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Correlation between nuclear DNA content and steroid receptor status in ovarian cancer
European Journal of Obstetrics & Gynecology and Reproductive
Biology, 38 (1990) 221-227
221
Elsevier EUROBS 01063
Correlation between nuclear DNA content and steroid receptor status in ovarian cancer Berend J. Slotman I, Jan P.A. Bask ’ and B. Ramanath Rao 1 Departments
of
’ Endocrinology and ’ Pathology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands Accepted for publication 24 April 1990
Summary
The nuclear DNA content of 47 malignant epithelial ovarian tumours and five ovarian tumours of borderline malignancy was correlated with estrogen (ER), progesterone (PR) and androgen receptor (AR) status. Aneuploidy was observed in 60% of the malignant tumours. The DNA index of poorly differentiated tumours was higher than that of well differentiated tumours (P < 0.05). Ploidy did not correlate with histological type, stage of disease and ER content. Of the diploid tumours, 74% was PR-positive, in contrast to 36% of the aneuploid tumours (P < 0.05). In addition, 89% of the diploid tumours had high AR levels ( r 30 fmol/mg cytosol protein), in contrast to only 54% of the aneuploid tumours (P < 0.05). These observations strengthen our previous findings on the prognostic importance of PR and also suggest that androgens may have a role in ovarian cancer. Ploidy; Steroid hormone receptor; Ovarian cancer
Introduction Previous studies have shown that most ovarian cancers contain steroid hormone receptors (see Ref. 1 for review). Estrogen (ER) and progesterone (PR) receptors are detected in about 50% of the tumours, while androgen receptors (AR) are found in 80 to 90% of them [1,2]. The results of studies on the correlation between steroid hormone receptor content and tumour histology are conflicting, with some showing [3-51 and others
Correspondence: B.R. Rao, Ph.D., Department of Endocrinology, BRUG 254, Academisch Ziekenhuis Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
0028-224?/90/$03.50
failing to show [6-111 a correlation between the grade of differentiation and the tumour ER and PR status. This controversy may be explained by differences in the assay methods and the criteria for positivity for steroid hormone receptors, the fact that within one tumour the population may be heterogenous with respect to receptor content [12,13], and there being a small number of cases in most studies. Furthermore, the lack of uniformity and poor reproducibility [14] of the various grading systems for ovarian cancer may contribute to this. Therefore, the assessment of more objective and reproducible techniques, such as the measurement of nuclear DNA content and morphological features in the histological examination of ovarian tumours has been advocated [14]. It has been demonstrated that the DNA content and quantita-
0 1990 Elsevier Science Publishers B.V. (Biomedicai Division)
222
tive morphological features of ovarian tumours are correlated with patient survival [15-241. Although the tumour PR level was found to be a prognostic indicator in ovarian cancer, independent of patients’ age, stage of disease, histological type and grade of differentiation [5,25], the biological functionality of any of the steroid hormone receptors in ovarian cancer is still to be demonstrated. In the present study we have correlated the tumour steroid hormone receptor status with the nuclear DNA content. Materials and Methods Patients and tissues Tumour specimens of 47 patients with newly diagnosed serous (n = 25), endometrioid (n = 13) and mutinous (n = 9) ovarian adenocarcinomas and tumours of five patients with ovarian tumours of borderline malignancy were investigated for steroid hormone receptors and nuclear DNA content. The 47 ovarian carcinomas comprised nine well differentiated (Grade l), eight moderately (Grade 2) and 30 poorly differentiated (Grade 3) tumours. Nine tumours were stage I, one was stage II, 30 were stage III and 7 were stage IV ovarian cancers. In 9 cases, the tumour specimen was taken from a metastatic site. The five tumours of borderline malignancy were obtained from patients with serous (n = 2) and mutinous (n = 3) tumours in stage Ia. The age of the patients varied between 26 and 86 years (mean & SD: 63 + 12 years). Nine patients were premenopausal and 38 were postmenopausal. Small pieces of tissue were frozen in liquid nitrogen and stored at - 70 ’ C until analysis. One part of tissue was taken for histological evaluation and determination of nuclear DNA content, while an adjacent part was taken for steroid hormone receptor determination. The histological classification was based on the W.H.O. criteria [26]. Steroid hormone receptors The methods used in our laboratory for the determination of steroid hormone receptors have
