Steroid hormone receptors in adenocarcinoma of the endometrium

GYNECOLOGIC

ONCOLOGY

10,

152-161(1980)

Steroid Hormone Receptors in Adenocarcinoma of the Endometrium RICHARD Division

E. HUNTER,

M. D., CHRISTOPHER LONGCOPE, AND V. CRAIG JORDAN, PH.D.

M.D.,

of Gyncologic Oncology, Department of Obstetrics and Gynecology, University Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01605; The Memorial Hospital, 119 Belmont Street, Worcester, Massachusetts 01605; and The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts

of

Received January 1, 1980 Estradiol cytosolic receptor levels were determined in tumor tissue of 73 patients with adenocarcinoma of the uterus. In 71% of the tumors, the estradiol receptor was >3 fmole/mg cytosol protein. An assay of 3 or more fmole/mg cytosol protein placed the tumor in the category of estrogen or progesterone positive. The levels of estrogen receptors were not related to the stage or histological grade of the tumor nor to the age of the patient. Progesterone cytosolic receptor levels were determined in 31 tumors. In 52% of the tumors the levels of progesterone receptor were >3 fmole/mg cytosol protein. No relationship could be demonstrated between the levels of progesterone receptor and the stage of the disease, the histological grade of the tumor, nor the age of the patient. Thirty-one cases were studied for the presence of both steroid hormone receptors and 52% of these tumors could be classified as both estrogen and progesterone receptor positive. Preoperative radiation appeared to interfere with the determination of the tumor’s receptor status.

INTRODUCTION

Steroid hormone receptor analysis in target tissues (i.e., uteri, vagina, breast tumors) of laboratory animals has been the subject of intense study during the last 20 years [l-6, 13, 141. The aim of the investigations has been to discover the control mechanisms of hormone-mediated cell division. During the last decade this knowledge has been applied to clinical medicine. Steroid hormone receptors have been the subject of much research in carcinoma of the breast but only a few studies have focused on adenocarcinoma of the endometrium. The presence of estrogen and progsterone receptors in human endometrial tissue has been demonstrated by numerous investigators [ 15, 16, 18, 191. Studies throughout the menstrual cycle have demonstrated that proliferative endometrium contains a high level of estrogen receptors but in secretory endometrium the level of estrogen receptors is markedly reduced [18, 251. For progesterone receptors the pattern is different with low levels in the proliferative phase, high levels at midcycle, and then lower levels in the secretory phase [16, 17, 19, 261. Whether there are changes in the levels of hormone receptor in endometrial 152

0090~8258/80/050152-lO$Ol.OO/O Copyright All rights

@ 1980 by Academic Press, Inc. of reproduction in any form reserved.

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carcinoma is the basis of this investigation. If endometrial carcinoma is under hormonal control then these tumors may be treated and possibly controlled by hormone or antihormone therapy. RECEPTORS

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Many investigators have reported that only 30 to 35% of patients with metastatic endometrial carcinoma will have an objective response to progestational agents [7-101. However, patients with metastatic carcinoma of the endometrium are now being treated unselectively with progestational agents and therefore two-thirds of these patients are being subjected needlessly to hormone therapy. Fortunately, progestational therapy has minimal side effects, but such empirical therapy leads to a delay in instituting other potentially effective modalities of treatment such as chemotherapy, irradiation, and surgery. Obviously, a search for a more rational means of selection of patients for endocrine therapy is needed. The relationship between steroid hormone receptors and carcinoma of the breast has been the subject of much basic and clinical research and enormous progress has been made in the use of steroid hormone receptors to select breast cancer patients for endocrine manipulative therapy [ 12, 291. During the past decade studies have been published sporadically about steroid hormone receptors and adenocarcinoma of the endometrium [ 19-281. However, such studies have lagged behind similar investigations relating to breast cancer. In the past 5 years we have measured estrogen receptor levels in 73 patients and progesterone receptor levels in 31 patients with adenocarcinoma of the endometrium, and the results of these studies are presented in this paper. MATERIALS

AND METHODS

Between November 1974 and July 1979, the uteri from patients undergoing a total abdominal hysterectomy for adenocarcinoma of the endometrium at the University of Massachusetts Teaching Hospital and the Worcester Memorial Hospital have been studied for the presence or absence of estrogen receptors, and in 1977 the study of progesterone receptors was added to the project. Seventythree patients ranging from 35 to 77 years of age were included in the study. Table 1 summarizes the clinical material in this study. TABLE ADENOCARCINOMA

1 OF THE ENDOMETRKJM

Stage Grade

0

Atypical endometrial hyperplasia

13

Grade I II III Total

13

IA

IB

II

III

Total 13

15 9 2

7 7 8

3 5 2

1

26 22 12

26

22

10

2

73

Note. Summary of clinical material in this study.

