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Relationship of Estrogen and Progesterone Receptors to Clinical

Relationship of Estrogen and Progesterone Receptors to Clinical

Gynecologic Oncology 106 (2007) 325–333 www.elsevier.com/locate/ygyno

Relationship of and receptors to clinical outcome in metastatic endometrial carcinoma: A Gynecologic Oncology Group Study ⁎ Meenakshi Singh a, Richard J. Zaino b, Virginia J. Filiaci c, Kimberly K. Leslie d,

a Department of Pathology, The University of Colorado Health Sciences Center, Denver, CO 80262, United States b Department of Pathology, Hershey Medical Center, Hershey, PA 17033, United States c Gynecologic Oncology Group Statistical and Data Center, Roswell Park Cancer Institute, Buffalo, NY 14263, United States d Departments of Obstetrics and Gynecology and Biochemistry and Molecular Biology, the University of New Mexico, Albuquerque, NM 87131, United States Received 18 January 2007 Available online 25 May 2007

Abstract

Introduction. The goal of this study was to explore the relationship between the expression of hormone receptors in metastatic endometrial tumors and clinical response to daily tamoxifen citrate and intermittent weekly acetate. Study design. Patients with measurable recurrent or advanced were enrolled on a clinical trial, Gynecologic Oncology Group Study 119. A pretreatment tumor biopsy was obtained and subjected to immunohistochemical analyses. Estrogen -α (ER-α) and (PR) were assessed on frozen tissues, and PR isoforms A and B were detected on fixed tissues. The receptors were scored using a semi-quantitative HSCORE, with a cut off greater than 75 considered positive. Results. Of the 60 eligible patients, 45 had evaluable tissues for all receptors. For ER, 40% of the cases were positive; for PR, 45% were positive. The sub-cellular distribution of PRA was exclusively nuclear, and 16% of the tumors demonstrated positive staining. PRB was nuclear and cytoplasmic, with 22% of the tumors staining for nuclear PRB and 36% of the tumors staining for cytoplasmic PRB. ER and PR from frozen tissues and PRA and cytoplasmic PRB from fixed tissues significantly decreased with increasing tumor grade. The co-expression of ER-α with PR from the frozen tissues (r = 0.68, p b 0.001) and PRA (r = 0.58, p b 0.001) from the fixed tissues was statistically significant. The ER HSCORE was related to both response and overall survival; there was no statistically significant correlation of PR with clinical response in this small number of patients. Conclusion. ER-α measured in metastatic endometrial carcinoma tissue prior to hormonal therapy was statistically significantly related to clinical response to daily tamoxifen and intermittent medroxyprogesterone acetate. © 2007 Elsevier Inc. All rights reserved.

Keywords: Uterus; Estrogen; Medroxyprogesterone acetate; Receptors; Tamoxifen; Endometrial; Cancer

Introduction terone mediates both proliferative and anti-proliferative effects [2,3]. Therefore, the study of progestin action in the endome- The role of progesterone in the glandular epithelium of the trium has particular importance because the epithelium relies on endometrium is primarily antagonistic to estrogen-mediated cell progesterone to induce cell differentiation and to counter uncon- proliferation [1]; this is in contrast to the breast, where proges- trolled growth. While progestins have been used with great success to reverse endometrial hyperplasia [4,5] they are not consistently effective in the treatment of primary endometrial cancer. Progestins as single agents have been used traditionally ⁎ Corresponding author. University of New Mexico, The Division of Maternal-Fetal Medicine, The Department of OB/GYN2211 Lomas Blvd., NE in the treatment of recurrent or metastatic endometrial adeno- ACC-4, Albuquerque, NM 87131, United States. carcinoma. However, the overall response rates range from only E-mail address: [email protected] (K.K. Leslie). 8% to 55% [6–11]. Progestin exposure results in a rapid and

