Estrogen Receptor and Natural Course of Breast Cancer AMNUAY SINGHAKOWINTA, M.D., HENRY G. POTTER, B. A., THOMAS R. BUROKER, D.O., BOHUMIL SAMAL, M.D., SAM C. BROOKS, PH.D., VAINUTIS K. VAITKEVICIUS, M.D.
The tumors from approximately 50% of patients with breast From the Department of Oncology, cancer contained estrogen receptor (ER). ER appeared more often Wayne State University School of Medicine and at higher levels in the tumors of postmenopausal women. and the Milton A. Darling Memorial Center Eleven out of 12 patients who had multiple ER assays from of the Michigan Cancer Foundation at The various metastatic sites showed no significant discrepancies in ER Grace Hospital, Detroit, Michigan values. ER level appears to decrease as the duration of metasta#.c cancer increase. Patients with ER in the tumor more frequently have bone metastases than those without ER. Visceral metastases only the activator for this receptor is not known at the occurred more often with ER negative patients and appeared to have a more malignant course with significant shorter survival. present time.
IOCHEMICAL MECHANISMS of hormone-target organ interaction have been recognized in the past several years. Polypeptide hormones and epinephrine react with
target tissue by activation of membrane-bound adenyl cyclase which result in formation of cyclic AMP.7 On the other hand steroid hormones appear to enter the target cells and bind to a specific extranuclear receptor.4 Among the various steroid hormones, estrogen was given the most attention since it has been shown for several years to be related to animal and human breast cancer. Jensen and DeSombre3 characterized estrogen receptor (ER) as a protein with the sedimentation coefficient of 8S which probably appears in the cytoplasm of target cell as 4S subunit. This estrogen receptor interacts with estradiol in the cytoplasm and undergoes transformation which is a temperature-dependent process. This transformed receptor-estrogen complex, which has a sedimentation coefficient of 5S, migrates to the nucleus and binds to the chromosome in the nucleus resulting in newly transcribed RNAs from the nucleus of target tissues. Whether this extranuclear receptor is merely a means of transporting the hormone into the nucleus or receptor protein itself is the active agent and hormone is
Breast cancer tissue, both primary and metastatic, has been shown by several investigators6 to contain ER. Some clinical aspects of human breast cancer associated with the presence or absence of ER in their tumor have been reported.6 We are reporting here additional information regarding the natural course of breast cancer and ER in the tumor. Materials and Methods
Breast cancer specimens both from primary sites and metastatic sites were obtained at surgery and frozen immediately; malignancy was verified by histological examination. All specimens were kept initially at -20 C, then transported to our laboratory in dry ice and stored at -70 C for longer periods. The tissues were processed and assayed for estrogen receptor by gel-filtration technique as previously described.2 One hundred milligrams of tissue were homogenized by a Willems Polytron in 10 mM Tris buffer, pH 7.4, containing 1.5 mM MgCl2, 10 mM KCI, and 5 mM reducing agent, dithiothreitol. After spinning in a Spinco model L ultracentrifuge at 15,000 g for 15 minutes, the resulting supernatant fluid was spun at 100,000 g for 65 minutes, 4 C, using a SW 50.1 rotor and buckets equipped with adaptors (Beckman 305527). Submitted for publication August 5, 1975. The supernatant fraction was diluted 1:1 with 10 mM Reprint Requests: A. Singhakowinta, M. D., Milton A. Darling Tris buffer, pH 8.5, containing 10 mM KCI, 5 mM Memorial Center, 4160 John R Street, Detroit, Michigan 48201. and mM EDTA (column buffer) and Dex1 dithiothreitol Supported in part by Clinical Cancer Research Center grant No. CA 07177 and grant No. T12 CA 08096 from the National Cancer Insti- tran Blue added. Protein was determined by the Folin tute, United States Public Health Service. method.5 Immediately following centrifugation, 0.4 ml of
84
Vol. 183 * No.