1

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HUNTER,

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JORDAN

The methods of treatment of adenocarcinoma of the endometrium included total abdominal hysterectomy and bilateral salpingo-oophrectomy for Stage IA, Grade I cases and preoperative irradiation followed by total hysterectomy and bilateral salpingo-oophorectomy for all other cases of Stage I. Stage II cases also received preoperative irradiation followed by the same surgery. Stage III and IV cases were individualized. Suspicious pelvic and para-aortic nodes were removed. Preoperative irradiation consisted of 5000 mg/hr of radium equivalent cesium- 137 in the uterine cavity and 4000 rad to the surface of the vagina followed in 48 hr by surgery. Stage II and Stage IB cases that consisted of uteri larger than 12 cm were treated with external radiation first followed by intracavitary implantation and the surgery was performed 6 weeks later. Postoperative radiotherapy was administered in those cases with greater than one-third myometrial invasion of the tumor and in those cases that had positive pelvic or para-aortic nodes. Immediately following surgical removal, the tumor was separated from the myometrium and frozen in liquid nitrogen. An additional sample was used for microscopic determination of pathology and the remainder of the tissue was assayed for estrogen receptors, as previously described [29]; using sucrose density gradient analysis and diethylstilbestrol as cold competitor. Protein concentration was measured by the method of Lowry [32]. The total sites are expressed as femtomoles per milligram of cytosal protein, and an assay of greater than 3 is considered positive. Progesterone receptors were measured using the method of Horwitz and McGuire [3 l] with minor modifications. The radiosteroid used was 3H-R5020 (17, a2 1-dimethyl- 19-nor-4, 9-pregnandine-3, 20-dione-( 17-methyl-3HO; 87 Ci/mmole) and nonradioactive R-5020 was used as “cold” competitor. Cortisol, 5.5 x 1O-6 M, and 5o-dihydrotestosterone, 2 x lo-‘, were added in excess to all tubes to occupy sites on cortisol-binding globulins and sex steroid-binding globulin and androgen receptors. These data were analyzed as follows: the relationship between the number of receptors and the age was analyzed using linear regression analysis [33, p. 1351; the grade and stage of the lesion were compared to receptor status positive >3 fmole/mg cytosol protein, or negative <3 fmole/mg cytosol protein by a multiple contingency test [34, p. 2281; the effect of preoperative radiation on positive or negative receptor status was determined by the x2 test [35, p. 2121. RESULTS

1. Estrogen Receptor Determination Figure 1 shows a sucrose density-gradient analysis with cpm plotted against tube number of an estrogen receptor-positive tumor containing 249 fmole/mg cytosol protein. Most of the sites sedimented at 8 S. The determination of estrogen receptor levels in the cytoplasm of 73 patients with adenocarcinoma of the endometrium indicated that 52 cases, 71%, were estrogen receptor positive, i.e., >3 fmole/mg. However, there was no specific relationship between the stage (Table 2) or grade (Table 3) of the tumor and estrogen receptor positivity. We could detect no relationship between the age of the patient and the number of estrogen receptors (Fig. 2).

RECEPTORS

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OF THE

155

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RECEPTOR

12W-

249 fm/mg

0 aoltm

10 FRACTlON*~“MBER

30

FIG. 1. Sucrose density gradient of tumor cytosol incubated with [3Hlestradiol plotted against fraction number.

TABLE 2 ESTROGEN RECEFTORLEVELSACCORDINGTO

STAGE

Estrogen receptor

Total

No. patients

Stage

Positive

Negative

13 48 9 3 0

0 I II III IV

11 (85) 32 (67) 7 (78) 2 (67) 0

2 16 2 1 0

52 (71)

21 (29)

73

Note. An assayof 3 or more fmolelmg of cytosol protein in the 8 S region is considered positive. ( ) = Percent.