0090-8258/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2007.03.042 326 M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333 predictable ligand-dependent loss of progesterone receptors Materials and methods (PR) in both normal and neoplastic endometrial glands [12]. Two recent publications of Gynecologic Oncology Group Patient population (GOG) studies, 119 and 153, reported attempts to circumvent Between 1991 and 1996, the GOG conducted a prospective phase II trial of PR down-regulation with a strategy combining tamoxifen, as an tamoxifen combined with intermittent MPA. This trial included patients with estrogen surrogate to induce PR, and intermittent progestin histologically-confirmed advanced, persistent or recurrent endometrial carcino- treatment [13,14]. However, neither of these papers provided the ma considered incurable by local therapy or refractory to local therapy. In expression data needed to correlate addition, all patients were to have disease that was measurable in 2 dimensions by clinical response with these variables. This report now provides palpation or imaging. Any lesion imaged by CT scan or ultrasound was to have a minimum diameter of 3 cm. The measurable tumor to be followed from entry information on the link between hormone receptor status and must not have received radiation therapy within 3 months prior to entry. Prior clinical response in one of these trials, GOG 119. therapy with cytotoxic drugs or hormonal therapy for endometrial cancer was not Two isoforms of PR, PRA and PRB, are expressed in humans: allowed. Normal hepatic, renal and hematologic functions, the absence of PRA encodes a 90 kDa protein, and PRB encodes a 120 kDa significant infection and GOG performance status 0–2 were required. Patients protein. Both forms arise from alternative promoters on the same with past or concomitant malignancy, other than non-melanoma skin cancer, were ineligible. Awritten informed consent conforming to federal, state and local and can form homo (A/A, B/B) or hetero (A/B) dimeric regulations was obtained from all participants prior to study entry. units. The isoforms are identical except that PRB has a longer N- terminus consisting of 164 amino acids not present in PRA. The Tissue requirements unique PRB N-terminus encodes a third activating domain, AF-3, that confers different functional characteristics to the isoforms. Pre-treatment tumor tissue from the metastatic or recurrent lesion was required for all patients for analysis of estrogen and progesterone receptors. A portion of the PRB is a stronger transcriptional activator of many com- fresh tumor, greater than 3×5×5 mm, was to be immediately placed in OCTor other pared to PRA [15,16]; PRA also has been reported to repress PRB embedding media into a foil cup or on cork or cardboard, rapidly frozen and [17]. A number of studies suggest that a relative change in PR maintained at a temperature of no more than −20 °C. The labeled vial of frozen tissue isoform expression may be as important as the total level of PR in was to be shipped on dry ice by overnight carrier to the initial testing laboratory. the genesis of endometrial cancer [15,16,18]. ER and PR immunohistochemistry on frozen tissue Tamoxifen is a complex compound, structur- ally similar to , which binds to the estrogen First, ER and PR were assessed directly on the frozen specimens. Frozen receptor (ER). The ability of tamoxifen-bound ER to activate sections were cut at 5 μM in a cryostat, and thaw mounted onto poly-L-lysine gene appears to be gene and tissue-specific and coated glass slides. The slides were placed in Zamboni's fixative at 4 ° C for 15 min determines whether the drug acts as an estrogen agonist then rinsed twice in PBS for 5 min. Immunohistochemistry was performed manually for both of the antibodies. (tamoxifen-bound ER is capable of activating gene transcrip- The primary antibody to ER was the D222 clone, which recognizes ER-α, with tion) or as an antagonist (competes for ER binding with other little or no affinity for ER-β. For ER, the reagents provided by the manufacturer of , but forms complexes that are incapable of inducing the ER-ICA kit (Abbott, Chicago, IL) were used as recommended for the blocking transcription). The estrogen-like effects of tamoxifen on steroid step and the control or the ER primary antibody, with avidin and biotin used for hormone receptor expression in the endometrium were amplification of the reaction, using a rat peroxidase kit (Vector, Burlingame, CA). For PR, the primary antibody used on the frozen tissue was mPRI. Mouse IgG exploited in this phase II clinical trial of tamoxifen plus was used in place of the primary antibody as a negative control. Endogenous intermittent weeks of MPA, GOG 119, where tamoxifen was peroxidase was blocked with 1% hydrogen peroxide. and biotin was used for used in theory as an estrogen surrogate to induce PR and amplification of the reaction using the Mouse IgG Elite Vectastain kit (Vector, maintain its up-regulation [13]. Patients were treated with Burlingame, CA). tamoxifen citrate, 20 mg, p.o. twice daily. On alternating (even- For immunolocalization of both ER and PR, 3,3-diaminbenizidine was used as the chromogen, and the slides were coverslipped with permount mounting numbered) weeks, they also received medroxyprogesterone solution. Non-gravid rabbit uterus was used as the positive control tissue for acetate (MPA), 100 mg, p.o., twice daily. The clinical results of both ER and PR, since there was a known, predictable variation in the intensity this study have been reported previously [13]. of staining in the glands, stroma and myometrium. Positive staining of the The original aim of GOG 119 was to determine clinical control tissue was limited to an intranuclear location for both ER and PR. response to this hormonal regimen; in addition, a pretreatment biopsy was obtained specifically to determine whether hormone Table 1 receptors correlated with response. Therefore, in the present Number and proportion of patients with positive or negative expression translational study, tissues from GOG 119 were analyzed to Receptor HSCORE 75 HSCORE greater Total determine, first, whether the expression of receptors on a pre- or less: negative than 75: positive treatment biopsy predicts for clinical outcome, and second, what ER 29 (60%) 19 (40%) 48 the relationships are between ER expression and the PR isoforms PR 27 (55%) 22 (45%) 49 A and B in advanced endometrial cancer. Interestingly, the PRA 42 (84%) 8 (16%) 50 relationship between hormone receptor expression and clinical PRB-nuclear 39 (78%) 11 (22%) 50 PRB-cytoplasm 32 (64%) 18 (36%) 50 response has yet to be demonstrated prospectively in a GOG trial; thus our hypothesis was that ER and/or PR expression would be ER and PR were measured from frozen tissue. The PR isoforms A and B were measured from formalin fixed tissues. Receptor data were assessed semi- required to achieve a clinical response. Also, as ER is generally quantitatively using the HSCORE. A cut-off of 75 was used to dichotomize the required to induce PR, we tested the hypothesis that tumors with HSCORE data into two groups: an HSCORE that was 75 or less was considered ER would also express PR. negative and an HSCORE over 75 was considered positive. M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333 327