NATURAL COURSE OF BREAST CANCER
the diluted supernatant fraction was incubated with 10 ,ul of (2,4,6,7-3 H)-estradiol-17,B (100 Ci/mmole) standard for 2 hours at 4 C. A duplicate sample was also incubated with tritiated standard plus 100-fold excess of unlabeled estradiol-17/3. Upon completion of incubation, each sample was chromatographed through a separate column containing 3-4 g Sephadex G-25 previously equilibrated in "column buffer." Flow rates were kept constant, and the void volume (5 ml, indicated by the Dextran Blue) was collected and extracted 3 times with ethyl acetate. An aliquot of the extract was counted in a scintillation spectrometer equipped with automatic external standardization. Routine saturation curves were constructed for incubations with tritiated estradiol-17,/ and with labeled estrogen plus excess unlabeled estradiol-17f3. Receptor levels were determined from the difference between total binding capacity at saturation of tritiated estradiol17,3 and non-specific binding (residual binding in the presence of unlabeled estrogen). Occasionally Scatchard plots were used to calculate the binding constants of the estrogen receptor.2 The level of 2.1 femtomoles (fmoles, 10-15 moles) estradiol bound per mg cytoplasmic protein or over was considered to be positive for estrogen receptor. This level or higher has been shown by us to correlate well with endocrine therapy response in disseminated breast cancer.6 Medical records of patients with diagnosis of breast cancer who had ER study on their tumor were reviewed. Patients with simultaneous bilateral breast cancer and patients with other active malignancy in addition to breast cancer were excluded. A total of 133 cases were found to be evaluable based on the above criteria. Most of the patients had an ER assay done only once; in 12 patients multiple ER assays were performed. In three of these 12 patients, the re-biopsy for ER test was carried out to study the effect of exogenous estrogen on the ER values. Specific information regarding date of onset of symptoms, menstrual status at the time of ER assay, and the date and cause of death were determined. The sites of metastasis were divided into three categories as follows: viscera (includes liver, lung, pleura, CNS, spinal cord, and all intra-abdominal and intrathoracic organs), bone, and soft tissues (skin, opposite breast, lymph nodes). The term dominant site of metastasis is used to denote the category which is most extensively involved by cancer. In case of equal degree of multiple system involvement, the following priority of assignment was used: visceral, bone, soft tissue. The disease free interval (DFI) is defined as the time interval between initial diagnosis of breast cancer and the clinical diagnosis of disseminated cancer. The duration of disease was measured from the onset of cancer to the time of death.
85
TABLE 1. ER Value in Pr-e tanid Postm7letiopaiusal Patients
Premenopause
Postmenopause
31 14 45.2 23.4 7.0 0
102 54 53 39.7 12.9 12
Total number of patients Positive ER patients Percentage of positive ER Mean ER value (femtomole)* Median ER value Patients with multiple assay *
P = 0.40 by Student's t-test.
Results
Of 133 patients, 102 were postmenopausal women and 31 were premenopausal at the time the assay for ER was done. Sixty-eight patients or 51% had tumor which contained ER. As shown in Table 1, 53% of postmenopausal and only 45% of premenopausal patients had tumors which were positive for ER. The level of ER also appeared to be higher in postmenopausal cases. The majority of patients under this study had metastatic breast cancer at the time ER assay was performed. Only 26 patients had ER study from primary breast cancer lesions while the disease was considered to be curable. However, 10 of them subsequently had recurrent carcinoma. Most of the patients had more than one organ involvement but the biopsy for ER study in general was carried out from the accessible lesions, i.e. skin, lymph nodes, breast masses. Biopsies from bones were possible when lesions were superficial such as from iliac crest area or more commonly when orthopedists operated on patients for various fractures. In many cases multiple visceral biopsies were obtained during surgery either for therapeutic purposes such as bilateral adrenalectomy or for complications arising from cancer such as intestinal obstruction. Table 2 indicates the sites from where the biopsy for each patient was obtained. It is not necessarily the only site of carcinoma involvement nor the dominant site of metastasis that the particular patient had. For example when a patient had recurrent breast cancer of the chest wall, bone, and with massive liver involvement, the biopsy for ER study was usually taken from the chest wall. The site of biopsy will be listed under soft tissue but the dominant site of metastasis will be classified as viscera. Twelve patients had multiple biopsies. All except one had no significant discrepancy in ER value from various TABLE 2. Resuilts of ER Values from Variou.s Sites Sites of Biopsy*
Positive
Negative
Primary breast lesions Soft tissue Bone Viscera
21 40 6 2
27 35 1 3
* See text.
