Objective
We sought to identify biomarkers associated with progestin therapy resistance and persistence/progression of endometrial hyperplasia.
Study Design
We performed a nested case-control study among women with complex (n = 73) and atypical (n = 41) hyperplasia treated with oral progestin, followed up 2-6 months for persistence/progression. We evaluated index endometrial protein expression for progesterone receptor isoform A, progesterone receptor isoform B (PRB), PTEN, Pax-2, and Bcl-2. Odds ratios and 95% confidence intervals (CIs) were estimated.
Results
Among women with atypical hyperplasia, high PRB expression was associated with 90% decreased risk of persistence/progression (95% CI, 0.01–0.8). High expression of progesterone receptor A and PRB suggested decreased risk of persistence/progression (odds ratio, 0.1; 95% CI, 0.02–1.0). These findings were not observed among women with complex hyperplasia. No associations were found with PTEN, Pax-2, and Bcl-2 protein expression.
Conclusion
PRB expression shows promise as a biomarker of progestin response. Further research is warranted to understand how PRB expression may guide treatment decisions.
Endometrial carcinoma is the most common reproductive tract cancer in the United States. Women with endometrial hyperplasia (EH), an abnormal proliferation of the endometrial glands, are at increased risk of developing carcinoma. EH is commonly managed with progestin therapy with surveillance or hysterectomy, depending on the severity of the histopathologic diagnosis and the perceived risk of progression to carcinoma. The World Health Organization (WHO) classifies EH severity as simple or complex, with or without cytological atypia, based on architectural and cytologic features. The likelihood of progression to carcinoma ranges by EH severity: simple hyperplasia without atypia (SH) often spontaneously regresses ; complex hyperplasia without atypia (CH) rarely progresses to endometrial cancer ; and atypical hyperplasia (AH) has the greatest risk of concomitant carcinoma and/or subsequent carcinoma. Therefore, hysterectomy is generally recommended for women with a diagnosis of AH who do not desire future fertility.
In this treatment paradigm, the medical and surgical treatment risks, costs, and quality-of-life issues differ substantially between a diagnosis of CH and AH. This is concerning given the low diagnostic reproducibility of the WHO classification scheme, particularly disagreement on key histologic features that distinguish AH from CH. Furthermore, new findings suggest that the majority of women with AH respond to progestin. In our prior report, 26.9% of women with AH treated with 2-6 months of progestin had persistence/progression of EH as compared to 66.7% of those not treated (relative risk, 0.39; 95% confidence interval [CI], 0.21–0.70). We found the long-term risk of endometrial carcinoma among women with AH who used progestin was 20.5 per 1000 woman-years as compared to 101.4 per 1000 woman-years among nonusers. Given the suboptimal reproducibility of WHO diagnostic criteria and the potential benefits of progestin therapy among women diagnosed with AH, there is a need for biomarkers that could predict progestin therapy resistance and EH persistence/progression, providing valuable information to guide treatment decisions.
Various immunohistochemical (IHC) biomarkers have been investigated for association with EH and/or endometrial carcinoma, in an attempt to distinguish between normal endometrium and endometrial neoplasm (preinvasive hyperplasia and invasive carcinoma). Relatively few longitudinal studies have explored IHC biomarker expression and EH persistence/progression with progestin therapy. The goal of most of these studies was to evaluate the change in IHC biomarkers before and after progestin therapy, with only 1 study seeking to identify baseline IHC biomarkers predictive of progestin therapy response in terms of EH progression.
The purpose of this study was to identify IHC biomarkers associated with progestin therapy resistance, defined as EH persistence/progression among women with CH or AH treated with oral progestin. We chose to investigate biomarkers of protein expression for which there is some evidence of: (1) early involvement in endometrial neoplasia; (2) a proposed steroid hormone receptor expression relationship; and (3) proven endometrial IHC methodologies. Specifically, we assessed expression of progesterone receptor isoforms A (PRA) and B (PRB), the tumor suppressor protein PTEN, the oncogenic protein Pax-2, and the antiapoptotic protein Bcl-2.