with 3% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase. Slides were rinsed in Tris buffer pH 7.6 for 10 min. The instrument (Ventana NexES) was programmed to incubate the tissue slides with primary monoclonal antibodies against PRA+B (to common regions of PRA and PRB, but preferentially identifying PRA) manufactured by Ventana (clone 1A6). This will be referred to as PRA in this report. A PRB-specific antibody, Ab-6 (NeoMarkers/Lab Vision), has also been tested with success on the Ventana NexES. The primary antibody was incubated for 32 min at room temperature, and the slides were washed. Secondary antibody, streptavidin, and chromogen incubations were programmed into the instrument following the manufacturer's instructions. Slides were rinsed and counterstained with hematoxylin prior to evaluation.

Cells and controls

To test the specificity of the PRA and PRB antibodies, cells were plated on chamber slides. The cell lines employed were well-differentiated Ishikawa endo- metrial cancer cells (that express PRA and PRB) and poorly differentiated Hec50co cells (that do not express or PR). Hec50co cells were infected with adenovirus vectors encoding PRA or PRB to obtain high PR isoform expression. Antibody specificity for PRA and PRB was tested using IHC and co-immunoprecipitation, as previously reported [19].

Semi-quantitation of IHC results

The results of immunostaining for all receptors were semi-quantified using the HSCORE, calculated based on estimates of percentages of positive stained epithelial cells in each of 5 intensity categories (0, 1+, 2+, 3+, and 4+). The HSCORE represents the sum of each of the percentages multiplied by the weighted intensity of staining, as follows: HSCORE = (i+1)π,wherei =1,2,3,4,andπ varies from 0 to 100%. A threshold of N75 as a positive result has previously been determined by McCarty et al. to provide the maximum sensitivity and specificity when compared with the biochemical assay for ER [20]. In addition to the nuclear staining clearly visible, cytoplasmic staining was also scored because of the recent report that some PR (particularly PRB) is located in the cytoplasm in the absence of ligand [19]. The specificity of PRB cytoplasmic staining was ascertained by the use of cell models expressing PRB compared to PRA, where 50% of the PRB introduced into cells remained cytoplasmic in the absence of progesterone but translocated into the nucleus when progesterone was added.

Clinical response and overall survival

A partial response was defined as a 50% or greater reduction in the product of the largest diameter and its perpendicular of each measurable lesion on two separate examinations at least 2 weeks apart. Additionally, there must have been no deterioration in performance status and no new lesions. If all gross evidence of disease disappeared for at least 1 month, this was classified as a complete response. All other patients were classified as non-responders. Overall survival is defined as the length of life measured from the date of entry on the study. Patients who were last known to be alive were censored at the date of last contact.