been described in detail [2,25]. Procedures in brief were as follows. Concentrations of ER, PR and AR were determined by saturation analysis. Cytosols were incubated for 16 h with tritiated steroids (0.1-1.0 nM) in the presence and absence of a 200-fold excess of unlabelled hormone. Bound and unbound steroids were separated by adding dextran-coated charcoal. The bound radioactivity was measured by liquid scintillation counting. The number of binding sites and dissociation constants were determined according to Scatchard [27]. Cytosols were designated as receptor-positive when specific high-affinity (& < 10e9 M) binding, with a binding capacity 2 10 fmol/mg cytosol protein was observed. Nuclear DNA content The method described by Vindelov et al. [28] was used in the preparation of nuclei for determination of DNA content. In brief, cells were trypsinised and stained using propidium iodide. Measurements were performed by using a Fats Star Plus cytometer (Becton & Dickinson, U.S.A.). The first peak in the DNA histogram was considered to represent normal cells and diploid tumour cells. The DNA index was calculated by comparing the different peaks with the diploid peak from human lymphocytes in the DNA histogram normalized to 1.0. The coefficient of variation of the G,/G, peak was I 5.5%. Statistics The cm-square test and the t-test were used for statistical analysis. Results Malignant tumours Of the 10 patients with early stage (stage I/II) ovarian cancer, 5 (50%) had well differentiated tumours, while 4 (11%) of the 37 patients with advanced disease (stage III/IV) had well differentiated tumours (Table I). This difference was statistically significant (P < 0.05). Mutinous tumours were seen more frequently in patients with early stage disease, while serous and endometrioid tumours were predominantly found among those with advanced disease (P -C0.05; Table I). Of the
223 TABLE
I
TABLE
Relationship between stage of disease and grade of differentiation and tumour histology in 47 ovarian carcinomas Stage I/II (I1 =lO)
II
Relationship between ploidy and grade of differentiation, histological type and stage of disease in 47 ovarian carcinomas
Stage III/IV (n = 37)
Diploid (n =19) ~ n R
Aneuploid (n-=28) ~ n %
~n
‘K
n
%
Well differentiated Moderately differentiated Poorly differentiated
5 2 3
50 20 30
4 6 27
11 16 73
Well differentiated Moderately differentiated Poorly differentiated
6 4 9
32 21 47
3 4 21
11 14 75
Serous Endometrioid Mutinous
3 2 5
30 20 50
22 11 4
59 30 11
Serous Endometrioid Mutinous
9 5 5
47 26 26
16 8 4
57 29 14
Stage I/II Stage III/IV
6 13
32 68
4 24
14 86
25 serous turnouts, 21 (84%) were poorly differentiated, in contrast to four (44%) of the nine mutinous tumours. This difference was statistically significant (P < 0.05). Twenty-two (47%) tumours were positive for ER, 24 (52%) for PR and 41 (87%) for AR. There were 10 (21%) tumours with ER-concentrations 2 30 fmol/mg, 14 (30%) tumours with PR concentrations 2 30 fmol/mg and 32 (68%) tumours with AR concentrations r 30 fmol/mg cytosol protein. The mean concentration of ER, after exclusion of zero-values, was 47 f 40 fmol/mg cytosol protein (mean + SD), for PR 67 f 99 fmol/mg and for AR 86 f 87 fmol/mg. Positivity for ER was seen more frequently in stage III/IV tumours (P < 0.05). Of the 10 stage I/II turnours, only one (10%) was ER-positive, whereas 21 (57%) of the 37 stage III/IV tumours were ER-positive. Stage of disease did not correlate with the tumour PR and AR status. There was no correlation between the histological type and the degree of differentiation and the steroid hormone receptor status. However, concentrations of ER were significantly higher in poorly differentiated turnours, as compared with well differentiated tumours (l-test: P -c 0.05). In addition, ER concentrations in serous and endometrioid tumours were significantly higher as compared to mutinous tumours (t-test: P < 0.05). Age and menopausal status did not correlate with the steroid hormone receptor status. Furthermore, there was no significant difference in steroid
hormone receptor content between tissues taken from ovarian and metastatic sites. Of the 47 malignant tumours, 19 (40%) were diploid and 28 (60%) were aneuploid. Among the aneuploid turnours, eight tumours were tetraploid. The DNA index of the aneuploid tumours ranged from 1.4 to 2.4. The relationship between ploidy and grade of differentiation, tumour type and stage of disease is shown in Table II. Of the 19 diploid turnours, 9 (47%) were poorly differentiated, whereas 21 (75%) of the 28 aneuploid tumours were poorly differentiated. This difference did not reach statistical significance. However, the DNA index of well differentiated tumours (mean f SD: 1.23 k 0.36) was significantly lower than that of poorly differentiated tumours (mean f SD: 1.52 + 0.37) (rtest: P < 0.05). There was no significant relationship between ploidy level and histological type and stage of disease (Table II). We did not find a correlation between ploidy and age or menopausal status as well (data not shown). In addition, no significant difference in ploidy was observed between tissues taken from the ovary and tissues taken from a metastatic site (data not shown). The nuclear DNA content did not correlate with the tumour ER status (Fig. 1). However, we found diploid tumours to be more frequently PRpositive, as compared to aneuploid tumours (P < 0.05). This difference was observed at cut-off values for positivity of both 10 and 30 fmol/mg
224 ,oo % positive 1
ER
+
ER ++
PR
AR
PR ++
+
AR ++
*
Fig. 1. Positivity for eStrogen (ER), progesterone (PR) and androgen receptors (AR) on the basis of cut-off values of 10 fmol/mg cytosol protein (EIk + ; PR + ; AR + ) and 30 fmol/mg cytosol protein (ER+ +; PR+ +; AR+ +). The data are given as percentage of the number of diploid (n = 19, open bars) and aneuploid (n = 28; hatched bars) ovarian carcinomas.