Materials and Methods
Study population and design
We performed a nested case-control study among women treated with progestin from a cohort with an index consensus diagnosis of CH or AH using the WHO classification scheme and followed up 2-6 months for persistence/progression of EH. Detailed Endometrial Hyperplasia Cohort study methods have been previously reported. Briefly, the study was conducted among female enrollees at Group Health (GH), an integrated health plan in Washington State. Women age >18 years diagnosed with CH and AH from Jan. 1, 1985, through April 1, 2005, were identified and were eligible if they had at least 1 additional endometrial pathology specimen (biopsy or hysterectomy) taken 8 weeks to 6 months after the index diagnosis with baseline tissue blocks available. This time interval corresponds with common clinical practice of progestin treatment for 2-6 months with pathology reassessment at treatment completion. Women were excluded if they had a hysterectomy, were diagnosed with endometrial cancer, or disenrolled from GH within 8 weeks of the index diagnosis. Histology slides were reviewed blindly, independently, and in random order by 2 University of Washington pathologists (R.L.G., K.H.A.) with a rigorous adjudication process. Pathology data were linked with automated enrollment, pharmacy, inpatient, and outpatient clinical databases.
For this study, 217 women with CH (n = 138) and AH (n = 79), confirmed by consensus review, were initially considered for inclusion, pending data on progestin treatment. We received approval from the GH and Fred Hutchinson Cancer Research Center Institutional Review Boards prior to the conduct of the study.
Progestin treatment
Automated pharmacy data were obtained on the dispensing of any progestin from 1 week before the index biopsy up to 6 months after the index biopsy, but prior to follow-up biopsy. Women were only included if >14 days of progestin was dispensed. The pharmacy database provided progestin type, dose, and duration dispensed.
Outcome ascertainment
The outcome was dichotomized into persistence/progression and regression of CH or AH by comparing the diagnosis at follow-up with the index diagnosis. Cases were women whose CH or AH persisted/progressed; controls were women whose CH or AH regressed. For example, among women with an index diagnosis of CH, follow-up diagnoses of CH, AH, or carcinoma were classified as CH persistence/progression and diagnoses of no hyperplasia or SH were classified as CH regression. We selected this outcome because failure to regress after 3-6 months of progestin therapy is commonly an indication for hysterectomy.
Exposure ascertainment, IHC methodologies
Exposures of interest were expression of PRA, PRB, PTEN, Pax-2, and Bcl-2 proteins as measured by IHC. IHC was conducted by the University of Washington Immunohistochemistry Research Laboratory. IHC stains for each biomarker were performed on unstained slides cut from formalin-fixed, paraffin-embedded tissue blocks. All tissues were deparaffinized followed by blockade of endogenous peroxidases and antigen retrieval using Antigen Unmasking Solution (Vector, Burlingame, CA). Pretreatment, antibody selection, dilution, incubation time, and positive control varied for each protein ( Table 1 ). The slides were counterstained in hematoxylin. Negative controls for each run were conducted using normal mouse or rabbit serum instead of the primary antibody on tissue.
| Procedure | PRA | PRB | PTEN | Pax-2 | Bcl-2 |
|---|---|---|---|---|---|
| Pretreatment | Citrate 18 min | Citrate 18 min | Citrate 18 min | EDTA 15 min | Citrate 15 min |
| Antibody | Mouse monoclonal | Mouse monoclonal | Mouse monoclonal | Rabbit polyclonal | Mouse monoclonal |
| Clone: 6H2-1 | Clone: Z-RX2 | Clone: 124 | |||
| Novocastra/Leica Microsystems, Buffalo Grove, IL | Novocastra/Leica Microsystems, Buffalo Grove, IL | Cascade Bioscience, Winchester, MA | Zymed, San Francisco, CA | Cell Marque, Rocklin, CA | |
| Dilution | 1:8000 | 1:1000 | 1:100 | 1:100 | 1:25 |
| Incubation time at RT, min | 40 | 40 | 40 | 40 | 40 |
| Positive control | Normal proliferative endometrium | Normal proliferative endometrium | Normal proliferative endometrium | Normal endometrial stroma | Tonsil |
IHC was run in a single batch for all samples and the antibody staining was scored without knowledge of the index or follow-up diagnoses. Nuclear antigens PRA and PRB were scored based on degree of nuclear staining whereas cytoplasmic antigen Bcl-2 was scored based on degree of cytoplasmic staining. PTEN is present in both cytoplasm and nuclei and both cytoplasmic and nuclear staining were considered in scoring. One of 2 study pathologists scored the percentage of cells with positive antibody staining, or for PTEN the loss of antibody staining, in the lesional tissue as 0%, 1-25%, 26-50%, 51-75%, or 76-100%.
