The relationship of steroid receptor expression to nuclear DNA distribution and clinicopathological characteristics in epithelial ovarian tumors

GYNECOLOGICONCOLOGY32, 184-190 (1989)

The Relationship of Steroid Receptor Expression to Nuclear DNA Distribution and Clinicopathological Characteristics in Epithelial Ovarian Tumors M. L. FRIEDLANDER,*'1 M. A. QUINN,'~D. FORTUNE,'~M. S. Foo,$ M. TOPPILA,§ C. N. HUDSON,§ AND P. RUSSELL~ *Ludwig Institute for Cancer Research (Sydney Branch), Blackburn Building, University of Sydney, N.S.W. 2006, Australia; ?Royal Womens Hospital, Melbourne, Australia; CRoyal Prince Alfred Hospital, Missenden Road, Camperdown, N.S.W. 2050, Australia; and §Westmead Hospital, Westmead, N.S.W. 2145, Australia Received May 27, 1987 Tumor specimens from 92 patients with ovarian carcinoma were analyzed for estrogen receptor (ER), progesterone receptor (PR), proliferative fraction, and ploidy. Seventy-one percent of tumors were either E R + (>5 fmole/mg protein) or P R + (>10 fmole/mg protein) with 27% of tumors overall being both ER + and PR + . There was no significant relationship between receptor expression and stage, grade, or histological subtype. Thirteen percent of diploid tumors were receptor negative in contrast to 38% of aneuploid tumors (P < 0.01). There was no significant association between ER status and ploidy, but 60% of diploid tumors were P R + in contrast to 33% of aneuploid tumors (P < 0.02). Eleven percent of tumors overall were both ER rich and PR rich and comprised 23% of diploid and 5% of aneuploid tumors (P < 0.01). Receptor-negative tumors had a median S phase of 18.8% which was significantly higher than the median S phase of 12% in receptor-positive tumors (P < 0.02). A similar analysis was also performed on specimens from 9 patients with borderline epithelial ovarian tumors and 12 with benign epithelial ovarian tumors. Up to 50% of benign and borderline epithelial tumors had measurable receptors, but all were diploid with a relatively low S phase fraction. The functional significance of steroid receptor expression in ovarian cancer is unclear, but the association with ploidy and proliferative activity particularly in patients with malignant ovarian tumors may allow better identification of prognostic subsets and aid in selection of patients for

hormonal therapy.

© 1989 Academic Press, Inc.

INTRODUCTION It has been recognized for many years that a small proportion of patients with epithelial ovarian cancer benefit from hormonal therapy but there have been no reliable means available to identify such individuals [1,2]. Recently, it has become apparent that over 50% of ovarian tumors I

To whom correspondence should be addressed. 184

0090-8258/89 $1.50 Copyright © 1989 by AcademicPress, Inc. All rights of reproduction in any form reserved.

contain measurable steroid hormone receptors [2,3-9] and it has been postulated that a situation analogous to that in breast cancer may exist whereby steroid receptor positivity is associated with a high likelihood of response to hormonal therapy [10]. There is, however, little direct evidence in ovarian cancer to demonstrate that these receptors are functional, although the estrogen receptor (ER) has been shown to share antigenic similarities and have similar binding characteristics to the receptor present in breast tumors [11-12]. The aim of this prospective study was to determine the incidence of estrogen and progesterone receptor (PR) expression in a large series of patients with ovarian epithelial tumors and to analyze their relationship with other possible determinants of biological behavior inluding tumor ploidy and proliferative activity. There is some evidence to suggest that patients with diploid prostatic cancers have a higher likelihood of responding to hormonal therapy than those with aneuploid tumors and it is tempting to speculate that this may also be the case in other tumor types [13,14]. Of particular interest to us was to determine whether there was an association between tumor ploidy and steroid receptor expression as we and others have demonstrated that tumor ploidy is a major prognostic determinant in patients with ovarian cancer, patients with diploid tumors having a significantly longer survival than patients with aneuploid tumors [15-18]. MATERIALS AND METHODS Tumor samples for flow cytometric analysis and receptor determination were obtained from 92 patients with ovarian carcinomas, 9 patients with borderline epithelial ovarian tumors (tumors of low malignant potential), and 12 patients