cytosol protein (Fig. 1). No correlation was found between the presence and absence of AR and ploidy when a cut-off value of 10 fmol/mg cytosol protein was used. However, we found 89% of the diploid tumours to have an AR concentration 2 30 fmol/mg cytosol protein, while this was seen in 54% of the aneuploid turnours. This difference was statistically significant (P < 0.05). TABLE III Relationship between ploidy and various combinations of estrogen (ER), progesterone (PR) and androgen receptors (AR) (based on cut-off values for positively of 10 fmol/mg cytosol protein) in 47 ovarian carcinomas Aneuploid (n = 28)
Diploid (n =19) n
%
n
%
ER+/PR+ ER+/PRER-/PR+ ER-/PR-
5 2 9 3
26 11 47 16
5 10 5 8
18 36 18 29
ER+,‘AR+ ER+/ARER-/AR+ ER-/AR-
6 1 12 0
32 5 29 0
13 2 10 3
46 I 36 11
PR+/AR+ PR+/ARPR-/AR+ PR-/AR-
14 0 4 1
14 0 21 5
9 1 14 4
32 4 50 14
The ploidy level was also investigated in relationship with various combinations of ER, PR and AR (Table III). Aneuploid tumours tended to be more frequently ER + /PR - , while diploid tumours tended to be more frequently ER - /PR + . Furthermore, we found 74% of the diploid tumours to be PR + /AR + , as compared to 32% of the aneuploid tumours. However, these differences did not attain statistical significance. Two tumours did not contain any of the three steroid hormone receptors. These tumours were both aneuploid. Borderline turnours
Of the five tumours of borderline malignancy, one contained ER and three were PR-positive, while AR were detected in all of them. Of the two serous tumours, one was tetraploid and the other was diploid. Among the three mutinous tumours, two were diploid and one was aneuploid. The two PR-negative tumours were both aneuploid, while the three PR-positive tumours were diploid. Discussion
Steroid hormone receptors have been detected in normal [29-311 and malignant ovarian tissue (see Ref. 1 for review). The finding of ER and PR in about 50% of the tumours and AR in almost 90% of the tumours is in agreement with the results from other investigations [1,2,5,7-10,251. The presence of steroid hormone receptors in ovarian cancers suggests that this cancer may be an endocrine-related tumour. Studies on the effect of hormonal therapy, mostly consisting of progestins or tamoxifen, have yielded an objective response rate of approximately 15% only [l]. However, it should be emphasized that endocrine therapy was almost invariably used on an empirical basis and as a last resort in patients with progressive disease who were unable to tolerate other therapies. In analogy to breast cancer, determination of steroid hormone receptors might be useful for selecting those ovarian cancer patients who are most likely to benefit from different forms of endocrine therapy. The relationship between steroid hormone receptors and the histological characteristics of
225
ovarian tumours is still not clear. This may be partly due to differences in methods used in the measurement of steroid hormone receptors, but also to differences in the histological characterization. It has been shown that there is a considerable variability in the grading of ovarian cancers P41. In the last years, analysis of DNA content by flow cytometry has been widely introduced, and it has been shown that quantitative changes in DNA content of ovarian tumours are of prognostic significance [15-241. In most investigations on tumour ploidy, paraffin-embedded tissue was being analysed. Recently, flow cytometric analysis has been performed on a large series of frozen breast-cancer tissues [32,33]. The use of frozen instead of paraffin-embedded tissue enabled these investigators to perform steroid hormone receptor assays and ploidy measurements on the same tissue. It was concluded that flow cytometric measurements of frozen breast-cancer tissue specimens can be indicators of prognosis in this tumour. We followed a similar approach to study the correlation between steroid hormone receptors and ploidy in ovarian cancer, and found 60% of the tumours to be aneuploid. This concurs with the results from other studies, reporting abnormalities in the DNA content in 50 to 80% of the epithelial ovarian cancers [15-24,34-361. Aneuploidy is most frequently found in poorly differentiated tumours [ 19,20,22,23,34-36). The results of the present study are in basic agreement with these findings. Some investigators also found a relationship between aneuploidy and advanced stage disease [22,35-371, while others did not detect such a correlation [20,24,38]. In the present series, 32% of the diploid tumours were in early stages, in contrast to 14% of the aneuploid tumours. This difference was not statistically significant. We did not observe a relationship between ploidy and the histological type of the tumour. Similar results have been reported by other investigators [24,3438]. We found the PR status to be correlated with the DNA content. Of the diploid tumours, 74% was PR-positive, in contrast to 36% of the aneuploid tumours (P < 0.05). These findings are in agreement with those reported by Friedlander
et al. [ll]. The tumour PR status did not correlate with grade of differentiation and tumour histology. The tumour ER status did not correlate with ploidy. Others reported similar results [ 11,381. However, in our study, higher concentrations of ER were observed in poorly differentiated and in serous and endometrioid tumours, as compared to tumours with other histological characteristics. We found tumours with relatively high AR-levels (L 30 fmol/mg cytosol protein) to be more frequently diploid (Fig. 1). This, together with the fact that the majority of tumours contains AR, suggests that androgens may have a role in ovarian cancer. It has been extensively demonstrated that ploidy is an important prognostic factor in ovarian cancer [15-241. The present observation strengthens the previous findings, on a prognostic advantage of PR-positive tumours, as compared to PR-negative tumours [5,25]. It can be hypothesized that the correlation between PR status and ploidy may contribute to this prognostic advantage of PRpositive tumours. Since the follow-up period of the patients in the present study has been of short duration, a definitive conclusion can not yet be drawn and it will be reported in the future.