TABLE 3 CORRELAT~ONOFHISTOLOGICALGRADEOFADENOCARC~NOMAOFTHEENDOMETRIUMAND ESTROGEN RECEPTORLEVELS

Estrogen receptor Grade Atypical endometrial hyperplasia Grade I II III

Total

No. patients

Positive

Negative

13

11 (85)

2

26 22 12

18 (67) 15 (68) 8 (73)

9 7 3

73

52 (71)

21 (29)

Note. An assay of 3 or more fmoleimg of cytosol protein in the 8 S region is considered positive. ( ) = Percent.

156

HUNTER,

LONGCOPE,

AND

JORDAN

.

heFIG. 2. Graph of estrogen receptor concentration in fmole/mg cytosol protein plotted against age of patient.

2. Progesterone

Receptor Determination

Figure 3 shows sucrose density gradient analysis with cpm plotted against tube number of a progesterone positive tumor containing 23 fmole/mg protein. As for the estrogen receptor most of the sites sedimented at 8 S. Thirty-one cases of adenocarcinoma of the endometrium were studied for the presence of progesterone receptors in the cytoplasm. Sixteen cases or 52% revealed progesterone receptor sites in excess of 3 fmole/mg of tissue protein and were classified as progesterone receptor positive (Table 4) but analysis of the cases by stage (Table 4) or grade (Table 5) indicated that neither was a factor in whether the tumor was positive or negative with regard to progesterone receptors. There was no correlation between the age of the patient and the number of sites (Fig. 4). Analyses of those cases that had both estrogen and progesterone receptor levels measured revealed no tumors with a positive progesterone receptor assay without a positive estrogen assay. Seventy-one percent of this group could be classified as estrogen receptor positive, 52% progesterone receptor positive; therefore, 52% PFifXESTERONE 8s

RECEPTOR

23 fm/mg

2

0 Worn

10

30 FRACTI&“N”MBER

40 TOP

FIG. 3. Sucrose density gradient of tumor sytosol incubated with 3H-R5020. cpm of 3H-R5020 plotted against fraction number.

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TABLE 4 ADENOCARCINOMA OF THE ENDOMETRIUM AND PROGESTERONE RECEPTOR LEVELS ACCORDING TO STAGE

Progesterone receptor Stage

Positive

No. patients

0 I II III IV

5 21 3 2 0

Total

31

Negative 1 12 1 1

4 9 2 I

52 (48)

15 (52)

Note. An assay of 3 or more fmole/mg cytosol protein in the 8 S region is considered positive. ( ) = Percent. TABLE 5 CORRELATION OF HISTOLOGICAL GRADE OF ADENOCARCINOMA OF THE ENDOMETRIUM AND PROGESTERONE RECEPTOR LEVEL

Progesterone receptor Grade Atypical endometrial hyperplasia

No. of patients

Positive

Negative

5

4 (80)

1

Grade I II III

7 16 3

3 (43) 8 (50) 1 (33)

4 8 2

Total

31

16 (52)

15 (48)

Note. An assay of 3 or more fmole/mg of cytosol protein in the 8 S region is considered positive. ( ) =

Percent.

FIG. 4. Graph of progesterone receptor concentration in fmoleimg cytosol protein plotted against age of patient.

158

HUNTER,

LONGCOPE, AND JORDAN TABLE 6

EFFECT OF PREOPERATIVE IRRADIATION ON STEROID HORMONE RECEPTOR DETERMINATIONS IN CARCINOMA OF THE ENDOMETRIUM

Estrogen receptor

Irradiated Nonirradiated

Progesterone receptor

Positive

Negative

Positive

Negative

22 (59) 30 (83)

15 (41) 6 (17)

6 (33) 10 (77)

12 (67) 3 (23)

Note. An assay of 3 or more fmole/mg of cytosol protein in the 8 S region is considered positive. ( ) = Percent.