STEROID RECEPTOR EXPRESSION IN OVARIAN TUMORS

with benign epithelial ovarian neoplasms. All patients were staged at laparotomy in accord with the FIGO recommendations. The WHO classification was used in the histopathological evaluation of tumors. Operative specimens were received fresh from theater and tumor tissue subdivided in the pathology department into adjacent sections for histological examination, flow cytometry, and receptor analysis. Analysis was performed either immediately or after tissue had been snap-frozen and stored in liquid nitrogen. Cytosol estrogen and progestone receptor analyses were performed as described previously [6,8]. The results of the receptor analyses from some of the patients in this study have been included in previous publications [6,8]. All tumors with ER values of greater than 5 fmole/mg cytosol protein and PR values of greater than 10 fmole/mg cytosol protein were considered to be receptor positive. ER-rich and PR-rich status was accorded to those tumors containing greater than 25 fmole/mg cytosol protein and 20 fmole/mg cytosol protein respectively which corresponded to the upper quartile of the distribution of receptor levels found in this study. These definitions are of necessity arbitrary and based on the sensitivity of the assays. Unlike the situation in breast cancer, there is insufficient clinical information available to establish what tissue level of receptor should be considered biologically significant in ovarian cancer. Flow cytometric analysis of DNA content and proliferative fraction was performed on a ICP22 flow cytometer using a rapid staining technique that has been described in detail previously [19,20]. Chicken red blood cells (CRBC) were used as an internal marker, as the ratio of the G~ DNA content of CRBC to the DNA content of human diploid cells is highly reproducible (2.9 _+ 0.17) under the staining conditions used [19]. In this way tumors could be classified as diploid or aneuploid (Fig. 1). The proportion of cells in S phase was estimated by computer analysis using a planimetric method devised by Milthorpe (1980) [201. RESULTS

Malignant Tumors Ninety-two invasive common epithelial ovarian carcinomas were analyzed by flow cytometry; 30 (33%) were diploid and 62 (63%) were aneuploid including 11 tumors which were multiploid. Multiploid tumors were analyzed together with single aneuploid tumors and classified simply as aneuploid in view of their relatively small numbers. Overlapping of subpopulations was the major reason for omitting tumor samples from analysis of S phase and while all diploid tumors could be analyzed, in only 50% of aneuploid tumors was S-phase analysis attempted.

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FIG. 1. Representative DNA histograms obtained from solid tumors analyzed by flow cytometry. Chicken red blood cells (CRBC) are used as an internal standard to identify the diploid Gt peak. Most tumors contain a population of cells with a diploid D N A content and when this is the only population present the tumor is classified as diploid. Tumors which have evidence of an additional G~ peak are classified as single aneuploid or multiploid.

Diploid tumors had a mean S phase of 10.4% _+ 4 which was significantly lower than the mean S phase of 16% _+ 4 in aneuploid tumors (P = 0.01). Seventy-one percent of the tumors fulfilled the criteria for either ER or PR positivity with 27% of tumors containing both estrogen and progesterone receptors. There was a wide range in levels, extending from 0 to a maximum of 245 fmole/mg cytosol protein for ER, and 0 to 1000 fmole/mg cytosol protein for PR (Fig. 2). The mean ER was 19.5 fmole/mg cytosol protein and median ER, 8 fmole/mg cytosol protein. The mean PR was 46 fmole/mg cytosol protein and median PR, 0.5 fmole/mg cytosol protein. Table 1 outlines the relationship of receptor status with

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stage, age, histological subtype, and histological grade. There was no significant relationship between receptor expression and tumor stage, or degree of histological differentiation. There was, however, an association between age and receptor expression in the ER-rich/PRrich group of tumors which were more common in women under 50 years old. The majority of serous tumors and endometrioid tumors, in contrast to the other histological subtypes, were receptor positive, but the overall numbers were too small in the latter group to draw any firm conclusions. The relationship between tumor ploidy and receptor expression is outlined in Table 2 and Fig. 3. There was no relationship between ER status and ploidy, but there was an apparent association between PR status and ploidy as 60% (18/30) of diploid tumors were PR+ in contrast to 33% (21/62) of aneuploid tumors (P < 0.02). In analyzing the relationship between ploidy and overall receptor expression (i.e., E R + or PR+), 13% of diploid tumors were receptor negative in contrast to 38% of aneuploid tumors (P < 0.02). There was no significant difference in the incidence of aneuploidy between the subgroups comprising ER + PR + , ER + P R - , or E R - PR + tumors. Eleven percent of tumors fulfilled criteria for ERrich and PR-rich status and comprised 23% of the diploid tumors and 5% of aneuploid tumors (P < 0.01). However, this relationship was not evident in tumors that were either ER rich or PR rich alone. Receptor negative tumors had a median S phase of 18.85% and a mean S-phase fraction of 15.8% (-+6.1%) which was significantly higher than the median and mean