Acknowiedgements We would like to thank the gynaecologists and pathologists of the following hospitals for providing tumour specimens: Academisch Ziekenhuis Vrije Universiteit, Onze Lieve Vrouwe Gasthuis and Sint Lucas Ziekenhuis, Amsterdam. Sint Elizabeth Ziekenhuis, Tilburg and Elisabeth Gasthuis, Haarlem, The Netherlands. The authors thank Mrs. C. van Galen, Department of Pathology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, for technical assistance in the determination of ploidy levels. This work was supported by grants from the Dutch Cancer Society (IKA 87-01) and The Praeventiefonds (28/834), The Netherlands.
References 1 Slotman BJ. Rao BR. Ovarian cancer (Review). Etiology. diagnosis. prognosis, surgery, radiotherapy. chemotherapy and endocrine therapy. Anticancer Res 1988;8:417-434.
226 2 Ktihnel R. De Graaff J, Rao BR. Stalk JG. Androgen receptor predominance in human ovarian carcinoma. J Steroid Biochem 1987;26:393-397. 3 Creasman WT. Sasso RA, Weed JC, McCarty KS. Ovarian carcinoma: histologic and clinical correlation of cytoplasmic estrogen and progesterone binding. Gynecol Oncol 1981; 12:319-327. 4 Quinn MA, Pearce P, Rome, R. Funder JW. Fortune D, Pepperell RJ. Cytoplasmic steroid receptors in ovarian turnours. Br J Obstet Gynaecol 1982;89:754-759. 5 Iversen OE. Skaarland E. Utaaker E. Steroid receptor content in human ovarian tumors: survival of patients with ovarian carcinoma related to steroid receptor content. Gynecol Oncol 1986;23:65-76. 6 Bibro MC, Schwartz PE, LiVolsi VA, Eisenfeld AJ. Estrogen binding macromolecules in human ovarian carcinoma. Lab Invest 1979;40:241-242. 7 Schwartz PE. LiVolsi VA, Hildreth N. MacLusky NJ. Naftolin FN. Eisenfeld AJ. Estrogen receptors in ovarian epithelial carcinoma. Obstet Gynecol 1982;59:229-238. 8 Jones LA. Edwards CL. Freedman RS, Tan MT, Gallager HS. Estrogen and progesterone receptor titers in primary epithelial ovarian carcinomas. Int J Cancer 1983:32:567571. 9 Pollow K. Schmidt-Matthiesen A. Hoffman G et al. [ ‘H]Estradiol and [ ‘HJR5020 binding in cytosols of normal and neoplastic human ovarian tissue. lnt J Cancer 1983;31:603-608. 10 Geyer H. Teufel G. De Gregorio G. Pfleiderer A. Steroidhormon-Rezeptoren beim Ovarialkarzinom und ihre klinische Bedeutung. Onkologie 1984;7(Suppl 2):4452. 11 Friedlander M. Quinn MA, Fortune D et al. The relationship of steroid receptor expression to nuclear DNA distribution and clinicopathological characteristics in epithehal ovarian tumors. Gynecol Oncol 1989;32:184-190. 12 Rao BR, Meyer JS. Estrogen and progestin receptors in normal and cancer tissues. In: McGuire WL. Raynaud J-P. Baulieu E-E. (eds). Progress in cancer research and therapy. Vol 4. Progesterone receptors in normal and neoplastic tissues. New York: Raven Press, 1977;155-169. 13 Quinn MA, Rome RM. Cauchi M, Fortune DW. Steroid receptors and ovarian tumors: Variation within primary tumors and between primary tumors and metastases. Gynecol Oncol 1988;31:424-429. 14 Baak JPA, Langley FA. Talerman A. Delemarre JFM. The prognostic variability of ovarian tumor grading by different pathologists. Gynecol Oncol 1987;27:166-172. 15 Erhardt K. Auer G, Bjorkholm E et al Prognostic significance of nuclear DNA wntent in serous ovarian tumors. Cancer Res 1984:44:2198-2202. I6 Friedlander ML, Hedley DW. Taylor IW. Russell P. Coates AS, Tattersall MHN. Influence of cellular DNA content on survival in advanced ovarian cancer. Cancer Res 1984;44:397-400. 17 Volm M, Btiiggeman A, Gunther M, Kleine W. Pfleiderer A. Vogt-Schaden M. Prognosttc relevance of ploidy. proliferation, and resistance-predictive tests in ovarian carcinoma. Cancer Res 1985;45:5180-5185.
