Flutamide and biomarkers in women at high risk for ovarian cancer: preclinical and clinical evidence

Abstract

We hypothesized that (i) preclinical biologic evidence exists for the role of androgens in ovarian cancer development and (ii) flutamide treatment of women at high risk for ovarian cancer may identify meaningful tissue biomarkers of androgen action and of ovarian cancer initiation. We showed that androgen ablation of male mice led to a 24-fold decrease in tumor burden from serous ovarian cells. In a phase II study, we studied the effect of preoperative flutamide treatment (125 mg/day × 6 weeks) in 12 women versus 47 controls, 47% with BRCA mutation. We analyzed immunohistochemical scores of candidate proteins CSF-1, CSF-1R, and ErbB4 in the epithelium and stroma of fallopian tube, ovary, and ovarian endosalpingiosis. Flutamide decreased the levels, notably, of CSF-1 and ErbB4 in ovarian stroma (P ≤ 0.0006) and ovarian endosalpingiosis (P ≤ 0.01), ErbB4 in ovarian epithelium (P = 0.006), and CSF-1R in ovarian endosalpingiosis (P = 0.009). Our logistic regression model clearly distinguished the flutamide patients from controls (P ≤ 0.0001). Our analysis of the precision of this model of CSF-1 and ErbB4 expression in ovarian stroma achieved 100% sensitivity and 97% specificity (AUC = 0.99). Thus, our data suggest that a short 6-week exposure of flutamide reversed elevated levels of CSF-1 and ErbB4 (both of which we had previously found correlated with high risk status). CSF-1 and ErbB4 in ovarian stroma led to a model with high predictive value for flutamide sensitivity. The effect of flutamide on marker expression in ovarian endosalpingiosis, previously associated with BRCA carrier status, suggests that ovarian endosalpingiosis may be a latent precursor to pelvic serous cancers.

Trial registration: ClinicalTrials.gov NCT00699907.

Conflict of interest statement

Conflicts of Interest: None of the authors have any conflicts to disclose.

©2014 American Association for Cancer Research.

Figures

Figure 1

A) Androgen treatment increased the directed motility of MCV152 cells toward fibronectin. MCV152 cells with or without 10 nM 5α-dihydrotestosterone (DHT) for 66h, were studied for directed motility compared to Hey ovarian cancer cells. DHT treatment led to a significant increase in cellular motility compared to vehicle control treated cells (5.16 ± 0.53 vs. 8.16 ± 0.65, P= 0.001), and matched the motility of the invasive Hey cells (8.24 ± 0.59). Each bar represents the mean value of 4separate experiments. Error bars represent +/- standard error of the mean (SEM). B) Androgen treatment increased, while the antiandrogen flutamide decreased, the invasiveness of NOSE.1 cells (P < 0.001). NOSE.1 cells were treated with vehicle (control), 5uM flutamide, or 10 nM DHT for 48h during the invasion assay. Treatment of NOSE.1 cells with DHT led to a significant increase in ability to invade through extracellular matrix by 3.6-fold compared to control (2.54 ± 0.32 vs. 9.20 ± 0.82). In contrast, flutamide treatment led to an inhibition in invasiveness (by 0.68-fold), vs. control. Hey ovarian cancer cells remained much more invasive (by 2.7-fold) than the DHT-treated NOSE.1 cells. Error bars represent +/- SEM.

Figure 2

A) Ovarian serous epithelial cells are tumorigenic in male nude mice. B) Extent of tumor burden in intact and castrate male nude mice, from ovarian serous epithelial cells. The total tumor volume was dramatically less, by 24-fold, when the cells were implanted in castrate male mice.

Figure 3

A) Shown are positive and negative immunohistochemical controls for CSF-1 (human cervical stromal cells), ErbB4 (cerebral cortex), and CSF-1R (placenta) expression. Representative examples of CSF-1, ErbB4, and CSF-1R expression in HR control and flutamide groups are shown in B) ovarian stroma and epithelium, with total scores for staining in parentheses (epithelium/stroma) and in C) ovarian endosalpingiosis, with total scores for staining in parentheses.

Figure 3

A) Shown are positive and negative immunohistochemical controls for CSF-1 (human cervical stromal cells), ErbB4 (cerebral cortex), and CSF-1R (placenta) expression. Representative examples of CSF-1, ErbB4, and CSF-1R expression in HR control and flutamide groups are shown in B) ovarian stroma and epithelium, with total scores for staining in parentheses (epithelium/stroma) and in C) ovarian endosalpingiosis, with total scores for staining in parentheses.

Figure 4

This receiver operating characteristic (ROC) curve resulting from our logistic regression model of CSF-1 and ErbB4 expression in ovarian stroma emphasizes the clear effect of flutamide in patients. The dashed diagonal line represents no logistic model effect. The ROC curve plots sensitivity which represents the true positive rate of all flutamide-treated women vs. (1-specificity) the rate of false positives of all flutamide-treated women at various thresholds of the logistic regression model. The area under the curve (AUC) is an indicator of the precision of the model. The point closest to the upper left corner defines the optimal cut point for the prediction. The model of CSF-1 and ErbB4 expression in the ovarian stroma as represented by the ROC curve demonstrates a sensitivity of 100% and a specificity of 97% (C = 0.99) when differentiating between flutamide and control patients.