Statistical analysis

HSCORE values for ER, PR, PRA and nuclear and cytoplasm PRB were Fig. 1. (A–C) Immunostaining for hormone receptors showing subcellular assessed both as continuous covariates and as dichotomous covariates using a distributions. ER (panel A) and PRA (panel B) are nuclear, while PRB (panel C) cutoff of 75 to categorize as negative versus positive receptor status. The Spearman are cytoplasmic and nuclear. correlation statistic was employed to measure the association of HSCORE data

Isoform Immunohistochemistry on fixed and paraffin embedded tissue Table 2 When discriminatory antibodies for PRA and PRB became available and Receptor HSCORE Spearman correlation statistics protocols derived for the analysis of formalin fixed tissues [19], the investigators ER PR PRA PRB-nuclear PRB-cytoplasm took portions of the original frozen tumor blocks and processed them for these ER 1.00 additional studies. The frozen tissues were thawed slowly to room temperature, PR 0.68 ⁎ 1.00 fixed in 4% neutral buffered formalin, processed through graded alcohols to PRA 0.58 ⁎ 0.63 ⁎ 1.00 acetone, and embedded in paraffin. Tissue slides were deparaffinized through 3 PRB-nuclear 0.09 −0.08 −0.15 1.00 changes of xylene to alcohol to water. Antigen retrieval was performed by micro- PRB-cytoplasm 0.12 0.14 0.29 ⁎⁎ 0.24 1.00 waving slides in 1 mM EDTA in a pressure cooker (Nordicware, MN) for 30 min. Following a cooling period at room temperature for 20 min, tissue was incubated ⁎ pb0.0001; ⁎⁎ p=0.04. 328 M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333