contained levels high enough to be considered both estrogen and progesterone receptor positive. 3. Effect of Preoperative Irradiation of Steroid Hormone Receptor Levels Many of our cases were subjected to radiation therapy before the hysterectomy was performed and the receptor status of these are shown in Table 6. There were significantly fewer estrogen and progesterone receptor-positive tumors in the irradiated than in the nonirradiated group. DISCUSSION Clinical investigation into hormonal therapy for endometrial cancer lags far behind breast cancer research. Estradiol receptor and progesterone receptor concentrations have not been measured in a large number of specimens and correlations with the effectiveness of hormonal therapy have not been established that can serve as a basis for the type of predictive test as used in breast cancer. In our group of patients with adenocarcinoma of the endometrium, 71% of the cases were classified as estrogen receptor positive, 52% as progesterone receptor positive, and 52% as both estrogen and progesterone positive and therefore 71% could be considered to have hormone-dependent tumors. These percentages are considerably higher than the 30-35% predicted on the basis of clinical results of the treatment of metastatic endometrial carcinoma with progestational agents [7-103. Well-differentiated adenocarcinomas of the endometrium are more likely to respond to progesterone therapy than poorly differentiated tumors. The next logical step would be to conclude that well-differentiated tumors are more likely to contain higher estrogen and/or progesterone receptor levels than poorly differentiated tumors. Our studies indicated, however, that the correlation between positive receptor tumors and tumor differentiation was not statistically significant although the atypical endometrial hyperplasia group did have more cases that were receptor positive. This conclusion differs with some other investigators [26] but may reflect the relatively small number of subjects in all series as well as differences in expressing differentiation. If this fact is true, then one would ask why do not poorly differentiated tumors respond as well to presterone therapy. Richart’s electron microscopy studies of endometrial carcinoma revealed that as a tumor cell becomes less well differentiated and further from the normal morphological structure of the normal cell, the less

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apt the cell is to respond to hormone therapy [34]. A poorly differentiated tumor may contain many steroid hormone receptors but the cancer cell will no longer respond because of the morphological changes that have occurred in becoming a poorly differentiated cell. Therefore, one cannot classify these tumors as hormone dependent or not, completely on the presence or absence of hormone receptors. Only future clinical investigation will determine the correlation between the presence of steroid hormone receptors and clinical response to hormone manipulation. Although for breast cancer tumors there appears to b some correlation between the age of the patient and the number of estrogen receptor sites, we could not find any significant correlation between age and number of estrogen or progesterone sites in these endometrial carcinoma cases. This may, in part, be related to the few endometrial carcinomas in the younger age groups. Studies with progesterone receptor levels may reveal the same clinical significance as in breast carcinoma. Breast carcinoma investigators have shown that breast tumors that are both estrogen and progesterone receptor positive are more likely to respond to hormone manipulation than if only estrogen receptors are present. Our studies indicated that only 52% of the endometrial cancers contained significant levels of both steroid hormone receptors, a figure much closer to the 30 to 35% progesterone responders with metastatic endometrial cancer than the figure of 71% of patients whose tumors contained only estrogen receptors. It would appear logical that only tumors with progesterone receptors would respond to progestational agents. However, other antiestrogenic compounds such as testosterone and tamoxifen can enter the cell by way of the estrogen receptor and exert deleterious effects on the cell reproductive cycle similar to progestational agents. Therefore, the measurement of both types of steroid hormone receptors is important and perhaps the hormone therapy for endometrial carcinoma should be both progestational compounds and other antiestrogenic compounds that enter the cell by a different pathway. Future clinical studies will answer these questions. All tumors that were progesterone receptor positive were also estrogen receptor positive, but there was no statistically significant correlation between the number of estrogen and progesterone receptors. Although the number of observations is small, no relationship could be shown between receptor-positive and -negative tumors and either the stage or histological grade. Preoperative radiation appeared to decrease the percentage of receptor positive tumors, both estrogen and progesterone. This apparent difference in tumor status may be due, in part, to the fact that atypical endometrial hyperplasia cases were not irradiated. However, even after these cases are removed from analysis, the difference remains. It would appear, therefore, that preoperative irradiation may interfere with receptor classification, and it may be advisable in the future to obtain tissue for receptor analysis prior to any irradiation whenever possible. Investigations are now underway to develop a technique of determining steroid hormone receptors on curettings which, when available, of course, will provide a more “pure” tumor for receptor analysis. The prognostic and therapeutic value of receptor analyses in endometrial carcinoma will have to await long-term follow-ups of large numbers of patients. From this and other [23-261 relatively small series it would appear that some 50 to 70%

