Elevated Serum 8-OHdG Is Associated with Poor Prognosis in Epithelial Ovarian Cancer

Patients and Methods

Patients. The study population consisted of 84 patients with ovarian cancer, diagnosed and treated from 1997-2005 at the Gynaecological Oncology Unit, Oulu University Hospital. Serum samples were available from all the patients and tumour samples from 78. The patients were treated by means of debulking surgery (with optimal or suboptimal results) and with platinum- and taxane-containing chemotherapy. Optimal surgery was defined as no macroscopic residual disease. The patient characteristics are shown in Table I. Blood samples were collected on the day before surgery and tumour samples during primary surgery. Serum samples were stored at −70°C until use. The tissue samples were fixed in neutral formalin, embedded in paraffin blocks and stored at the Department of Pathology, Oulu University Hospital. Histological diagnoses of the tumours were determined according to the criteria of the most recent WHO classification of ovarian cancer (16).

Enzyme-linked immunosorbent assay (ELISA). Serum levels of 8-OHdG were measured by using an ELISA (Highly Sensitive 8-OHdG Check ELISA kit; Gentaur Europe, Brussels, Belgium). Assays were carried out by following the manufacturer's instructions, with a few modifications. First all the serum samples were pre-processed using Millipore Microcon filters (Millipore, Billerica, MA, USA). The filters were dampened with 100 ml of distilled water and then centrifuged at 14,000 ×g for 5 min. Then the filters were turned around and centrifuged for another 5 min to remove any remaining water. The damp filters were moved to new tubes and 200 ml of serum sample was added into each tube and then centrifuged for 30 min at 14,000 ×g. The primary antibody was reconstituted with the primary antibody solution then 50 ml of sample or standard were added to the wells of a well plate each in duplicate before adding 50 ml of reconstituted primary antibody to each well. The plate was shaken and covered with adhesive strip and then incubated at 4°C for overnight. After incubation, the contents of the wells were poured off and each well was washed with 250 ml of washing solution three times. Secondary antibody was subsequently reconstituted with the secondary antibody solution; 100 ml of reconstituted secondary antibody was added to each well. The plate was then shaken, covered with adhesive strip and incubated for 1 h at room temperature. Washing was repeated at the end of the incubation period. After that, a substrate solution was prepared and 100 ml was added to each well and the plate was shaken. The plate was incubated in the dark for 15 min at room temperature then 100 ml of reaction terminating solution was added to each well and the plate was shaken. Absorbance was measured at 450 nm in a plate reader and a standard curve was used to determine the level of 8-OHdG in the samples. Duplicates of each sample were assayed and also the outermost wells of the microtitre plates were used. Forty samples were assayed in one plate. If the measured values in duplicate samples differed by more than 10%, the assay was repeated.

Immunohistochemistry (IHC). Four-micron-thick sections were cut from a representative paraffin block and placed on SuperFrostPlus glass slides (Menzel-gläser, Braunschweig, Germany). The sections were first de-paraffinized in xylene and rehydrated in a descending series of ethanol concentrations, incubated in 10 mM citrate buffer (pH 6.0), boiled in a microwave oven for 10 min, and cooled at room temperature. The slides were then immersed in 3% hydrogen peroxide in methanol for 15 min to consume the endogenous peroxide. The sections were incubated with a 1:125 primary antibody dilution against 8-OHdG (mouse monoclonal 8-OHdG antibody, Gentaur, Europe) overnight at 4°C. Both ELISA kit and immunostainings were based on the same 8-OHdG antibody (N45.1).

Immunostaining was carried out using a biotinylated secondary antibody 1:400 dilution with an avidin–biotin–peroxidase complex (Dakopatts, Glostrup, Denmark). Aminoethyl carbazole (Zymed Laboratories Inc., South San Francisco, CA, USA) was used as a chromogen. Immersed in 2% ammonia water, Meyer's haematoxylin was used for counterstaining and finally, the sections were mounted with Immu-Mount (Shandon, Pittsburgh, PA, USA). Negative controls were prepared using the same procedure except that the primary antibodies were replaced with PBS or serum isotype controls (Zymed Laboratories Inc.).

Immunoreactivity in the samples was assessed semi-quantitatively by assessing both the staining intensity in the nuclei of the tumour cells and the proportion of positively stained nuclei in the tumour cells. The immunoreactivity was evaluated by three independent investigators (MP, SK, PK), who divided the staining reactions into four groups: −, no staining intensity and no positive or only a few positive cells; +, weak staining intensity (>20-49% of positive cells); ++, moderate staining intensity (>50-89% of positive cells); +++, strong staining intensity (>90% of positive cells). If the evaluation of immunoreactivity differed between investigators, the sample was re-evaluated and consensus was reached. The investigators were blinded to the clinical data. For statistical analysis, the immunoreactivity results were separated into two groups: −/+ and ++/+++. Tumour grade was separated into groups of 1-2 and 3, and stages into I-II and III-IV.

Statistical analyses. SPSS 17.0 for Windows (IBM Corporation, Somers, NY, USA) and R-language (R Foundation for Statistical Computing, Vienna, Austria) were used for statistical analysis. The significance of associations was defined using the Mann–Whitney U-test and Pearson's Chi-square test; the reported values are two-sided. Survival was analysed by means of Kaplan–Meier curves with log-rank, Tarone–Ware and Breslow tests. Cox multivariate regression analysis was used for multivariate analysis. In the survival analysis, only confirmed death caused by ovarian cancer was considered as an event. Probability values <0.05 were considered significant. The statistician (AS) confirmed the data analysis.