Lenhard et al. BMC Cancer 2012, 12:2 http://www.biomedcentral.com/1471-2407/12/2 was quenched by dipping in 3% hydrogen peroxide (Merck, Darmstadt, Germany) in methanol for 20 min. Non-specific binding of the primary antibodies was blocked by incubating the sections with “diluted normal serum” (10 ml PBS containing 150 μl horse serum; Vec- tor Laboratories, CA) for 20 min at room temperature. Then, slides were incubated with the primary antibodies at room temperature for 60 min. After washing with PBS, slides were incubated in “diluted biotinylated serum” (10 ml PBS containing 50 μl horse serum; Vec- tor Laboratories, CA) for 30 min at room temperature. After incubation with the avidin-biotin-peroxidase com- plex (diluted in 10 ml PBS, Vector Laboratories, CA) for 30 min and repeated PBS washing, visualization was conducted using substrate and chromagen 3,3’-diamino- benzidine (DAB; Dako, Glostrup, Denmark) for 8-10 min. Slides were then counterstained with Mayer’s acidic hematoxylin and dehydrated in ascending concen- trations of alcohol (50-98%). After xylol treatment, slides were covered. Placental tissue at 3rd trimenon served as a positive control for the hCG and LH-R staining, accordingly. For negative controls, primary antibody was replaced with normal control serum rabbit IgG (BioGenex, San Ramon, USA). Positive staining resulted in brownish color, negative controls as well as unstained cells in blue color. Immunohistochemical analysis Slides were evaluated and digitalized with a Zeiss photo- microscope (Axiophot, Axiocam, Zeiss, Jena, Germany). Immunohistochemical staining was assessed using a semiquantitative score according to Remmele and Stegner [20], comprising optical staining intensity (graded as 0 = no, 1 = weak, 2 = moderate, and 3 = strong staining) and the percentage of positively stained cells (0 = no, 1 = < 10%, 2 = 11-50%, 3 = 51-80% and 4 = > 81% cells). According to previously published data, we scored the tumor tissue as positive if more than 10% of cells were scored with an immunoreactive score (IRS) higher than 2 [15]. The slides were reviewed in a blinded fashion by two independent observers, including a gynecological pathologist (D.M.). Statistical analysis Statistical analysis was performed using SPSS 18.0 (PASW Statistic, SPSS Inc., IBM, Chicago, IL). Correla- tion analysis of the receptor expression was performed for the histological subtype, tumor stage, grading and clinical data using the non-parametric Kruskal-Wallis rank-sum test and the non-parametric Spearman corre- lation coefficient. For the comparison of survival times, Kaplan-Meier curves were drawn. The chi-square statis- tic of the log-rank test was calculated to test differences Page 3 of 8 between survival curves for significance. P values below 0.05 were considered statistically significant. Results Patient characteristics Sera of 123 patients diagnosed with either benign (n = 83) or malignant (n = 40) ovarian tumors were obtained before surgery to test for serum hCG levels. Among the patients with benign ovarian tumors were cystadenomas (n = 12), simple ovarian cysts (n = 25), endometriosis (n = 9), teratomas (n = 10), fibromas (n = 8) and other tumors (n = 18). Patients with ovarian carcinomas mostly presented at stage III or IV (FIGO I: 15.4%, FIGO II: 11.5%, FIGO III: 53.8% and FIGO IV: 19.2%). Patients with borderline tumors of the ovary are neither included in the benign nor in the ovarian cancer patient group. Paraffin embedded tissue of 156 ovarian cancer patients was available. Median age at primary diagnosis was 58 years (range 18-88). Most patients presented with progressed disease at primary diagnosis [FIGO I: n = 35 (22.6%), FIGO II: n = 9 (5.8%), FIGO III: n = 109 (70.3%), FIGO IV: n = 2 (1.3%)]. Patient characteristics are detailed in Table 1. Median follow-up time was 7.3 years (range 0.3-16.8) with 26 documented relapses and 91 deaths. Median relapse free survival was 2.1 years (range 0.9-7.2) and median overall survival 5.9 years (range 0.3-16.6) (Table 1). hCG ELISA In serum analysis, we found hCG-positive sera in 26.7% of patients with benign ovarian tumors and 67% positive sera in patients with malignant ovarian tumors. In addi- tion, we identified significant differences in hCG con- centration in benign compared to malignant diseases of the ovaries (p = 0.000). The median calculation has been done using all samples, i.e. negative samples were also included in the calculation. Median hCG concentra- tion in patient sera with benign ovarian tumors was 0.1 mU/ml and 4 mU/ml in patients with malign ovarian tumors (Figure 1). hCG expression in ovarian cancer tissue Immunohistochemical analysis revealed hCG positive tumors in 68% of all cancer tissues investigated (Figure 2a, b). Only slight differences in hCG expression could be observed with respect to the histological subtype, with lowest expression in clear cell carcinomas and highest in mucinous ovarian carcinomas (Figure 3a). Regarding tumor grade, we identified significant differ- ences in hCG expression among G1, G2 and G3 carci- nomas (Figure 3b, p = 0.022). With respect to tumor stage, a significant difference was observed in mucinous tumors at stage FIGO I compared FIGO II and FIGO III (p = 0.018, Figure 3c).