86
SINGHAKOWINTA AND OTHERS TABLE 3. Disease Free Interval
Free of disease Inoperable cases Mean DFI* Median DFI *
Positive ER 68 pts
Negative ER 65 pts
8 10 37.6 27.5
9 16 31.2 months 21.5 months
P = 0.4 by Student's t-test.
sites of metastatic lesions, i.e. when the lesion from the skin was positive for ER, the lesions from lymph node and bone also were positive although the ER value from various sites may not be exactly the same. The one patient with a discordant ER value had ER of 31.6 fmoles from her primary breast lesion. Fifty-four months later the ER value obtained from a bone lesion was 3.5 fmoles but a negative ER value was found in a metastatic hepatic nodule. In Table 2 she was counted three times for the site of biopsy: primary, bone, viscera. The rest of the patients were counted once based on the first site of biopsy. Disease Free Interval
Ann.
Surg. * January
1976
tient had ER assay prior, during and 4 months after DES therapy. The ER values were 3.3, 0 and 11.5 fmoles/mg protein respectively. The other two patients each had ER assays while on DES and repeat assay after DES treatment was discontinued. One patient's ER level rose from 0 to 2.2 and the other went from 4.8 to 61 fmoles/mg protein after discontinuation of DES for 4 and 7 months respectively.
Survival Data
Forty-eight patients had expired as of December 15, 1974; 21 were positive for ER and 27 were negative.
Table 4 demonstrates the total duration of disease from
onset of cancer to death in each category. All but two
died of metastatic breast cancer; both had ER in their tumor. One died of toxicity from chemotherapy and the other died from pulmonary embolism 2 weeks after androgen therapy was initiated. Nine patients with negative ER had rapidly progressive disease which compelled the managing physicians to bypass endocrine treatment and initiate chemotherapy as the first systemic treatment for disseminated cancer. The difference in survival between ER positive and ER negative patients is statistically significant in favor of the ER positive group.
Seventeen patients are still free of recurrent cancer. Twenty-six patients presented with inoperable breast Comment cancer; 16 were ER negative patients. Therefore, in a Lower ER level in the premenopausal group is probably total of 43 patients the DFI cannot be determined. In due to partial saturation of the receptor sites by endogthe remaining 90 patients DFI could be calculated but no significant difference was observed between the positive and negative ER groups as illustrated in Table 3. 100POSITIVE ER NEGATIVE ER
90-Q
Dominant Site of Metastasis
80A total of 116 patients had recurrent or inoperable breast cancer. Fig. 1 shows a preponderance for bone 70metastasis in the positive ER group while in the negative ER group visceral metastasis appears dominant. The difference is highly significant with the P-value of 0.0001. PERCENT 60-
Patients with Multiple ER Assay Of 12 patients with multiple biopsies, 11 had concordant ER value regardless of the site of the biopsy. Three were negative for ER and 8 were ER positive. There were 6 patients who were ER positive subsequently rebiopsied after a period of 6 to 54 months. All were found to have decreasing values of ER except for one who displayed an increase in ER 11 months after bilateral adrenalectomy as shown in Table 5. None of these 6 patients received estrogen or androgen during the 6 months preceding the second biopsy. There were three patients who had ER assay performed while on Diethylstilbestrol (DES) 15 mg/day. One pa-
OF PATIENTS
49
0
4030an
J
16
SOFT TISSUE
r
SONE
VISCERA FIG. 1. Comparison of dominant site of metastasis between ER positive and ER negative group. More bone metastasis occurs in ER positive
and more visceral metastasis in ER negative groups. Fisher test for P = 0.001 for bone vs viscera and P = 0.01 for soft tissue vs viscera.