We defined high expression of PRA or PRB and no loss of Pax-2 and Bcl-2 biomarker expression as >75% of cells staining positive. We defined low expression of PRA or PRB and loss of Pax-2 and Bcl-2 expression as ≤75% of cells staining positive. For PTEN, we defined no loss of expression as ≤25% loss of staining and loss of expression as >25% loss of staining. Cut points that were conservative with regard to high or no loss of expression were chosen to minimize misclassification of biomarker expression and were informed by review of the distribution of expression.
Covariate ascertainment
Age, race, parity, height, weight, personal history of breast or colon cancer, diabetes, and smoking status at index diagnosis, as well as oral contraceptive and postmenopausal hormonal therapy use in the 6 months prior to index diagnosis, were abstracted from the GH medical record.
Statistical analyses
To evaluate the relationship between individual biomarkers at index and the risk of EH persistence/progression at follow-up, we estimated the odds ratio (OR) as the measure of association and exact 95% CI using the epitab command in Stata 11.0 software (Stata Corp, College Station, TX) and conducted Fisher exact test with α = 0.05 significance level. Each IHC biomarker was analyzed as a dichotomous variable, high or low expression (PRA and PRB) and loss or no loss of expression (PTEN, Pax-2, Bcl-2). Analyses were performed separately among women with CH and AH and the role of age (<50 and ≥50 years) and body mass index (BMI) (<30 and ≥30 kg/m 2 ) as confounding factors in the association were evaluated. To assess if the association between individual biomarkers and progestin therapy resistance varies by duration of progestin therapy, we repeated the analyses among women with at least 8 weeks of progestin treatment.
Results
We excluded 77 of the 217 women with CH and AH considered for IHC analyses because: material was not available for IHC at index (n = 54); histologic presentation of the IHC stained sample was different from the original diagnostic slide (n = 4); or material was insufficient for accurate IHC scoring (n = 19). We restricted our analyses to 114 women with CH (n = 73) and AH (n = 41).
Among women with an index diagnosis of CH, a greater proportion of cases than controls were younger (≤39 years), current smokers, and overweight (BMI ≥25 kg/m 2 ). Similarly, among women with AH, cases tended to be younger (≤49 years), current smokers, and obese (BMI ≥30 kg/m 2 ) compared to controls. Overall, cases were less likely than controls to be nulliparous. Among women with an index diagnosis of CH, the time intervals between index and follow-up biopsies and weeks of progestin use, on average, were generally comparable between cases and controls. While the time interval and weeks of progestin use were greater for controls compared to cases among women with an index diagnosis of AH, the percent time on progestin treatment, or the estimated proportion of weeks between index and follow-up diagnoses in which progestin was taken, was similar for both groups. The majority of women with an index diagnosis of CH (cases 61.9%, controls 75.0%, P = .271) and AH (cases 75.0%, controls 89.7%, P = .334) were treated with progestin for at least 8 weeks between index and follow-up biopsies. The type and dose of progestin treatment were similar between cases and controls with the exception that, among women with an index diagnosis of AH, controls were less likely than cases to receive low-dose progestin treatment, defined as medroxyprogesterone acetate (MPA) <10 mg/d or norethindrone acetate <1 mg/d ( Table 2 ).