18 Baak JPA, Chan KK, Stalk JG. Kenemans P. Prognosttc factors in borderline and invasive ovarian tumours of the common epithelial type. Path Res Pratt 1987;182:755-774. 19 Rodenburg CJ. Comelisse CJ. Heintz APM, Hermans J. Fleuren GJ. Tumor ploidy as a major prognostic factor in advanced ovarian cancer. Cancer 1987:59:317-323. 20 Rutgers DH, Wils IS. Schaap AHP. Van Lindert ACM. DNA flow cytometry, histological grade. stage. and age as prognostic factors in human epithelial ovarian carcinomas. Pathol Res Pratt 1987;182:207-213. 21 Baak JPA. Schipper NW, Wisse-Brekelmans ECM et al. The prognostic value of morphometrical features and cellular DNA content in cis-platin-treated late ovarian cancer patients. Br J Cancer 1988;57:503-508. 22 Iversen OE. Prognostic value of the flow cytometric DNA index in human ovarian carcinoma. Cancer 1988;61:971975. 23 Rodenburg CJ. Cornelisse CJ, Hermans J. Fleuren GJ. DNA flow cytometry and morphometry as prognostic indicators in advanced ovarian cancer: a step fotward m predicting the clinical outcome. Gynecol Oncol 1988;29:176187. 24 Murray K. Hopwood L. Volk D. Wilson JF. Cytofluorometric analysis of the DNA content in ovarian carcinoma and its relationship to patient survival. Cancer 1989;63: 2456- 2460. 25 Slotman BJ. KUhnel R. Rao BR. Dijkhuizen GH. De Graaff J. Stolk JG. Importance of steroid receptors and aromatase activity in the prognosis of ovarian cancer: High tumor progesterone receptor levels correlate with longer survival. Gynecol Oncol 1989:33:76-81. 26 Serov SF. Scully RE. Sobin LH. Histological typmg of ovarian tumors. International hrstological classification of tumors, No. 9. Geneva: World Health Organization, 1973. 27 Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:660-672. 28 Vmdelov LL, Christensen IJ. Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 1983;3:323-327. 29 Bergqvist A. Kullander S, Thorell J. A study of estrogen and progesterone cytosol receptor concentration in benign and malignant ovarian tumors and a review of mahgnant ovarian tumors treated with medroxy-progesterone acetate. Acta Obstet Gynecol Stand (Suppl) 1981;101:75-81. 30 Vierikko P, Kauppila A. Vihko R. Cytosol and nuclear estrogen and progestin receptors and 17 beta-hydroxysteroid dehydrogenase activity in non-diseased tissue and in benign and malignant tumors of the human ovary. Int J Cancer 1983;32:413-422. 31 Lantta M. Estradiol and progesterone receptors in normal ovary and ovarian tumors. Acta Ohstet Gynecol Stand 1984:63:497-503. 32 Dressler LG. Seamer LC, Owens MA. Clark GM, McGuire WL. DNA flow cytometry and prognostic factors in 1331 frozen hreast cancer specimens. Cancer 1988;61:420-427. 33 Clark GM, Dressler LG. Owens MA, Pounds G. Oldaker T, McGuire WL. Prediction of relapse or survival in pa-
tients with nodenegative breast cancer by DNA flow cytometry. N EngJ J Med 1989;320:627-633. 34 Christov K, Vassilev N. Flow cytometric analysis of DNA and cell proliferation in ovarian tumours. Cancer 1987;60:121-125. 35 Iversen OE, Skaarland E, Ploidy assessment of benign and malignant ovarian tumors by flow cytometry. Cancer 1987;60:82-87. 36 Kallioniemi O-P, Matilla J, Punnonen R, Koivula T. DNA
ploidy level and cell cycle distribution in ovarian cancer: relation to histopathological features of the tumor. Int J Gynecol Path01 1988;7:1-11. 37 Erba E, Ubezio E, Pepe S et al. Flow cytometric analysis of DNA content in human ovarian cancers. Br J Cancer 1989;60:45-50. 38 Jakobsen A, Hansen V, Pot&en HS. DNA profile and steroid receptor content of human ovarian cancer. Eur J Gynaecol Oncol 1988;9:461-463.