Vol. 183 . No. I
NATURAL COURSE OF BREAST CANCER
estrogen. The study of ER values in three patients while on and off therapy with DES seemed to be in agreement with this hypothesis, although the dose of DES used was much higher than the physiological amount of estrogens present in normal premenopausal women. It has been suggested that the critical level of ER in postmenopausal women to be considered as positive should be higher than premenopausal patients. On the other hand, several investigators6 reported good correlation of response to endocrine therapy in patients with borderline ER value. The interpretation of an ER value in a patient with disseminated cancer who is receiving or recently received estrogen therapy must be interpreted with caution because of the possibility of a false negative ER value. The binding between estrogen and receptor protein is strong. This tight binding appears to result from a slow rate of dissociation.8 How much time is required for the receptor sites to be freed up again after discontinuation of estrogen treatment is not known. The three patients who had re-biopsies for ER assays after interruption of DES for periods of 4 to 7 months showed increased ER levels. It is quite conceivable that less than 4 months is required for the receptor sites to become unsaturated. The fact that 11 out of 12 patients who had multiple ER determinations which were concordant would suggest that in most cases single biopsy of an accessible lesion is adequate for ER study. However when a change in endocrine therapy is contemplated, a repeat biopsy of an accessible lesion, when available, should be performed. This is to insure the presence of hormonal dependent cells in the tumor prior to institution of a new form of endocrine therapy. This is particularly important when major surgery such as bilateral adrenalectomy or hypophysectomy are involved. The fact that 5 of6 patients in this study showed a progressive decrease in ER value as the duration of metastatic disease increased, as shown in Table 5, appears to support the hypothesis that a patient whose tumor contains ER actually has both hormonal dependent cells and independent cells but in different proportions. After receiving endocrine manipulation, the hormonal dependent cells are destroyed allowing the hormonal independent cells to proliferate. The alternative explanation is the continuous existence of hormonal dependent cells, but ability to produce ER is lost after endocrine treatment. On the other hand, a patient with no ER in the tumor probably has few or none of hormonal dependent cells in their tumors. The clinical course of disseminated breast cancer, in general, correlates with the sites of metastasis and DFI. Patients with a long DFI or metastatic involvement of skin, lymph nodes, or bone without visceral metastasis are found to have a longer survival compared to those with visceral metastasis.1 Our ER study seems to coincide with the above clinical observations, with the exception of the prognostic role of DFI for which there was no
87
TABLE 4. Duration of Disease
enous
Positive ER 21
Mean* Median
56.7 54 1 5 15 3
Chemotherapy only
Endocrine therapy only Endocrine and chemotherapy Inoperable case at initial diagnosis *
cases
Negative ER 27
cases
29.6 months 23 months 9 3 15 8
P < 0.01 by Student's t-test.
difference between positive and negative ER groups. Patients with ER negative tumors more frequently have visceral involvement, whereas those with ER positive have predilection for bone involvement. The survival was significantly shorter in the ER negative group. Because the inoperable breast cancer in the ER negative group outnumbered those in the ER positive at a ratio of 8 to 3, one may raise the question that the poorer prognosis in the ER negative group could have been the result of excessive patient procrastination in seeking medical treatment. However, in review of the medical records in those 11 cases who presented with inoperable breast carcinoma, the median delay in getting medical attention was 5 months (range 1-12) in the ER negative group as contrasted with 12 months (range 1-13) in the positive ER group. Moreover, 5 out of 8 patients in the negative ER group, at the time of initial diagnosis, had visceral metastases in addition to breast lesions. However, only one out of 3 in the positive ER group had visceral involvement at the time of diagnosis of breast cancer. These findings tend to support the hypothesis that the biological behavior rather than procrastination by the patients is the reason for shorter survival in ER negative patients. We feel that ER study should be done in every patient with breast cancer regardless of operability. A significant number of patients who undergo potentially curative mastectomy will eventually have recurrent cancer. If cancer recurs in bones or viscera, frequently the biopsy for ER assay cannot be done either because of technical TABLE 5. ER Level in Patients with Multiple Sequential Assay
Name
1st and 2nd ER level fmoles
E.F. W.B. V.T. H.B. G.G. G.T.