Table 3A Table 4 Jonckheere–Terpstra test results of receptor and tumor grade Frequency of response within dichotomized receptor HSCORE category Receptor p-value a J–T test Standardized test Responders Non-responders Total number treated statistic statistic b with Receptor data ER (n=48) 0.051 292.5 −1.63 ER HSCORE≤75 7 (26%) 20 (74%) 27 PR (n=49) 0.024 286.0 −1.99 ER HSCOREN75 9 (47%) 10 (53%) 19 PRA (n=50) 0.027 310.0 −1.96 PR HSCORE≤75 8 (32%) 17 (68%) 25 PRB-nuclear (n=50) 0.391 394.0 −0.29 PR HSCOREN75 9 (41%) 13 (59%) 22 PRB-cytoplasm (n=50) 0.045 323.5 −1.65 PRA HSCORE≤75 13 (32%) 27 (68%) 40 PRA HSCOREN75 4 (50%) 4 (50%) 8 a Monte Carlo estimate for exact J–T test one-sided p-value using 10,000 PRB-nuclear 13 (35%) 24 (65%) 37 samples. HSCORE≤75 b Provides information on the direction of the alternative hypothesis: a small PRB-nuclear 4 (36%) 7 (64%) 11 p-value for a negative value of the standardized test statistic supports the HSCOREN75 alternative of decreasing HSCORE with increasing grade. PRB-cytoplasm 10 (32%) 21 (68%) 31 HSCORE≤75 between pairs of receptors. The Jonckheere–Terpstra (JT) test [23] was used to test PRB-cytoplasm 7 (41%) 10 (59%) 17 N if HSCORE values varied with increasing tumor grade. A negative standardized JT HSCORE 75 test statistic indicates receptor HSCORES decreased with increasing tumor grade. This table summarizes the response data for each receptor with an HSCORE Monte Carlo estimates of the exact p-values are reported. The JT test is a non- greater that 75 considered positive. parametric test for ordered alternatives. When ordered alternatives are of interest, the JT test is preferred to a more general test such as the Kruskal–Wallis test that tests for class differences without any regard to ordering. Monte Carlo estimates cluded the primary tumor was not endometrial carcinoma. (using 10,000 samples) of the exact test p-values are reported since given the Tissue was never submitted for five patients and was inevalu- sample size, an exact test was preferred over an asymptotic test, but the exact test able for all analyses in two patients. Additionally, tissue from was computationally intensive [24]. The relationship between receptors, tumor four patients was inevaluable in the initial analysis of ER-α and grade and clinical response was analyzed using logistic regression. The effect of PR, and tissue from three patients was inevaluable for isoform each receptor HSCORE on the odds of response was estimated as an odds ratio α (OR) and reported with a 95% Wald confidence interval (CI). A receiver operator analysis. One additional patient was inevaluable for ER- .Two curve (ROC) was constructed to assess the utility of the ER HSCORE as a test to patients were never treated and, thus, not evaluated for re- predict clinical outcome. The value of c was used to estimate the area under the sponse. Forty-five patients had enough available tissues to ROC [25].TheKaplan–Meier method was used to estimate the probability of perform each of the immunoassays, and of these, 43 had survival within ER positive and ER negative subgroups. Cox proportional hazards clinical response data for correlation. models were used to estimate the relative effect of receptor HSCOREs and tumor grade on the hazard of death. Hazard ratio (HR) estimates are reported with 95% Wald confidence intervals. Martingale residuals calculated from a model without Percent of cells staining for receptors the ER HSCORE covariate were plotted against ER HSCORE. Residuals greater than zero suggest worse than expected survival and residuals less than zero suggest Table 1 summarizes the receptor status of the pre-treatment better than expected survival. A smoothing spline function was fit to evaluate the tumor tissues. Using an HSCORE of greater than 75, 40% of the functional form of the dependence of the residuals on ER HSCORE [26].All α statistical tests were two-sided, unless otherwise stated, with a p-value less than frozen tumors demonstrated positive staining for ER- ,while 0.05 considered as statistically significant. These analyses are exploratory; no 45% had positive staining for PR using the antibody mPRI. adjustment was made to any p-value to account for multiple comparisons. Receptor isoforms were evaluated on formalin fixed tissues: PRA was found to be exclusively nuclear in distribution, while PRB Results was both nuclear and cytoplasmic. Some tumors demonstrated nuclear and cytoplasmic PRB, while other tumors demonstrated Patient evaluability only cytoplasmic PRB. Sixteen percent of the tumors were positive for PRA, 22% for nuclear PRB, and 36% for cytoplasmic Sixty-one patients were entered onto the study. One patient was ineligible following central pathology review which con- Table 5 Associations between receptor HSCORE and response or overall survival Table 3B Frequency of tumor grade category Response Overall survival Grade 1 Grade 2 Grade 3 or not specified Receptor OR a 95% CI C b HR c 95% CI ER HSCORE ≤75 7 8 14 ER 1.007 1.001 to 1.014 0.708 0.996 0.993 to 0.999 ER HSCORE N75 5 7 7 PR 1.002 0.997 to 1.008 0.573 0.997 0.994 to 1.000 PR HSCORE ≤75 6 7 14 PRA 1.003 0.997 to 1.009 0.630 0.998 0.994 to 1.002 PR HSCORE N75 6 9 7 PRB-nuclear 0.999 0.993 to 1.005 0.463 0.999 0.996 to 1.002 PRA HSCORE ≤75 9 13 20 PRB-cytoplasm 1.001 0.995 to 1.006 0.507 0.999 0.996 to 1.001 PRA HSCORE N75 3 4 1 a Change in relative odds of response per unit increase in receptor HSCORE. PRB-nuclear HSCORE ≤75 8 14 17 b Estimates the area under the ROC curve; values closer to 1 would indicate PRB-nuclear HSCORE N75 4 3 4 better classification of responders; values closer to 0.5 indicate a useless PRB-cytoplasm HSCORE ≤75 4 14 14 discriminator of responders versus non-responders. PRB-cytoplasm HSCORE N75 8 3 7 c Change in relative hazard of death per unit increase in receptor HSCORE. M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333 329

Table 6 Association between HSCORE and response or overall survival adjusted for tumor grade Response Overall survival Factor OR 95% CI C HR 95% CI ER (continuous) 1.008 1.001 to 1.015 0.692 0.996 0.993 to 1.000 ER (dichotomous) 2.589 0.737 to 9.093 0.626 0.474 0.244 to 0.919 This table provides a summary of the tumor grade-adjusted associations between ER HSCORE and response and overall survival. Adjustment was accomplished by including two indicator variables into the models: one for grade 1 and one for grade 2 tumors.