Biology, 38 (1990) 221-227
221
Elsevier EUROBS 01063
Correlation between nuclear DNA content and steroid receptor status in ovarian cancer Berend J. Slotman I, Jan P.A. Bask ’ and B. Ramanath Rao 1 Departments
of
’ Endocrinology and ’ Pathology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands Accepted for publication 24 April 1990
Summary
The nuclear DNA content of 47 malignant epithelial ovarian tumours and five ovarian tumours of borderline malignancy was correlated with estrogen (ER), progesterone (PR) and androgen receptor (AR) status. Aneuploidy was observed in 60% of the malignant tumours. The DNA index of poorly differentiated tumours was higher than that of well differentiated tumours (P < 0.05). Ploidy did not correlate with histological type, stage of disease and ER content. Of the diploid tumours, 74% was PR-positive, in contrast to 36% of the aneuploid tumours (P < 0.05). In addition, 89% of the diploid tumours had high AR levels ( r 30 fmol/mg cytosol protein), in contrast to only 54% of the aneuploid tumours (P < 0.05). These observations strengthen our previous findings on the prognostic importance of PR and also suggest that androgens may have a role in ovarian cancer. Ploidy; Steroid hormone receptor; Ovarian cancer
Introduction Previous studies have shown that most ovarian cancers contain steroid hormone receptors (see Ref. 1 for review). Estrogen (ER) and progesterone (PR) receptors are detected in about 50% of the tumours, while androgen receptors (AR) are found in 80 to 90% of them [1,2]. The results of studies on the correlation between steroid hormone receptor content and tumour histology are conflicting, with some showing [3-51 and others
Correspondence: B.R. Rao, Ph.D., Department of Endocrinology, BRUG 254, Academisch Ziekenhuis Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
0028-224?/90/$03.50
failing to show [6-111 a correlation between the grade of differentiation and the tumour ER and PR status. This controversy may be explained by differences in the assay methods and the criteria for positivity for steroid hormone receptors, the fact that within one tumour the population may be heterogenous with respect to receptor content [12,13], and there being a small number of cases in most studies. Furthermore, the lack of uniformity and poor reproducibility [14] of the various grading systems for ovarian cancer may contribute to this. Therefore, the assessment of more objective and reproducible techniques, such as the measurement of nuclear DNA content and morphological features in the histological examination of ovarian tumours has been advocated [14]. It has been demonstrated that the DNA content and quantita-
0 1990 Elsevier Science Publishers B.V. (Biomedicai Division)
222
tive morphological features of ovarian tumours are correlated with patient survival [15-241. Although the tumour PR level was found to be a prognostic indicator in ovarian cancer, independent of patients’ age, stage of disease, histological type and grade of differentiation [5,25], the biological functionality of any of the steroid hormone receptors in ovarian cancer is still to be demonstrated. In the present study we have correlated the tumour steroid hormone receptor status with the nuclear DNA content. Materials and Methods Patients and tissues Tumour specimens of 47 patients with newly diagnosed serous (n = 25), endometrioid (n = 13) and mutinous (n = 9) ovarian adenocarcinomas and tumours of five patients with ovarian tumours of borderline malignancy were investigated for steroid hormone receptors and nuclear DNA content. The 47 ovarian carcinomas comprised nine well differentiated (Grade l), eight moderately (Grade 2) and 30 poorly differentiated (Grade 3) tumours. Nine tumours were stage I, one was stage II, 30 were stage III and 7 were stage IV ovarian cancers. In 9 cases, the tumour specimen was taken from a metastatic site. The five tumours of borderline malignancy were obtained from patients with serous (n = 2) and mutinous (n = 3) tumours in stage Ia. The age of the patients varied between 26 and 86 years (mean & SD: 63 + 12 years). Nine patients were premenopausal and 38 were postmenopausal. Small pieces of tissue were frozen in liquid nitrogen and stored at - 70 ’ C until analysis. One part of tissue was taken for histological evaluation and determination of nuclear DNA content, while an adjacent part was taken for steroid hormone receptor determination. The histological classification was based on the W.H.O. criteria [26]. Steroid hormone receptors The methods used in our laboratory for the determination of steroid hormone receptors have