31.6 80.4 32 41.3 14.6 14.2
3.5 8.3 0.9 5.4 6.5 80.7
Treatment between 2 ER
Duration between 2 ER (in mos.)
Adr., chemo.* Adr. Adr. Adr. Adr. Adr.
* Adr. = bilateral surgical adrenalectomy. t L.N. = lymph node.
54 7 6 8 7 11
M M M M M M
Sites of biopsy
Primary, bone Skin, skin Skin, skin L.N., L.N.t L.N., L.N. L.N., L.N.
Ann. Surg. * Januzu-% 1976
SINGHAKOWINTA AND OTHERS
88
or ethical reasons. The only tissue available for ER assay in these individuals would be the primary lesions. It is proposed that ER study not only can guide the physician in selecting the appropriate type of therapy for disseminated cancer but in our opinion it can provide additional information regarding prognosis and probable future sites of metastasis.
Addendum Since the submission of this article, 8 more patients whose tumors contained ER were subjected to repeat biopsies for ER after termination of endocrine therapy (and in some cases after both endocrine and chemotherapy). All 8 patients showed decrease in ER value in the second ER assays. Not all patients responded to endocrine therapy.
Acknowledgment The authors wish to thank Mrs. Cynthia Van De Walle for her statistical assistance in this study.
References 1. Cutler, S. J.. Asire. A. J. and Taylor. S. G.: Classification of Patients with Disseminated Ciancer of the Breast. Cancer. 24: 861-869. 1969. 2. Godefroi. V. C. and Brooks. S. C.: Impr-oved Gel-Filtration Method for Analysis of Estrogen Receptor Binding. Anal. Biochem.. 51:335-344. 1973. 3. Jensen. E. V. and DeSombre. E. R.: Estrogen-Receptor- Interaction. Science, 182:126-134. 1973. 4. Jensen. E. V.. Nemato. M.. Brecher. P. I. and DeSombre. E. R.: The Biochemistry of Steroid Hormone Action. Edited by R. M. S. Smillie. London. Academic Press. 1971. 5. Lowry. 0. H.. Rosebrough. N. J.. Farr. A. L. and Randall. R. J.: Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.. 193:265-275. 1951. 6. McGuire. W. L.. Carbone. P. P. and Vollmer, E. P.: Estrogen Receptors in Human Breast Cancer. New York. Raven Press. 1975. 7. Robison. G. A.. Butcher. R. W. and Sutherland. E. W.: Cyclic AMP. New York. Academic Press. 1971. 8. Truong. H.. and Baulien. E. E.: Interaction of Uterus Cytosol Receptor with Estradiol. Equilibrium and Kinetic Studies. Biochim. Biophys. Acta. 237:167-172. 1971.
The tumors from approximately 50% of patients with breast From the Department of Oncology, cancer contained estrogen receptor (ER). ER appeared more often Wayne State University School of Medicine and at higher levels in the tumors of postmenopausal women. and the Milton A. Darling Memorial Center Eleven out of 12 patients who had multiple ER assays from of the Michigan Cancer Foundation at The various metastatic sites showed no significant discrepancies in ER Grace Hospital, Detroit, Michigan values. ER level appears to decrease as the duration of metasta#.c cancer increase. Patients with ER in the tumor more frequently have bone metastases than those without ER. Visceral metastases only the activator for this receptor is not known at the occurred more often with ER negative patients and appeared to have a more malignant course with significant shorter survival. present time.