PRB using antibodies 1A6 for PRA and Ab-6 for PRB. Figs. 1A and B illustrate the typical nuclear staining for ER-α and PRA, while 1C demonstrates PRB in the nucleus and the cytoplasm. Table 2 displays the Spearman correlation statistic for each pair of receptors. The expression of ER from frozen tissues, PR from frozen tissues, and PRA from formalin fixed tissues are mode- rately correlated, with statistically significant (p b 0.0001) values between 0.58 and 0.68; there is a weak correlation between PRA Fig. 3. Martingale residuals for ER HSCORE. and cytoplasmic PRB on fixed tissues (r =0.29,p =0.04). tive tumors. For frozen tissues with positive PR (HSCORE N 75) Relationship between tumor grade and receptor expression 0, 41% responded. From fixed tissues, 50% of patients with PRA responded. Response among patients with nuclear PRB Tables 3A and 3B illustrate that as tumor grade increased, (HSCORE N 75) was 36%; whereas, among patients with cyto- expression of ER, PR, PRA and cytoplasmic PRB decreased, plasm PR, 41% responded. indicating an inverse relationship between receptor expression Except for ER, no correlation between receptor expression and and grade. clinical response was found. The positive correlations between ER HSCORE and response and between ER HSCORE and Clinical response rates and association with receptors survival are summarized in Table 5. Adjusted for tumor grade, there is a significant effect of ER HSCORE on the log odds of Overall, the clinical study observed a 10% complete response response when analyzed as a continuous covariate (OR: 1.008, rate and a 22% (13/58) partial response rate for a total of 33% 95% CI 1.001 to 1.015) (Table 6). A change in ER HSCORE of (19/58). These results have been previously reported and dis- 100 translates into a predicted 222% increase in the odds of cussed [13]. Table 4 provides the frequency of response cate- response. As the HSCORE varies from 0 to 500, an increase in the gorized by receptor HSCORE. For ER-positive tumors, the score of 100 would roughly translate into a 20% up-regulation of clinical response rate was 47% compared to 26% for ER-nega- receptor staining. Fig. 2 is a receiver operator curve for the ER

Fig. 4. Kaplan–Meier Survival plot grouped by ER status. An HSCORE cut off Fig. 2. ER receiver operator curve (ROC) for classifying response. of 75 was used to classify tissues as ER-positive or ER-negative. 330 M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333