been described in detail [2,25]. Procedures in brief were as follows. Concentrations of ER, PR and AR were determined by saturation analysis. Cytosols were incubated for 16 h with tritiated steroids (0.1-1.0 nM) in the presence and absence of a 200-fold excess of unlabelled hormone. Bound and unbound steroids were separated by adding dextran-coated charcoal. The bound radioactivity was measured by liquid scintillation counting. The number of binding sites and dissociation constants were determined according to Scatchard [27]. Cytosols were designated as receptor-positive when specific high-affinity (& < 10e9 M) binding, with a binding capacity 2 10 fmol/mg cytosol protein was observed. Nuclear DNA content The method described by Vindelov et al. [28] was used in the preparation of nuclei for determination of DNA content. In brief, cells were trypsinised and stained using propidium iodide. Measurements were performed by using a Fats Star Plus cytometer (Becton & Dickinson, U.S.A.). The first peak in the DNA histogram was considered to represent normal cells and diploid tumour cells. The DNA index was calculated by comparing the different peaks with the diploid peak from human lymphocytes in the DNA histogram normalized to 1.0. The coefficient of variation of the G,/G, peak was I 5.5%. Statistics The cm-square test and the t-test were used for statistical analysis. Results Malignant tumours Of the 10 patients with early stage (stage I/II) ovarian cancer, 5 (50%) had well differentiated tumours, while 4 (11%) of the 37 patients with advanced disease (stage III/IV) had well differentiated tumours (Table I). This difference was statistically significant (P < 0.05). Mutinous tumours were seen more frequently in patients with early stage disease, while serous and endometrioid tumours were predominantly found among those with advanced disease (P -C0.05; Table I). Of the
223 TABLE
I
TABLE
Relationship between stage of disease and grade of differentiation and tumour histology in 47 ovarian carcinomas Stage I/II (I1 =lO)
II
Relationship between ploidy and grade of differentiation, histological type and stage of disease in 47 ovarian carcinomas
Stage III/IV (n = 37)
Diploid (n =19) ~ n R
Aneuploid (n-=28) ~ n %
~n
‘K
n
%
Well differentiated Moderately differentiated Poorly differentiated
5 2 3
50 20 30
4 6 27
11 16 73
Well differentiated Moderately differentiated Poorly differentiated
6 4 9
32 21 47
3 4 21
11 14 75
Serous Endometrioid Mutinous
3 2 5
30 20 50
22 11 4
59 30 11
Serous Endometrioid Mutinous
9 5 5
47 26 26
16 8 4
57 29 14
Stage I/II Stage III/IV
6 13
32 68
4 24
14 86
25 serous turnouts, 21 (84%) were poorly differentiated, in contrast to four (44%) of the nine mutinous tumours. This difference was statistically significant (P < 0.05). Twenty-two (47%) tumours were positive for ER, 24 (52%) for PR and 41 (87%) for AR. There were 10 (21%) tumours with ER-concentrations 2 30 fmol/mg, 14 (30%) tumours with PR concentrations 2 30 fmol/mg and 32 (68%) tumours with AR concentrations r 30 fmol/mg cytosol protein. The mean concentration of ER, after exclusion of zero-values, was 47 f 40 fmol/mg cytosol protein (mean + SD), for PR 67 f 99 fmol/mg and for AR 86 f 87 fmol/mg. Positivity for ER was seen more frequently in stage III/IV tumours (P < 0.05). Of the 10 stage I/II turnours, only one (10%) was ER-positive, whereas 21 (57%) of the 37 stage III/IV tumours were ER-positive. Stage of disease did not correlate with the tumour PR and AR status. There was no correlation between the histological type and the degree of differentiation and the steroid hormone receptor status. However, concentrations of ER were significantly higher in poorly differentiated turnours, as compared with well differentiated tumours (l-test: P -c 0.05). In addition, ER concentrations in serous and endometrioid tumours were significantly higher as compared to mutinous tumours (t-test: P < 0.05). Age and menopausal status did not correlate with the steroid hormone receptor status. Furthermore, there was no significant difference in steroid
hormone receptor content between tissues taken from ovarian and metastatic sites. Of the 47 malignant tumours, 19 (40%) were diploid and 28 (60%) were aneuploid. Among the aneuploid turnours, eight tumours were tetraploid. The DNA index of the aneuploid tumours ranged from 1.4 to 2.4. The relationship between ploidy and grade of differentiation, tumour type and stage of disease is shown in Table II. Of the 19 diploid turnours, 9 (47%) were poorly differentiated, whereas 21 (75%) of the 28 aneuploid tumours were poorly differentiated. This difference did not reach statistical significance. However, the DNA index of well differentiated tumours (mean f SD: 1.23 k 0.36) was significantly lower than that of poorly differentiated tumours (mean f SD: 1.52 + 0.37) (rtest: P < 0.05). There was no significant relationship between ploidy level and histological type and stage of disease (Table II). We did not find a correlation between ploidy and age or menopausal status as well (data not shown). In addition, no significant difference in ploidy was observed between tissues taken from the ovary and tissues taken from a metastatic site (data not shown). The nuclear DNA content did not correlate with the tumour ER status (Fig. 1). However, we found diploid tumours to be more frequently PRpositive, as compared to aneuploid tumours (P < 0.05). This difference was observed at cut-off values for positivity of both 10 and 30 fmol/mg
224 ,oo % positive 1
ER
+
ER ++
PR
AR
PR ++
+
AR ++
*
Fig. 1. Positivity for eStrogen (ER), progesterone (PR) and androgen receptors (AR) on the basis of cut-off values of 10 fmol/mg cytosol protein (EIk + ; PR + ; AR + ) and 30 fmol/mg cytosol protein (ER+ +; PR+ +; AR+ +). The data are given as percentage of the number of diploid (n = 19, open bars) and aneuploid (n = 28; hatched bars) ovarian carcinomas.