IOCHEMICAL MECHANISMS of hormone-target organ interaction have been recognized in the past several years. Polypeptide hormones and epinephrine react with
target tissue by activation of membrane-bound adenyl cyclase which result in formation of cyclic AMP.7 On the other hand steroid hormones appear to enter the target cells and bind to a specific extranuclear receptor.4 Among the various steroid hormones, estrogen was given the most attention since it has been shown for several years to be related to animal and human breast cancer. Jensen and DeSombre3 characterized estrogen receptor (ER) as a protein with the sedimentation coefficient of 8S which probably appears in the cytoplasm of target cell as 4S subunit. This estrogen receptor interacts with estradiol in the cytoplasm and undergoes transformation which is a temperature-dependent process. This transformed receptor-estrogen complex, which has a sedimentation coefficient of 5S, migrates to the nucleus and binds to the chromosome in the nucleus resulting in newly transcribed RNAs from the nucleus of target tissues. Whether this extranuclear receptor is merely a means of transporting the hormone into the nucleus or receptor protein itself is the active agent and hormone is
Breast cancer tissue, both primary and metastatic, has been shown by several investigators6 to contain ER. Some clinical aspects of human breast cancer associated with the presence or absence of ER in their tumor have been reported.6 We are reporting here additional information regarding the natural course of breast cancer and ER in the tumor. Materials and Methods
Breast cancer specimens both from primary sites and metastatic sites were obtained at surgery and frozen immediately; malignancy was verified by histological examination. All specimens were kept initially at -20 C, then transported to our laboratory in dry ice and stored at -70 C for longer periods. The tissues were processed and assayed for estrogen receptor by gel-filtration technique as previously described.2 One hundred milligrams of tissue were homogenized by a Willems Polytron in 10 mM Tris buffer, pH 7.4, containing 1.5 mM MgCl2, 10 mM KCI, and 5 mM reducing agent, dithiothreitol. After spinning in a Spinco model L ultracentrifuge at 15,000 g for 15 minutes, the resulting supernatant fluid was spun at 100,000 g for 65 minutes, 4 C, using a SW 50.1 rotor and buckets equipped with adaptors (Beckman 305527). Submitted for publication August 5, 1975. The supernatant fraction was diluted 1:1 with 10 mM Reprint Requests: A. Singhakowinta, M. D., Milton A. Darling Tris buffer, pH 8.5, containing 10 mM KCI, 5 mM Memorial Center, 4160 John R Street, Detroit, Michigan 48201. and mM EDTA (column buffer) and Dex1 dithiothreitol Supported in part by Clinical Cancer Research Center grant No. CA 07177 and grant No. T12 CA 08096 from the National Cancer Insti- tran Blue added. Protein was determined by the Folin tute, United States Public Health Service. method.5 Immediately following centrifugation, 0.4 ml of
84
Vol. 183 * No.
