastasis (n = 387) Post-operative therapy (n = 377) Female Male Left-sided Right-sided pT1-2 pT3-4 pN0 pN1-2 G1-2 G3 Absent Present Pushing Infiltrating Absent Present Absent Present Absent Present No Yes 5-year (%) (95%CI) Frequency n (%) 175 (45.9) 206 (54.1) 242 (65.6) 127 (34.4) 86 (23.1) 287 (76.9) 178 (49.9) 179 (50.1) 289 (77.3) 85 (22.7) 267 (71.4) 107 (28.6) 195 (52.1) 179 (47.9) 308 (82.4) 66 (17.7) 208 (55.2) 169 (44.8) 313 (80.9) 74 (19.1) 289 (76.7) 88 (23.3) Survival time (n = 377) 58.9 (52-65) *G1-G2: well and moderately differentiated tumours; G3: poorly differentiated tumors dewaxed and rehydrated in dH2O. Following pressure cooker mediated antigen retrieval in 0.001 M ethylene- diaminetetraacetic acid (EDTA) pH 8.0, endogenous peroxidase activity was blocked using 0.5% H2O2 and the sections were incubated with 10% normal goat serum (Dako Cytomation, Carpinteria, CA) for 20 min. The colorectal cancers were then incubated with pri- mary antibody for for VEGF-A (Santa Cruz; USA; 1:300), VEGF-B (Santa Cruz; USA; 1:100), VEGF-C (Santa Cruz; USA; 1:100), VEGF-D (Santa Cruz; USA; 1:100), VEGFR1 (LabVision; USA; 1:100), VEGFR2 Page 3 of 10 (Santa Cruz; USA; 1:1000) and VEGFR3 (Chemicon; USA; 1:200). Subsequently, sections were incubated with HRP-conjugated secondary antibody (DakoCytomation) for 30 min at room temperature. For visualization of the antigen, the sections were immersed in 3-amino-9-ethyl- carbazole+substrate-chromogen (DakoCytomation) for 30 min, and counterstained with Gill’s Haematoxylin. Negative controls underwent the identical procedure with primary antibody omitted. Positive controls con- sisted of tumours known to contain the protein of inter- est. Additionally, internal positive controls for VEGF ligands, such as inflammatory cells, needed also to be present for immunohistochemistry to be considered valid. The use of tissue for this study was approved by the local Ethics Committee of the University Hospital of Basel. Evaluation The evaluation of immunohistochemistry was performed by an expert gastro-intestinal pathologist blinded to patient outcome. Using a semi-quantitative scoring method which describes the percentage of positive tumour cells assigned to each case on 5% intervals (from 0% to 100%). This scoring system has previously been found to lead to significant inter-observer agree- ment between independent pathologists, particularly using tissue microarrays. Staining intensity for most VEGF ligands and receptors was homogeneous and therefore not assessed. For VEGF ligands, cytoplasmic staining was assessed whereas receptor expression was scored in both cytoplasm and membrane. Representative immunostains for VEGF ligands and receptors are illu- strated in Figure 1. VEGF ligand/VEGF receptor ratio VEGF ligand to receptor ratios were determined by uti- lizing the percentage of immunoreactivity in both cases and dividing expression of the ligand by that of the receptor. The following ratios were explored: VEGF-A/ VEGFR1, VEGF-A/VEGFR2, VEGF-B/VEGFR1, VEGFR- C/VEGFR2, VEGFR-C/VEGFR3, VEGFR-D/VEGFR2 and VEGF-D/VEGFR3. In the event that the expression of the receptor was 0% which occurred in 40 cases for VEGFR1, 4 cases for VEGFR2 and 40 cases for VEGFR3, cases were removed from the study. Ratios >1.0 indicate a higher expression of ligand compared to receptor while ratios <1.0 describe tumours with greater expres- sion of receptor compared to ligand. Study Design The study design is outlined in Figure 2. Resection spe- cimens from 489 patients treated between 1988 and 1996 were collected, retrospectively. A tissue microarray containing all cases was constructed. Immunohisto- chemistry for VEGF ligands and receptors was carried out and staining evaluated. Immunohistochemistry for