Progesterone Receptor Status Predicts Response to Progestin Therapy in Endometriosis

Valerie A. Flores; Arne Vanhie; Tran Dang; Hugh S. Taylor

Disclosures

J Clin Endocrinol Metab. 2018;103(12):4561-4568. 

In This Article

Methods

We conducted a retrospective cohort study utilizing endometriotic lesions obtained from women undergoing surgical evaluation for endometriosis at Yale New Haven Hospital. The study was approved by Yale University's Institutional Review Board. Inclusion criteria were: women with histologically confirmed endometriosis, available information regarding endometriosis-associated pain symptoms, and information regarding response to progestin-based therapy. Subjects were excluded if data regarding response to progestin-based therapy were missing or insufficient. Data regarding subject characteristics and response to progestin-based therapies were determined from the electronic medical record. Fifty-seven subjects met the inclusion criteria. Five subjects were excluded due to insufficient data on response to progestin-based therapy. As such, 52 subjects were included in the analysis. Of the 52 subjects, 21 had more than one lesion collected at the time of surgery. In 17 subjects, eutopic endometrium was also collected at the time of surgery. Each subject was considered the biologic variable using the lesion with the highest Histo (H)-score for further analysis.

Immunohistochemistry

Tissue was embedded in paraffin, cut into 5-mm sections, and mounted onto slides. Immunohistochemical analysis of PR expression was performed as previously described.[15–17] Briefly, slides were deparaffinized and dehydrated through a series of xylene and ethanol washes. After a 5-minute rinse in distilled water, slides were steamed in 0.01 M sodium citrate buffer for 15 minutes and cooled for 45 minutes. Slides were rinsed for 5 minutes in PBS with 0.1% Tween 20 (PBST), and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 minutes followed by a 5-minute PBST wash. Nonspecific binding was blocked with 5% normal goat serum in PBST for 1 hour at room temperature. The primary antibody used was PR H-190 (sc _7208; 1:800) and was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Slides were incubated with the primary antibody overnight at 4°C. Normal goat IgG (Santa Cruz Biotechnology) was used as a negative control; normal day-14 endometrium was used as a positive control. Goat antirabbit biotinylated secondary antibody was used for PR (Vector Laboratories, Burlingame, CA) and applied for 1 hour at room temperature. Slides were washed in 1× PBS, incubated in ABC Elite (Vector Laboratories) for 30 minutes at room temperature, washed in 1× PBS, and incubated for 41 seconds with diaminobenzidine (Vector Laboratories). A 30-second exposure to hematoxylin was used as a counterstain. Slides were rehydrated through 5-minute ethanol and xylene washes and mounted with Permount (Thermo Fisher Scientific, Waltham, MA).

An H-score for immunohistochemical staining was determined for each slide. Each slide was scored independently by two investigators blinded to subject response, and H-scores were averaged. The H-score was calculated using a modified H-score: the percentage of negative (score 0), weakly positive (score 1), positive (score 2), and strongly positive (score 3) cells were estimated, and the percentages were multiplied with the scores and summed.[18]

Statistical Analysis

Subject characteristics from the response and no response group were compared with an unpaired Student t test (continuous variables), Fisher exact test, or χ 2 test (categorical variables). H-scores were compared using an unpaired Student t test. R Programming version 3.5.1 and prebuilt irr (Various Coefficients of Interrater Reliability and Agreement) package (https://cran.r-project.org/web/packages/irr/index.html) were used to calculate the intraclass correlation coefficient between the two blinded investigators. Thresholds for categorization of PR status were based on receiver operating characteristic (ROC) curve analysis. A χ 2 test was used for analysis of the final contingency table for prediction of response to progestin therapy. Spearman rank correlation coefficient was used for analysis of matched eutopic and ectopic lesions. All analyses were two-sided, and a P value of ≤ 0.05 was considered significant.

Comments

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