Journal of Experimental & Clinical Cancer Research 2009, 28:120 http://www.jeccr.com/content/28/1/120 80°C, maintaining this temperature and keeping sections in this solution for 10 min in a microwave pressure cooker. After allowing the sections to cool to room tem- perature, the slides were rinsed in PBS (pH 7.4). Endog- enous peroxidase activity was blocked by incubation of the tissue samples for 10 min in 3% hydrogen peroxide. Samples were incubated for 45 min with the primary anti- bodies at room temperature in a moisture chamber. VEGF determination and analysis Samples were incubated with mouse anti-VEGF mono- clonal antibody (1:100) (Abcam, Cambridge MA, USA) in BSA 1% in PBS for 45 min. After washing with PBS, bind- ing of the primary antibodies was revealed by incubation for 20 min with LSAB+ System Link (DAKO, Carpinteria, CA, USA) and LSAB+ HRP, (Streptavidin HRP kit, DAKO). The slides were rinsed with PBS and exposed to diami- nobenzidine for 5 min. After washing with PBS and coun- ter-staining with hematoxylin, the slides were dehydrated by graduated alcohols and xylol, and mounted with Poly- mount. Numerical proportions of stained cells were estab- lished by analyzing 10 high-power fields (400×) in each section. Only cytoplasmic staining was considered posi- tive. Intensity was graded on a semi-quantitative scale from 0–3. Graduation of expression was considered nega- tive if fewer than 5% of cells were stained. Determination of vascular density The samples were incubated for 45 min with mouse anti- CD34 monoclonal antibody (1:200) (Biocare Medical, Concord, CA, USA) as a marker for vascular endothelial cells. Three separated, highly vascularized areas ("hot spots"), previously identified in high-power fields (100×, then 400×), were analyzed by two pathologists by means of optic microscopy without previous knowledge of hCG determinations. Any immunostained vessel clearly sepa- rated from adjacent vessels with no muscular wall and within the optic field was considered a neovascularization vessel. Vascular density (VD) was considered as the aver- age of the three evaluated zones. Statistical analysis For descriptive purposes, continuous variables were sum- marized as arithmetic means, medians, and standard devi- ations (SDs), while categorical variables were expressed as proportions and confidence intervals (CIs). Inferential comparisons were carried out using the Student t or the Mann-Whitney U test, according to data distribution determined by the Kolmogorov-Smirnov test. Chi square or Fisher exact test was used to assess significance between categorical variables. Statistically significant and border- line-significant variables (p < 0.1) were included in the multivariate logistic regression analysis. Overall survival time was measured from day of surgery to date of death or last follow-up visit and analyzed with the Kaplan-Meier method, and comparisons among sub-groups were per- formed with the log-rank test. For survival curve analysis, all variables were dichotomized. Adjustment for potential confounders was performed by multivariate regression analysis. Statistical significance was determined as p < 0.05 with a two-sided test. SPSS software package version 16 (SPSS Inc., Chicago, IL, USA) was employed to analyze the data. Results A total of 109 patients were included. Two patients were excluded due to insufficient biopsy material and six because a different method to measure hCG was used. General patient characteristics are shown in Table 1. From a total of 101 tumors, non-seminomas corresponded to 54%, and seminomas to 46%. Diagnosis was confirmed by the pathologists, independent of the general character- istics of the patients. The most frequent histological sub- types were endodermal sinus tumors and mature ter- atoma in 21.8 and 14.9% of cases, respectively. Median age was 26 ± 7.7 years. The majority of patients (70.7%) had good risk according to the international risk (IGCCCG). hCG median and mean serum levels were 25.0 (range, 0–479000) and 14772 ± 71503, respectively. Only 10% of patients had hCG levels >5,000 mIU/mL, as shown in Table 2, percentiles for hCG, AFP and DHL val- ues are also stated in this table. Table 1: Patient characteristics (101 patients) Characteristic Age (years) Histology Seminoma Non-seminoma Endodermal sinus Choriocarcinoma Embryonal cell carcinoma Mature teratoma Immature teratoma Teratocarcinoma TNM stage I II III Metastasis (N or M) Absent Present International consensus risk Good Intermediate Poor % Median ± SD 26 ± 7.7 46 54 21.8 5.0 8.9 14.9 2.0 1.0 46.5 27.3 26.3 48.5 51.5 70.7 16.2 13.1 SD = standard deviation; TNM = Tumor, Node, Metastasis Page 3 of 7 (page number not for citation purposes)