TABLE 1

Relationship between Receptor S t a t u s a n d Clinical Stage, P a t i e n t Age, Histological S u b t y p e , a n d G r a d e in P a t i e n t s w i t h Invasive Epithelial Ovarian Cancer Receptor status Patient characteristic Stage Early Late Age <50 years >50 years Histological type Serous Endometrioid Mucinous Clear cell Undifferentiated Mixed Histological grade b Well differentiated Moderately differentiated Poorly differentiated

ER + PR +

ER + P R -

10 (40)" 15 (40)

5 21

2 12

8 19

6 (100) 19 (21)

11 15

8 6

12 15

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19 4 1 -1 1

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8 10 2 3 2 2

4 (75) 9 (11) 11 (45)

1 4 18

2 2 9

4 3 11

Figures in parentheses refer to percentage of patients with ER-rich and PR-rich tumors. b Degree of histological differentiation in tumors that were graded.

ER-

PR +

ER-

PR-

187

STEROID RECEPTOR EXPRESSION IN OVARIAN TUMORS TABLE 2 Relationship of Receptor Status with T u m o r Ploidy a n d Proliferative Fraction Tumor characteristics

ER + PR +

Ploidy Diploid Aneuploid Proliferative fraction (%) Median Means (4-SD)

ER + PR -

ER - PR +

ER - PR -

11 (7)a 14 (3)

8 18

7 7

4 23

12.5 (9.8) 11.9 -4- 4

12.15 11.4 4- 3.5

11.7 12.7 4- 5.6

18.85 15.8 4- 6.1

Figures in parentheses refer to patients with ER-rich and PR-rich tumors.

S phase in receptor positive tumors of 12 and 11.9% (+ 4.5%), respectively (P < 0.02). Analysis of the S phase within the various subgroups of receptor positivity revealed no other significant differences (Table 2).

Benign and Borderline Tumors Nine borderline (7 early stage and 2 late stage) and 12 benign ovarian tumors were analyzed with FCM; all were 100

90

diploid and had a similar mean S-phase fraction of 6.9% _+ 2.7 and 5.6% _+ 2.7, respectively. Twelve benign and 9 borderline ovarian tumors were assayed for ER and PR and the findings are outlined in Table 3. Fifty percent of benign tumors were PR positive and 25% ER positive with a total of 25% being both ER and PR positive. Twenty-two percent of borderline tumors were PR positive, 30% ER positive, and 11% both ER and PR positive. The numbers are however too small to allow meaningful assessment of the relationship between histological subtype and receptor expression, although it is interesting to note that all three Brenner tumors were PR positive.

80

TABLE 3 Receptor Expression in Benign a n d Borderline Ovarian Tumors 70

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A. Benign 1. Mucinous 2. Mncinous 3. Mucinous 4. Mucinous 5. Mucinous 6. Serous 7. Serous 8, Serous 9. Serous 10. Brenner 11. Brenner 12. Brenner B. Borderline 1. Mucinous 2. Mucinous 3. Mucinous 4. Mucinous 5. Mucinous 6. Mucinous 7. Mucinous 8. Serous 9. Serous

188

FRIEDLANDER ET AL.

DISCUSSION Despite a large number of reports claiming effectiveness for various chemotherapeutic regimens in advanced ovarian cancer, overall survival rates have not changed appreciably with the introduction of combination chemotherapy, and therapy is given with palliative rather than curative intent to the majority of patients with advanced disease [22]. Hormonal therapy is associated with minimal toxicity and has been reported to have objective response rates in 0-65% of patients with epithelial ovarian cancer [1,2,9,23-26]. There still remains considerable doubt however as to the efficacy of hormonal therapy in patients with advanced ovarian cancer as most studies have reported a generally low objective response to hormonal treatment. Patients who receive hormonal treatment almost invariably have had extensive prior chemotherapy and hormonal treatment is usually delayed until tumor progression has occurred and no further systemic therapy is possible. It is not surprising therefore that the reported response rates to hormonal therapy are so low and it is of interest that there does appear to be a higher response rate in patients who have only received a single alkylating agent prior to commencing hormonal treatment [26]. This has led the Clinical Oncology Society of Australia (COSA) recently to initiate a randomized study of chlorambucil + Tamoxifen as first-line treatment in patients with bulky Stage III disease to answer the question whether the early introduction of hormonal therapy offers any increase in objective response rates and overall survival. The presence of estrogen and progesterone receptors in 40-70% of ovarian cancers has led a number of investigators to suggest that they may help identify those patients likely to respond to hormonal therapy [2,3-8]. The functional significance of these hormone receptors in ovarian tumors is still not clear, nor is their relationship with hormonal responsiveness or other determinants of biological behavior such as tumor stage, histological subtype, and degree of differentiation established beyond doubt. The 70% incidence of ER and PR in malignant ovarian tumors found in this study is in keeping with the published literature. As others have also reported, serous and endometrioid carcinomas were more likely to be receptor positive than the other histological subtypes [5,10]. There was no demonstrable relationship between receptor expression and clinical stage or histological grade. However, there are conflicting data with respect to the association between grade and receptor levels and this probably reflects the general lack of uniformity and poor reproducibility of the various grading systems currently in use [7,27,28]. Up to 50% of benign and borderline tumors had measurable receptors but the numbers included in this study, as in others, are too small to draw any definite conclusions.