HSCORE as it relates to response. The estimated area under the The development of monoclonal antibodies to ER and PR and curve is 0.708. Fig. 3 exhibits the relationship between ER their isoforms has permitted immunohistologic localization of HSCORE and the hazard of death using martingale residuals. A hormone receptors in endometrial cancers and demonstrated their Kaplan–Meier plot of OS categorizing ER HSCORE as above heterogeneous distribution within primary tumors and when pri- or below 75 is shown in Fig. 4. The estimated median survival mary tumors are compared with their metastases [38]. While the among the patients with ER HSCORES equal to or below 75 is presence of antigenicity does not guarantee the functionality of 8 months compared with 19 months for patients with the receptor or its downstream pathways, a number of studies HSCORES above 75. The estimated reduction in the death have shown that ER, PR, or both predict for an improved clinical hazard ratio associated with ER HSCORES greater than 75 outcome compared to receptor negative tumors [39–43]. A recent relative to HSCORES 75 or less adjusted for tumor grade is evaluation of ER and PR in 164 endometrial carcinomas found a 53% (HR: 0.47, 95% CI 0.24 to 0.92). When evaluating the trend towards improved survival for patients with ER and PR effect of ER HSCORE as a continuous covariate on the positive tumors, but the level did not reach statistical significance hazard of death, there is a predicted 33% [calculated as follows: [44]. Another report failed to find a relationship between the ER (1−(0.996100))×100%] reduction in the death hazard associat- and PR status of tumors and recurrence in 62 high risk endo- ed with an increase of 100 in ER HSCORE (HR 0.996, 95% CI metrial cancers [45]. These studies have limitations in that the 0.99 to 1.00). treatments were not uniform; therefore, the strength of the cor- relation of receptor status with clinical outcome was confounded Discussion by different therapies. Nevertheless, despite the early enthusiasm for directing care based upon the presence of ER and/or PR, the Single agent progestins have been used in the treatment of receptor status of endometrial tumors is not routinely evaluated in recurrent and metastatic endometrial carcinoma. Upon treat- clinical practice in the United States. ment with a progestin, there is activation of PR as a transcription In the present study, ER and PR were assessed initially in factor, and the induction of genes associated with endometrial frozen tissue using a method available in the early years of the differentiation follows [21–23]. However, the ligand-dependent study. Later, it became possible to discern two isoforms of PR, A activation of PR is closely linked to the ligand-dependent down- and B, by immunostaining on formalin-fixed tissues [19,46]. regulation of PR, a process that occurs upon PR phosphoryla- Both methods are reported in this study and correlated with tion that is both a prerequisite for transcriptional activation and clinical response. Also, cytoplasmic as well as nuclear PRA and a preliminary step for PR ubiquitination and destruction in the B staining was evaluated because of the recent report that PRB is proteasome [12,24,25]. Therefore, long-term continuous expo- often cytoplasmic as well as nuclear, particularly in the absence sure to progestins sets the stage for loss of PR effects. of ligand [19]. Previous immunohistochemical studies often The strategy chosen in this study, GOG 119, was to treat ignored cytoplasmic staining because the potential non-genomic continuously with tamoxifen, a partial estrogen agonist, which in functions of such receptors and their ability to rapidly translocate a model system stimulated PR as potently as but with to the nucleus with the addition of ligand were not understood. less growth stimulation. Since continuous treatment with pro- Despite the fact that this study used two immunohistochem- gestin alone down-regulates PR, it was hypothesized that inter- ical methodologies to detect receptors, concordance between PR mittent treatment with MPAwould permit tamoxifen to induce PR (frozen tissues, assessed by the antibody mPRI) and PRA (for- and enhance the effect of progestin therapy. This phase II clinical malin-fixed tissues, assessed by the antibody 1A6) was reas- trial demonstrated the tolerability and activity of combining the suringly high and statistically significant. However, PR from estrogen-like drug tamoxifen with intermittent progestin. Patients frozen tissues did not correlate well with PRB, identified using were treated with tamoxifen citrate, 20 mg, p.o. twice daily. On the antibody Ab-6 with an epitope in the unique N-terminus of B. alternating (even-numbered) weeks, they also received MPA, This indicates that, like many of the currently available PR 100 mg, p.o., twice daily [13]. GOG 119 also allows the eva- antibodies raised against shared regions of PRA and PRB, mPRI luation of pretreatment tumor receptor expression and permits an does not have a high affinity for PRB. Therefore, to reliably analysis of whether ER or PR expression correlates with each identify this isoform, an antibody to the unique N-terminus of other, with tumor grade, and with clinical outcome. These results PRB must be employed, as previously reported [19,47]. are reported in this study. The single best predictor of response in the present study was The presence of hormone receptors in endometrial cancer ER, which was significantly associated with clinical response and the link to clinical response has been studied extensively. and survival. Also, ER expression was highly correlated with the Initial studies utilized cytosolic ligand binding assays in which expression of PRA, but not PRB. Nevertheless, 26% of the protein extracts from ground tissue, including contaminating tumors without ER and 32% of the tumors without PRA or PRB, surrounding stroma, were analyzed. These early techniques had as determined on a pre-treatment biopsy, responded to therapy. limitations, as previously reviewed [26,27]. Despite the draw- The HSCORE cutoff of 75 recommended by McCarty et al. backs of the early technology, significant correlations between [20] to categorize those with positive versus negative receptor receptors and clinical outcome, survival, and response to expression appears reasonable within this study. The fitted spline progestins were documented for patients with early and advanced function in Fig. 3 crosses from positive to negative residuals very endometrial cancer in many studies [28–36]; however, others close to 75. However, the relationship appears linear in HSCORE. failed to show that receptors predicted for improved survival [37]. Additionally, the difference in survival between ER receptor M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333 