160

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of patients will be receptor positive, a percentage close to that reported for breast carcinoma. However, only 30 to 35% of patients have been reported to respond favorably to progestational therapy [7-lo]. It thus appears that approximately 50% of receptor-positive patients will be unresponsive to such therapy and, as for breast cancer patients, major efforts should be directed to identify this population. The only practical value of steroid receptors at the present time is that with this knowledge of the original tumor if a recurrence or a metastases occurs, then therapy can be planned upon the presence or absence of steroid hormone receptors. If the tumor is hormone dependent, then one would treat the patient immediately with hormone therapy such as progesterone, tamoxifen, testosterone, etc. If the tumor is not hormone dependent then one should treat immediately with chemotherapy. That is not to say, of course, that in any case of metastatic or recurrent endometrial carcinoma, the case should not be studied individually for the possibility of using radiation or surgical therapy as well as hormone and chemotherapy. Knowledge of the steroid hormone receptors in any event may help us plan therapy more intelligently. ACKNOWLEDGMENTS

The authors would like to acknowledge the excellent assistance of Isabel Rafkind and Kim Crisfield in the receptor analysis and Joyce Sirois in the correlation of clinical and laboratory data. A portion of this study was supported by Grant AG-00058. REFERENCES 1. Jensen, E. V., and Jacobson, H. I., in Biological activities of steroids in relation to cancer (G. Pincus and E. P. Vollmer, Eds.), Academic Press, New York, pp. 161-178 (1960). 2. Gorski, J., Taft, D., Shyamala, G., Smith, D., and Notides, A. Hormone receptors. Studies on the interaction of estrogens with the uterus, Rec. Progr. Harm. Res. 24, 45-80 (1968). 3. Jensen, E. V., Suzuki, T., Kawashima, T., Stumpf, W. E., Jungblut, P. W., and De Sombre, E. R. A two-step mechanism for the interaction of estradiol with rat uterus, Proc. Nat. Acad. Sci. USA 59, 632-638 (1968). 4. Jensen, E. V., Suzuki, T., Numata, M., Smith, S., and De Sombre, E. R. Estrogen-binding substances of target tissues, Steroids 13, 417-427 (1%9). 5. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and De Sombre, E. R., in Estrogen target tissues and neoplasia (T. L. Dao, Ed.), Chicago Univ. Press, Chicago, p. 23 (1971). 6. Shyamala, G., and Gorski, J. Interrelationship between estrogen receptors in the nucleus and cytosol, J. Cell. Biol. 35, 125A (1%7). 7. Anderson, D. G. Management of advanced endometrial adenocarcinoma with medroxyprogesterone acetate, Amer. J. Obstet. Gynecol. 92, 87-99 (1%5). 8. Kelley, R. M., and Baker, W. H. The role of progesterone in human endometfial cancer, Cancer Res. 25, 1190 (1965). 9. Kennedy, B. J. Progestogens in the treatment of carcinoma of the endometrium, Surg. Gyneco/. Obstet. 127, 103 (1968). 10. Reifenstein, E. C. Hydroxyprogesterone caproate therapy in advaced endometrial cancer, Cancer 27, 485-502 (1971). 11. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and DeSombre, E. R. Estrogen receptors and breast cancer response to adrenalectomy, Nat. Cancer Inst. Monogr. 34, 55 (1971). 12. Jensen, E. V. Estrogen receptors in hormone-dependent breast cancers, Cancer Res. 35, 3362 (1975). 13. Shyamala, G., and Gorski, J. Estrogen receptors in the rat uterus. Studies on the interaction of cytosol and nuclear binding sites, J. Biol. Chem. 224, 1097-l 103 (1969).