| Complex (n = 73) | Atypia (n = 41) | |||
|---|---|---|---|---|
| Cases | Controls | Cases | Controls | |
| Characteristics | (n = 21) | (n = 52) | (n = 12) | (n = 29) |
| Age, y | ||||
| ≤39 | 4 (19) | 2 (4) | 1 (8) | 3 (10) |
| 40-49 | 4 (19) | 17 (33) | 4 (33) | 3 (10) |
| 50-59 | 6 (29) | 13 (25) | 5 (42) | 12 (41) |
| 60-69 | 6 (29) | 13 (25) | 0 (0) | 5 (17) |
| ≥70 | 1 (5) | 7 (13) | 2 (17) | 6 (21) |
| Caucasian a | 18 (95) | 45 (90) | 8 (73) | 20 (80) |
| Diabetes a | 2 (11) | 4 (8) | 0 (0) | 3 (12) |
| Personal history of breast or colon cancer a | 1 (6) | 1 (2) | 1 (9) | 1 (4) |
| Current smoker a | 3 (16) | 4 (9) | 2 (18) | 2 (8) |
| BMI, a kg/m 2 | ||||
| <25 | 3 (16) | 16 (32) | 2 (18) | 7 (29) |
| 25-29.9 | 7 (37) | 7 (14) | 3 (27) | 7 (29) |
| ≥30 | 9 (47) | 27 (54) | 6 (55) | 10 (42) |
| Nulliparous a | 2 (11) | 10 (20) | 2 (18) | 10 (40) |
| Oral contraceptive b | 2 (10) | 1 (2) | 0 (0) | 0 (0) |
| HT b , c | 3 (14) | 6 (12) | 2 (17) | 3 (10) |
| Unopposed estrogen b , d | 3 (14) | 7 (13) | 0 (0) | 3 (10) |
| Progestin only b | 1 (5) | 1 (2) | 0 (0) | 0 (0) |
| Weeks between index and follow-up biopsy, mean (SD) | 15.6 (4.9) | 16.8 (4.3) | 12.8 (2.5) | 17.0 (4.3) |
| Weeks of progestin treatment between index and follow-up, mean (SD) | 9.7 (5.4) | 11.3 (5.2) | 9.7 (3.7) | 13.5 (5.2) |
| Percent time on progestin treatment, mean (SD) | 64.1 (30.0) | 68.5 (27.7) | 75.3 (24.3) | 79.3 (21.1) |
| Progestin treatment dose e | ||||
| Low | 4 (19) | 6 (12) | 2 (17) | 1 (4) |
| Medium/high | 12 (57) | 33 (65) | 6 (50) | 23 (82) |
| Maximum | 5 (24) | 12 (24) | 4 (33) | 4 (14) |
| Progestin treatment type f | ||||
| MPA | 11 (52) | 22 (42) | 5 (42) | 12 (41) |
| MEGA | 7 (33) | 21 (40) | 5 (42) | 13 (45) |
| NETA | 0 (0) | 6 (12) | 2 (17) | 1 (3) |
| Mixed class | 3 (14) | 3 (6) | 0 (0) | 3 (10) |
| Index biopsy year | ||||
| 1985 through 1989 | 0 (0) | 4 (8) | 0 (0) | 0 (0) |
| 1990 through 1994 | 6 (29) | 12 (23) | 4 (33) | 7 (24) |
| 1995 through 1999 | 7 (33) | 17 (33) | 7 (58) | 14 (48) |
| 2000 through 2005 | 8 (38) | 19 (37) | 1 (8) | 8 (28) |
a Missing data on: complex: race, diabetes, BMI–4; history of breast/colon cancer, parity–5; smoking–7; atypia: race, diabetes, history of breast/colon cancer, smoking, parity–5, BMI–6;
b Dispensed in the 6 mo preceding diagnosis of endometrial hyperplasia and dispensed for at least 2 mo;
c Postmenopausal estrogen plus progestin hormone therapy where progestin was dispensed for at least a third of time that estrogen was dispensed;
d Postmenopausal estrogen therapy where estrogen dispensed alone or with progestin if progestin was dispensed less than a third of time that estrogen was dispensed;
e low = MPA <10 mg/d, NETA <1 mg/d; medium/high = MPA ≥ 10 mg/d, NETA ≥ 1 mg/d, MEGA < 40 mg/d; maximum = MEGA≥40 mg/d;
f Progestin type received ≥80% days progestin dispensed; mixed type if several types of progestin dispensed and predominant progestin dispensed <80% days or combined oral contraceptive dispensed.