331 expression groups, after adjusting for tumor grade, is apparent. trial cancer. In this study, ER expression was significantly and Furthermore, the maximum difference between sensitivity and 1- positively associated with PR and PRA expression, as was PRA to specificity along the ROC for predicting response is associated cytoplasmic PRB. No relationship was appreciated between ER with ER HSCORE equal to 40 (sensitivity = 0.75, specifici- and PRB expression, indicating an uncoupling of the expected co- ty = 0.63). The difference at ER HSCORE equal to 70 is similar expression of ER with both PR isoforms in these advanced (sensitivity = 0.69, specificity = 0.67). tumors. In addition, receptor expression was down-regulated as An interesting question to consider is why some tumors tumor grade increased, confirming our previous observations and without receptors on the pre-treatment biopsy responded. There those of others [15,16,57]. are two possibilities: first, tamoxifen induced the steroid receptors The principal strength of the study is the availability of pre- as expected based upon the study design, or second, tamoxifen treatment tissue receptor data linked to clinical response to a and/or MPA worked through non-receptor mediated pathways. standardized hormonal regimen; however, there were limitations The core rationale for this clinical trial was that tamoxifen, as an to the study, including its relatively small size and the lack of estrogen surrogate, would induce ER and PR [48,49] Tamoxifen post-treatment biopsies to follow the effects of therapy on is well known to act as an estrogen agonist in endometrial cancers receptors. Nevertheless, this study demonstrated that ER [50]. Therefore, as shown in previous work in the rat uterus [51],it receptor status, as reported by the semi-quantitative HSCORE, is likely that ER and PR expression was induced in some tumors was related to clinical response and survival in patients treated initially found to be devoid of receptors on the pre-treatment with tamoxifen and intermittent MPA, but PR expression did not biopsy. This could explain the findings that 26% of ER negative significantly correlate with clinical outcome. However, it is tumors and 32% of PR negative tumors (as determined prior to possible that the small number of tumors in this study that treatment) responded to therapy. While this trial required a pre- expressed PRA, only 16%, may have prevented us from detec- treatment biopsy for entrance, no post-treatment biopsy was ting PRA as a prognostic marker even though it was statistically obtained — such a biopsy would have been for research purposes linked to ER expression. only and is difficult to justify to human subject review boards. Importantly, the median survival of patients with an ER Therefore, we do not know whether tamoxifen treatment did HSCORE above 75 on this hormonal regimen was 19 months, indeed up-regulate PR and/or ER. The alternative explanation is which is comparable to the 15- to 18-month median survival time that tamoxifen or MPA had receptor independent anti-tumor reported with doxorubicin, cisplatin and paclitaxel with granulo- effects. Particularly in the case of tamoxifen, such effects are cyte colony-stimulating factor support and paclitaxel and cisplatin proposed to occur through inhibition of protein kinase C, which is with granulocyte colony-stimulating factor support [58].Fromthe the rationale for the use of high dose tamoxifen in the treatment of GOG 119 cohort, 40% of the patients expressed ER at levels malignant glioma [52]. above an HSCORE of 75, indicating that many patients could A second question surrounds the significance of the cyto- potentially benefit from hormonal therapy. These data demon- plasmic PRB found in many tumors. The presence of PR in the strate the potential value of routine characterization of endome- cytoplasm indicates a potential non-genomic function for this trial tumors with respect to receptor expression. Therefore, the receptor. PR is known to interact with signal transduction path- authors recommend ER and PR testing for all endometrial tumors ways that initiate or enhance responses in conjunction with because the results may impact the choice of therapy. For tumors membrane-bound growth factor receptors and G-protein recep- with high ER expression, it is possible that hormonal therapy may tors. For example, PR associates with p42 MAPK and is involved achieve response rates equal to that of chemotherapy with fewer in phosphatidylinositol 3-kinase signaling in Xenopus oocytes expected side effects, thereby improving quality of life. Further [53]. In addition, PRs contain a proline-rich motif within the investigation is warranted to determine whether this result is shared A domain of PRA and PRB which interacts directly with specific to the use of tamoxifen combined with intermittent MPA, SH3 domains of c-Src and its family member Hck [54]. Both or is applicable to hormonal therapy in general. In addition, it will PRA and PRB demonstrate this interaction in vitro, but since be important to confirm these findings in larger studies. PRA is largely restricted to the nucleus in endometrial cancer cells, it is predicted that cytoplasmic signaling events depend Acknowledgments upon PRB. It has been suggested that a dynamic situation exists whereby receptors diffuse into the cytoplasm and are constantly Further support was provided by the Dean and Alice Irvin and actively transported back to the nucleus [55].The Family Foundation and Mrs. Shirley Leslie, who made contribu- mechanism(s) underlying PR cytoplasmic to nuclear shuttling tions to the research activities of the GOG Core Laboratory for are currently under investigation and hopefully will shed new Receptors. Administrative support was provided by Ms. Anne light on the functions of the PRA compared to the PRB isoform. Reardon, the Gynecologic Oncology Group, and Ms. Loretta Given the difference in localization of PRA compared to PRB Campbell, the University of New Mexico. noted on clinical specimens in this study, these investigations are This study was supported by National Cancer Institute grants particularly relevant. to the Gynecologic Oncology Group Administrative Office (CA In conclusion, recent calls for the incorporation of translational 27469) and the Gynecologic Oncology Group Statistical and studies in clinical trials have been heard [56]; this study inves- Data Center (CA 37517). This work was also supported by NIH tigated the relationship between ER and PR expression, tumor R01CA 99908-1 (KL), by NIH CA 27469 to the Gynecologic grade, and clinical response in patients with advanced endome- Core Laboratory for Receptors, by the Cory/Beach Family Fund 332 M. Singh et al. / Gynecologic Oncology 106 (2007) 325–333

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