Simultaneous expression of steroid sulfatase and androgen receptor reduced overall survival of patients with epithelial ovarian tumors

STS was detected in 65% of the tumors. No association was observed between the expression of STS and the histological subtypes of the tumors (p = 0.20) or between the expression of STS and the clinical stage (p = 0.84) (Tables 2 and 3).

The androgen receptor was detected in the nucleus of epithelial tumor cells in 58% of the samples. However, there was no association found between the expression of the androgen receptor to the histological subtypes (p = 0.39) or the clinical stage (p = 0.96). When comparing serous versus non-serous subtypes, we observed a higher frequency of AR expression in serous subtypes (p˂ 0.05) (Tables 2 and 3).

The survival curves obtained by Kaplan–Meier analysis displayed in Fig. 2A showed that the presence of STS in epithelial ovarian tumors is related to a reduced survival of patients (log-rank test, p = 0.032), whereas the presence of androgen receptor did not modify the patients´ survival during a the 6-years follow-up (Fig. 2B).

Based on Cox proportional hazards regression model the age at diagnosis, the clinical stage (FIGO III and IV), and a suboptimal debulking surgery showed a significant increase in hazard ratio (HR) values in univariate analysis; whereas STS and androgen receptor presence HR values were not significant. In the multivariate analysis, considering age and clinical stage as covariates, HR values for STS and AR remained non-significant, p = 0.08 and 0.68, respectively (Table 4).

The total of the patients included in the study were stratified considering the presence or absence of AR in the ovarian tumor evaluating their overall survival. The survival curves demonstrated that the presence of STS represents a worse prognosis for the patients with AR positive tumors (p = 0.011); whereas in AR negative tumors, STS expression did not modify the overall survival of patients (p = 0.998) (Fig. 3). The proportional hazard regression analysis displayed the following values for STS in AR positive tumors: HR = 3.46, 95%CI 1.00–11.92, p = 0.049 and HR = 5.92, 95%CI 1.34–26.09, p = 0.019 univariate and multivariate analysis, respectively. The HR values in AR negative tumors were not significant (Table 5).

The study group comprised 154 samples of patients with pathological diagnosis of primary ovarian tumor from the Military Hospital for Women’s Specialties and Neonatology, SEDENA (Mexico’s Defense Ministry), and the National Institute of Cancerology in Mexico City spanning a time period of 10 years between 2008 and 2018. Written consent was obtained from each patient in order to participate in this study, none of whom had received chemotherapy or radiotherapy prior to surgery, per inclusion criteria. The tissue processing was done in accordance with the protocols established for international tumor banks and was approved by the Ethics Committee of the School of Medicine of the National Autonomous University of Mexico (UNAM-108/2015) as well as the Military Hospital of Women’s Specialties and Neonatology (310–18) and the National Institute of Cancerology (INCan) (019/060/OMI).

The study samples were obtained through intraoperative biopsy for diagnosis by the pathology department, after which whole sections of paraformaldehyde-fixed and paraffin-embedded tissue samples were serial sectioned at 3 μm thickness and placed on coated glass slides (Biocare Medical, Pacheco, CA, USA) which were deparaffinized by incubation in xylol and rehydrated through graded concentrations of ethanol, with the antigens being retrieved with Diva Decloaker citrate buffer (Biocare Medical) in a pressurized cooker at 110 °C for 15 min. Endogenous peroxidase was blocked by hydrogen peroxide at 1% H₂O₂ for 10 min. (Biocare Medical) after rising in PBS (pH 7.3) incubating the slides overnight at 4 °C with the following polyclonal rabbit primary antibody to AR, diluted 1:50 (Sc816, Santa Cruz Biotechnology, Santa Cruz, CA), using Mach2 anti-rabbit HRP as a secondary antibody for 1 h at room temperature (Biocare Medical) and signal detection was achieved using a diaminobenzidine chromogen kit (DAB) (Biocare Medical). The negative controls were tissue samples in which the primary antibody was substituted with PBS including the positive control tissues (placenta for STS and mice testis for AR) as well in each immune reaction, rinsing the slides with water after counterstaining them with hematoxylin. To assess the positivity ratio of reactions we evaluated the intensity of the staining and the percentage of labeled cells in the tissue samples. Thus the samples with no noticeable staining were rated as 0, while cells exhibiting a light staining were labeled as 1, cells with a moderate staining were marked as 2, and cells with a strong staining were designated as 3. The percentage of labeled cells was considered as: 1 = 10–25%, 2 = 26–50%, 3 = 51–80%, and 4 ˃ 80%. Positive reaction was considered whenever the index obtained from multiplying intensity and percentage was ≥ 2. Sample analyses were performed by three independent observers (MJG, MAA, and EP).

Slides were incubated overnight at room temperature with the following polyclonal rabbit primary antibodies: anti-STS, diluted 1:200 (GTX105498, GeneTex, Inc.) and further incubated with a secondary antibody goat-anti-rabbit Alexa 488, diluted 1:1000 (Thermo Fisher Scientific, Waltham. MA, USA). In order to identify epithelial cells, the slides were secondarily incubated overnight at 4 °C, with mouse monoclonal anti pan-cytokeratin AE1/AE3 + 8/18, diluted 1:100 (CM162C, Biocare Medical). The samples were washed with PBS and incubated with the secondary antibody Alexa Fluor 647 donkey anti-mouse, diluted 1:500 (A31571, Thermo Fisher Scientific). Placenta was used as a positive control in each immune reaction. The nucleus was stained with 4′, 6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Sigma-Aldrich, Inc). The slides were mounted with VectaShield mounting medium (Vector Laboratories) and the immunolabeled slices were observed by confocal laser microscope Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) using excitation spectral laser lines at 488 and 647 nm.