CH persisted/progressed at follow-up in 21 of the 73 women with an index diagnosis of CH treated with progestin: 2 with adenocarcinoma, 8 with AH, and 11 with CH. Among the 41 women with an index diagnosis of AH treated with progestin, AH persisted/progressed in 12 women: 5 with adenocarcinoma and 7 with AH.
Among women with CH at index, no associations were found between PRA, PRB, PTEN, Bcl-2, and Pax-2 protein expression and persistence/progression. Among women with AH at index, high expression of PRB was associated with a 90% decreased risk of persistence/progression (95% CI, 0.01–0.8). Additionally, there was a suggestion of a decreased risk of persistence/progression with high expression of PRA and PRB compared to low expression of both receptors (OR, 0.1; 95% CI, 0.02–1.0). No associations were found between PTEN, Pax-2, and Bcl-2 protein expression measured at index biopsy and AH persistence/progression ( Table 3 ). When the analyses were repeated adjusting for age and BMI, the magnitude of the associations were similar. Additionally, when we repeated the analyses among women with at least 8 weeks of progestin treatment, we observed the same pattern of associations as in the main analyses (data not shown).
| Complex (n = 73) | Atypia (n = 41) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Biomarkers | Cases (n = 21) n (%) | Controls (n = 52) n (%) | OR | 95% CI | P value | Cases (n = 12) n (%) | Controls (n = 29) n (%) | OR | 95% CI | P value |
| PRA expression | ||||||||||
| Low | 5 (28) | 19 (43) | 1.0 | Reference | 6 (55) | 7 (30) | 1.0 | Reference | ||
| High | 13 (72) | 25 (57) | 2.0 | 0.5–8.3 | .390 | 5 (45) | 16 (70) | 0.4 | 0.06–2.0 | .262 |
| PRB expression | ||||||||||
| Low | 6 (32) | 15 (34) | 1.0 | Reference | 8 (73) | 6 (25) | 1.0 | Reference | ||
| High | 13 (68) | 29 (66) | 1.1 | 0.3–4.3 | 1.000 | 3 (27) | 18 (75) | 0.1 | 0.01–0.8 | .011 |
| PRA and PRB expression a | ||||||||||
| PRA low, PRB low | 5 (28) | 13 (30) | 1.0 | Reference | 6 (55) | 4 (17) | 1.0 | Reference | ||
| PRA low/PRB high, PRA high/PRB low | 1 (6) | 8 (18) | 0.3 | 0.03–3.6 | 2 (18) | 4 (17) | 0.3 | 0.03–3.2 | ||
| PRA high, PRB high | 12 (67) | 23 (52) | 1.4 | 0.4–4.8 | .464 | 3 (27) | 15 (65) | 0.1 | 0.02–1.0 | .059 |
| PTEN | ||||||||||
| No loss | 6 (30) | 27 (52) | 1.0 | Reference | 4 (33) | 4 (14) | 1.0 | Reference | ||
| Loss | 14 (70) | 25 (48) | 2.5 | 0.8–9.2 | .118 | 8 (67) | 24 (86) | 0.3 | 0.05–2.3 | .211 |
| Pax-2 | ||||||||||
| No loss | 3 (14) | 3 (6) | 1.0 | Reference | 0 (0) | 2 (7) | 1.0 | Reference | ||
| Loss | 18 (86) | 48 (94) | 0.4 | 0.05–3.1 | .348 | 12 (100) | 26 (93) | NE | NE | 1.000 |
| Bcl-2 | ||||||||||
| No loss | 13 (62) | 23 (46) | 1.0 | Reference | 2 (18) | 8 (28) | 1.0 | Reference | ||
| Loss | 8 (38) | 27 (54) | 0.5 | 0.2–1.7 | .300 | 9 (82) | 21 (72) | 1.7 | 0.3–19.5 | .696 |
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