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14. O’Malley, B. W., Means, A. R., and Sherman, M. R. Mechanism o action of progesterone: Regulation of gene transcription, in The sex steroids (K. W. McKems, Ed.), AppletonCentury-Crofts, New York, p. 315 (1971). 15. Cracker, S. G., Milton, P. J., and King, R. J. B. Uptake of (6,7-3H) oestradiol-178 by normal and abnormal human endometrium, J. Endocrinol. 62, 145 (1974). 16. Dyer, R. D., Sarto, G. E., and Colas, A. E. Binding of progesterone by human uterine cytosol, J. Clin. Endocrinol. Metabol. 43, 1211 (1976). 17. Bayard, R., Louvet, J. P., Monrozies, M., Boulard, A., and Pontonnier, G. Endometrial progesterone concentrations during the menstrual cycle, J. Clin. Endocrinol. Metabol. 41,412 (1975). 18. Hunter, R. E., and Jordan, V. C. Detection of the 8s oestrogen-binding component in human endometrium during the menstrual cycle, J. Endocrinol. 65, 757 (1975). 19. Bayard, F., Kreitman, B., and Derache, B. Measurement of the progesterone receptor in human endometrium using progesterone and R 5020, in Progesterone receptors in normal and neoplastic tissues (W. L. McGuire, J. P. Raynaud, and E. -E. Baulieu, Eds.), Raven Press, New York (1977). 20. Ehrlich, C. E., Cleary, R. E., and Young, P. C. M. The use of progesterone receptors in the management of recurrent endometrial cancer, in Endometrial cancer (M. G. Brush, R. J. B. King, and R. W. Taylor, Eds.), Bailhere-Tindall, London, p. 258 (1978). 21. Gurpide, E., Gusberg, S. B., and Tseng, L. Estradiol binding and metabolism in human endometrial hypernlasia and adenocarcinoma. .I. of Steroid Biochem. 7, 891 (1976). 22. Gurpicte, E., and Welch, M. Dynamtcs of uptake of estrogens and androgens in human enaometrium, J. Biol. Chem. 244, 5159 (1969). 23. Gustafsson, J. -A., Einhom, N., Elfstrom, G., Nordenskjold, B., and Wrang, 0. Progestin receptor in endometrial carcinoma, in Progesterone receptors in normal and neoplastic tissues (W. C. McGuire, J. P. Raynaud, and E. -E. Baulieu, Eds.), Raven Press, New York (1977). 24. Haukkamaa, M., Karjalainen, O., and Luukkainen, T. In vitro binding of progesterone by the human endometrium during the menstrual cycle and by hyperplastic, atrophic and carcinomatous endometrium, Amer. J. Obstet. Gynecol. 111, 205 (1971). 25. Pollow, K., Lubbert, H., Boquoi, E., Kreuzer, G., and Pollow, B. Characterization and comparison of receptors for 17/3-estradiol and progesterone in human proliferative endometrium and endometrial carcinoma, Endocrinology %,3 19 (1975). 26. Pollow, K., Schmidt-Gollwitzer, M., and Nevinny-Stickel, J. Progesterone receptors in normal human endometrium and endometrial carcinoma, in Progesterone receptors in normal and neoplastic tissues (W. L. McGuire, J. P. Raynaud, and E. -E. Baulieu, Eds.), Raven Press, New York, pp. 313-338 (1977). 27. Terenius, L., Lindell, A., and Persson, B. H. Binding of estradiol-17/3 to human cancer tissue of the female genital tract, Cancer Res. 31, 1895 (1971). 28. Young, P. C. M., Ehrlich, C. E., and Cleary, R. E. Progesterone binding in human endometrial carcinomas, Amer. J. Obstet. Gynecol. 125, 353 (1976). 29. McGuire, W. L. Estrogen receptors in human breast cancer, J. Clin. Invest. 52, 73-77 (1973). 30. McGuire, W. L., and DeLaGarza, M. Improved sensitivity in the measurement of estrogen receptor in human breast cancer. J. Clin. Endocrinol. Metabol. 37, 986-989 (1973). 31. Horwitz, K. B., and McGuire, W. L. Specific progesterone receptors in human breast cancer, Steroids 25, 497 (1975). 32. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 33. Snedecor, G. W., and Cochran, W. G. (Eds.), Statistical methods, 6th ed. Iowa State Univ. Press, Ames, pp. 135, 212, 228 (1967). 34. Richart, R. M., and Ferenczy, A. Endometrial morphologic response in hormonal environment, Gynecol. Oncol. 2, 180-197 (1974).