cytosol protein (Fig. 1). No correlation was found between the presence and absence of AR and ploidy when a cut-off value of 10 fmol/mg cytosol protein was used. However, we found 89% of the diploid tumours to have an AR concentration 2 30 fmol/mg cytosol protein, while this was seen in 54% of the aneuploid turnours. This difference was statistically significant (P < 0.05). TABLE III Relationship between ploidy and various combinations of estrogen (ER), progesterone (PR) and androgen receptors (AR) (based on cut-off values for positively of 10 fmol/mg cytosol protein) in 47 ovarian carcinomas Aneuploid (n = 28)
Diploid (n =19) n
%
n
%
ER+/PR+ ER+/PRER-/PR+ ER-/PR-
5 2 9 3
26 11 47 16
5 10 5 8
18 36 18 29
ER+,‘AR+ ER+/ARER-/AR+ ER-/AR-
6 1 12 0
32 5 29 0
13 2 10 3
46 I 36 11
PR+/AR+ PR+/ARPR-/AR+ PR-/AR-
14 0 4 1
14 0 21 5
9 1 14 4
32 4 50 14
The ploidy level was also investigated in relationship with various combinations of ER, PR and AR (Table III). Aneuploid tumours tended to be more frequently ER + /PR - , while diploid tumours tended to be more frequently ER - /PR + . Furthermore, we found 74% of the diploid tumours to be PR + /AR + , as compared to 32% of the aneuploid tumours. However, these differences did not attain statistical significance. Two tumours did not contain any of the three steroid hormone receptors. These tumours were both aneuploid. Borderline turnours
Of the five tumours of borderline malignancy, one contained ER and three were PR-positive, while AR were detected in all of them. Of the two serous tumours, one was tetraploid and the other was diploid. Among the three mutinous tumours, two were diploid and one was aneuploid. The two PR-negative tumours were both aneuploid, while the three PR-positive tumours were diploid. Discussion
Steroid hormone receptors have been detected in normal [29-311 and malignant ovarian tissue (see Ref. 1 for review). The finding of ER and PR in about 50% of the tumours and AR in almost 90% of the tumours is in agreement with the results from other investigations [1,2,5,7-10,251. The presence of steroid hormone receptors in ovarian cancers suggests that this cancer may be an endocrine-related tumour. Studies on the effect of hormonal therapy, mostly consisting of progestins or tamoxifen, have yielded an objective response rate of approximately 15% only [l]. However, it should be emphasized that endocrine therapy was almost invariably used on an empirical basis and as a last resort in patients with progressive disease who were unable to tolerate other therapies. In analogy to breast cancer, determination of steroid hormone receptors might be useful for selecting those ovarian cancer patients who are most likely to benefit from different forms of endocrine therapy. The relationship between steroid hormone receptors and the histological characteristics of
225
ovarian tumours is still not clear. This may be partly due to differences in methods used in the measurement of steroid hormone receptors, but also to differences in the histological characterization. It has been shown that there is a considerable variability in the grading of ovarian cancers P41. In the last years, analysis of DNA content by flow cytometry has been widely introduced, and it has been shown that quantitative changes in DNA content of ovarian tumours are of prognostic significance [15-241. In most investigations on tumour ploidy, paraffin-embedded tissue was being analysed. Recently, flow cytometric analysis has been performed on a large series of frozen breast-cancer tissues [32,33]. The use of frozen instead of paraffin-embedded tissue enabled these investigators to perform steroid hormone receptor assays and ploidy measurements on the same tissue. It was concluded that flow cytometric measurements of frozen breast-cancer tissue specimens can be indicators of prognosis in this tumour. We followed a similar approach to study the correlation between steroid hormone receptors and ploidy in ovarian cancer, and found 60% of the tumours to be aneuploid. This concurs with the results from other studies, reporting abnormalities in the DNA content in 50 to 80% of the epithelial ovarian cancers [15-24,34-361. Aneuploidy is most frequently found in poorly differentiated tumours [ 19,20,22,23,34-36). The results of the present study are in basic agreement with these findings. Some investigators also found a relationship between aneuploidy and advanced stage disease [22,35-371, while others did not detect such a correlation [20,24,38]. In the present series, 32% of the diploid tumours were in early stages, in contrast to 14% of the aneuploid tumours. This difference was not statistically significant. We did not observe a relationship between ploidy and the histological type of the tumour. Similar results have been reported by other investigators [24,3438]. We found the PR status to be correlated with the DNA content. Of the diploid tumours, 74% was PR-positive, in contrast to 36% of the aneuploid tumours (P < 0.05). These findings are in agreement with those reported by Friedlander
et al. [ll]. The tumour PR status did not correlate with grade of differentiation and tumour histology. The tumour ER status did not correlate with ploidy. Others reported similar results [ 11,381. However, in our study, higher concentrations of ER were observed in poorly differentiated and in serous and endometrioid tumours, as compared to tumours with other histological characteristics. We found tumours with relatively high AR-levels (L 30 fmol/mg cytosol protein) to be more frequently diploid (Fig. 1). This, together with the fact that the majority of tumours contains AR, suggests that androgens may have a role in ovarian cancer. It has been extensively demonstrated that ploidy is an important prognostic factor in ovarian cancer [15-241. The present observation strengthens the previous findings, on a prognostic advantage of PR-positive tumours, as compared to PR-negative tumours [5,25]. It can be hypothesized that the correlation between PR status and ploidy may contribute to this prognostic advantage of PRpositive tumours. Since the follow-up period of the patients in the present study has been of short duration, a definitive conclusion can not yet be drawn and it will be reported in the future.
Acknowiedgements We would like to thank the gynaecologists and pathologists of the following hospitals for providing tumour specimens: Academisch Ziekenhuis Vrije Universiteit, Onze Lieve Vrouwe Gasthuis and Sint Lucas Ziekenhuis, Amsterdam. Sint Elizabeth Ziekenhuis, Tilburg and Elisabeth Gasthuis, Haarlem, The Netherlands. The authors thank Mrs. C. van Galen, Department of Pathology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, for technical assistance in the determination of ploidy levels. This work was supported by grants from the Dutch Cancer Society (IKA 87-01) and The Praeventiefonds (28/834), The Netherlands.
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