Tumor ploidy has been demonstrated to be a major determinant of outcome in patients with ovarian cancer and patients with diploid tumors have a significantly longer survival than those with aneuploid tumors [15-18]. It is therefore relevant that a significant relationship between ploidy and receptor expression was demonstrated with diploid malignant ovarian tumors having a higher incidence of receptor positivity (ER+ or P R + ) than aneuploid tumors. There was in particular a significant association between PR positivity and ploidy with diploid tumors being more commonly PR + than aneuploid tumors. This may be relevant in the light of recent reports on the value of the progesterone receptor as a factor of major prognostic importance in breast cancer [29,30]. Progesterone receptor expression is believed to reflect a "functional" ER and is possibly of greater importance than the ER alone in determining outcome. Receptor-rich ovarian tumors have been reported to have a relatively favorable prognosis [5] as have receptor-rich breast tumors [31-35]. It was therefore appropriate to find that 70% of ER-rich and PR-rich malignant ovarian tumors were diploid. The numbers involved were small and this should be studied further in a larger group of patients. It has been speculated that DNA measurements could be of value in predicting the endocrine sensitivity of breast tumors and an association between ploidy and steroid receptor status has been reported with near-diploid tumors tending to be E R + [36]. This relationship, however, does not achieve statistical significance in all studies [37-41]. There is also some evidence that in patients with prostatic cancer those with diploid tumors have a higher likelihood of response to estrogen therapy than those with aneuploid tumors [13,14]. A similar situation may exist in ovarian cancer and although this question would be best addressed in prospective studies, the flow cytometric analysis of the DNA content of paraffinembedded tumor tissue from patients known to have had hormonally sensitive tumors would be an effective approach [42]. A number of investigators have reported that diploid tumors have a significantly lower S phase than aneuploid tumors and that receptor-positive breast tumors have a lower S phase than receptor-negative tumors (37-38), a finding supported by thymidine labeling studies [43]. This study of ovarian tumors indicates a similar relationship between ploidy and proliferative fraction, with both diploid and receptor-positive tumors having a significantly lower S phase than aneuploid and receptor-negative tumors. However, it should be recalled that there are a number of problems inherent in the S-phase determination. The S phase can be determined in all diploid tumors, but only in approximately 50% of aneuploid tumors because of substantial overlapping of tumor populations. Diploid tumors have a lower S phase than aneuploid tumors, but this may be artificially reduced by the presence of normal

STEROID RECEPTOR EXPRESSION IN OVARIAN TUMORS

noncycling cells (e.g., lymphocytes, fibroblasts). In contrast, the estimation of the aneuploid S-phase population could be falsely elevated by the presence of diploid doublets and tetraploid normal cells [43]. This study confirms the high incidence of receptor positivity in ovarian tumors and demonstrates an association between steroid receptor levels with other determinants of biological behavior. However, prolonged follow-up of patients is required before the clinical significance of ER and PR expression in ovarian cancer can be fully elucidated. Given the overall high incidence of receptor positivity and the relatively low incidence of hormonal responsiveness reported in ovarian tumors, it seems unlikely that receptor levels alone will accurately predict hormonal sensitivity. This question should be answered when the results of randomized studies which include hormonal therapy as part of first-line treatment of patients with ovarian cancer become available. ACKNOWLEDGMENTS We are extremely grateful to the many gynecologists who allowed us to study their patients. The receptor analysis on patients from the Westmead Centre was supported by a grant from the N.S.W. State Cancer Council. We thank Judy Hood for secretarial assistance and for typing the manuscript.

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