Downregulation of FOXO1 mRNA levels predicts treatment failure in patients with endometrial pathology conservatively managed with progestin-containing intrauterine devices

Abstract

Objective: To examine hormone receptor expression levels and downstream gene activation in pre-treatment and post-treatment biopsies in a cohort of patients with endometrial pathology who were being conservatively managed with a progestin-containing intrauterine device (IUD). A molecular signature of treatment failure is proposed.

Methods: A retrospective analysis of pre- and post-treatment biopsy specimens from 10 women treated with progestin-containing IUD for complex atypical hyperplasia (CAH) or grade 1 endometrioid adenocarcinoma was performed. Expression of estrogen receptor (ER), progesterone receptor (PR) and PR target genes was examined by immunohistochemistry (IHC) and quantitative RT-PCR.

Results: The mean treatment duration was 14.3 months. Four CAH patients had stable disease or regressed after treatment, and four progressed to endometrioid adenocarcinoma. Both patients with an initial diagnosis of endometrioid adenocarcinoma regressed to CAH or no disease. In general, hormone receptor levels diminished post-treatment compared to pre-treatment biopsies; however, we noted unexpected higher expression of the B isoform of PR (PRB) as well as ER in those patients who progressed to frank cancer. There was a trend towards a non-nuclear cytoplasmic location of PRB in these patients. Importantly, the differentiating impact of PR signaling, as determined by the expression of the progestin-controlled tumor suppressor FOXO1, was lost in individuals who progressed on therapy.

Conclusions: FOXO1 mRNA levels may serve as a biomarker for response to therapy and an indicator of PR function in patients being conservatively managed with a progestin-containing IUD.

Keywords: Endometrial cancer; Endometrial hyperplasia; FOXO1; Progesterone receptor; Progestin-containing intrauterine devices.

Figure 1. Study Design

CAH, complex atypical hyperplasia; CH, complex hyperplasia; EA, endometrioid adenocarcinoma.

Figure 2. Effect of progestin – containing intrauterine device (IUD) on hormone receptor protein levels in endometrial biopsies

A. Representative IHC images in endometrial biopsy samples before (Pre) and after (Post) insertion of the IUD. Modified H-scores (glandular) take into account the percentage of positive glandular cells and the staining intensity. The percent (%) nuclear staining indicates the percentage of nuclei that stained positive for the receptor. B. Scatter plots of hormone receptor levels in endometrial biopsies before and after treatment using paired data. **p<0.01 vs. pre-IUD insertion. C. Average modified H-score values based on data in panel (B). D. Pearson correlation analysis of the relationship between expression of PR (left panel) and PRB (right panel) with ER in post-IUD insertion biopsies.

Figure 3. Hormone receptor protein expression in endometrial biopsies from patients that progressed vs. patients that regressed or had stable disease after conservative management with progestin – containing IUD

A, B. Modified H-scores (glandular) by IHC in patients that either had stable disease/regressed (“No progression,” A) or progressed (“Progression,” B). Hormone receptor levels (modified H-scores) pre- and post-IUD insertion are presented as scatter plots using paired data. * p<0.05, ** p<0.01 vs. pre-IUD insertion. C, D. Average modified H-score (glandular) values pre-IUD insertion (C) or post-IUD insertion (D) based on data in panels A, B.

Figure 4. Downregulation of FOXO1 in patients who progressed after conservative management with progestin – containing IUD

A. mRNA expression of hormone receptors and PR target genes was determined by RT-qPCR, normalized to 18S, and data displayed as the fold change in the “Progression” group relative to the “No progression” group. Comparisons of normalized expression values (ΔCt) employed the conventional ΔΔCt fold change method. B. mRNA levels of FOXO1 in post- relative to pre-IUD insertion biopsies. C. Representative IHC images of FOXO1 staining in endometrial biopsies D, E. Average modified FOXO1 H-score (glandular) values (D) and % nuclear FOXO1 staining (E). In A and B, N=5 for “No progression” and N=2 for “Progression” in the pre-IUD insertion biopsies; N=4 for “No progression” and N=4 “Progression” in the post-IUD insertion biopsies. ‡‡ p = 0.002 vs. “No progression” in A; ‡‡ p = 0.006 vs. pre-treatment levels in B. In C and D, N=6 for “No progression” and N=4 for “Progression”; paired pre-and post-IUD insertion samples were available and analyzed for all subjects.

Figure 5. Nuclear and cytoplasmic distribution of hormone receptors in endometrial biopsies from patients that progressed vs. patients did not progress on therapy

A. Average % nuclear staining. See Figure 2A for representative images. N=10; paired pre- and post-IUD insertion samples were available and analyzed for all subjects. B, C. Average % nuclear hormone receptor staining pre-IUD insertion (B) or post-IUD insertion (C). D. Scatter plot of cytoplasmic PRB levels in endometrial biopsies before (Pre) and after treatment (Post) using paired data. Modified H-score (cytoplasmic) take into account the percentage of glandular cells with positive cytoplasmic staining and the staining intensity. p=0.07 vs. pre-IUD insertion. E. Average modified H-score (cytoplasmic) values post-IUD insertion. p=0.183 vs. “No Progression.” In B, C, and E, N=6 for “No progression” and N=4 for “Progression” groups.