NATURAL COURSE OF BREAST CANCER
the diluted supernatant fraction was incubated with 10 ,ul of (2,4,6,7-3 H)-estradiol-17,B (100 Ci/mmole) standard for 2 hours at 4 C. A duplicate sample was also incubated with tritiated standard plus 100-fold excess of unlabeled estradiol-17/3. Upon completion of incubation, each sample was chromatographed through a separate column containing 3-4 g Sephadex G-25 previously equilibrated in "column buffer." Flow rates were kept constant, and the void volume (5 ml, indicated by the Dextran Blue) was collected and extracted 3 times with ethyl acetate. An aliquot of the extract was counted in a scintillation spectrometer equipped with automatic external standardization. Routine saturation curves were constructed for incubations with tritiated estradiol-17,/ and with labeled estrogen plus excess unlabeled estradiol-17f3. Receptor levels were determined from the difference between total binding capacity at saturation of tritiated estradiol17,3 and non-specific binding (residual binding in the presence of unlabeled estrogen). Occasionally Scatchard plots were used to calculate the binding constants of the estrogen receptor.2 The level of 2.1 femtomoles (fmoles, 10-15 moles) estradiol bound per mg cytoplasmic protein or over was considered to be positive for estrogen receptor. This level or higher has been shown by us to correlate well with endocrine therapy response in disseminated breast cancer.6 Medical records of patients with diagnosis of breast cancer who had ER study on their tumor were reviewed. Patients with simultaneous bilateral breast cancer and patients with other active malignancy in addition to breast cancer were excluded. A total of 133 cases were found to be evaluable based on the above criteria. Most of the patients had an ER assay done only once; in 12 patients multiple ER assays were performed. In three of these 12 patients, the re-biopsy for ER test was carried out to study the effect of exogenous estrogen on the ER values. Specific information regarding date of onset of symptoms, menstrual status at the time of ER assay, and the date and cause of death were determined. The sites of metastasis were divided into three categories as follows: viscera (includes liver, lung, pleura, CNS, spinal cord, and all intra-abdominal and intrathoracic organs), bone, and soft tissues (skin, opposite breast, lymph nodes). The term dominant site of metastasis is used to denote the category which is most extensively involved by cancer. In case of equal degree of multiple system involvement, the following priority of assignment was used: visceral, bone, soft tissue. The disease free interval (DFI) is defined as the time interval between initial diagnosis of breast cancer and the clinical diagnosis of disseminated cancer. The duration of disease was measured from the onset of cancer to the time of death.
85
TABLE 1. ER Value in Pr-e tanid Postm7letiopaiusal Patients
Premenopause
Postmenopause
31 14 45.2 23.4 7.0 0
102 54 53 39.7 12.9 12
Total number of patients Positive ER patients Percentage of positive ER Mean ER value (femtomole)* Median ER value Patients with multiple assay *
P = 0.40 by Student's t-test.
Results
Of 133 patients, 102 were postmenopausal women and 31 were premenopausal at the time the assay for ER was done. Sixty-eight patients or 51% had tumor which contained ER. As shown in Table 1, 53% of postmenopausal and only 45% of premenopausal patients had tumors which were positive for ER. The level of ER also appeared to be higher in postmenopausal cases. The majority of patients under this study had metastatic breast cancer at the time ER assay was performed. Only 26 patients had ER study from primary breast cancer lesions while the disease was considered to be curable. However, 10 of them subsequently had recurrent carcinoma. Most of the patients had more than one organ involvement but the biopsy for ER study in general was carried out from the accessible lesions, i.e. skin, lymph nodes, breast masses. Biopsies from bones were possible when lesions were superficial such as from iliac crest area or more commonly when orthopedists operated on patients for various fractures. In many cases multiple visceral biopsies were obtained during surgery either for therapeutic purposes such as bilateral adrenalectomy or for complications arising from cancer such as intestinal obstruction. Table 2 indicates the sites from where the biopsy for each patient was obtained. It is not necessarily the only site of carcinoma involvement nor the dominant site of metastasis that the particular patient had. For example when a patient had recurrent breast cancer of the chest wall, bone, and with massive liver involvement, the biopsy for ER study was usually taken from the chest wall. The site of biopsy will be listed under soft tissue but the dominant site of metastasis will be classified as viscera. Twelve patients had multiple biopsies. All except one had no significant discrepancy in ER value from various TABLE 2. Resuilts of ER Values from Variou.s Sites Sites of Biopsy*
Positive
Negative
Primary breast lesions Soft tissue Bone Viscera
21 40 6 2
27 35 1 3
* See text.
86
SINGHAKOWINTA AND OTHERS TABLE 3. Disease Free Interval
Free of disease Inoperable cases Mean DFI* Median DFI *
Positive ER 68 pts
Negative ER 65 pts
8 10 37.6 27.5
9 16 31.2 months 21.5 months
P = 0.4 by Student's t-test.
sites of metastatic lesions, i.e. when the lesion from the skin was positive for ER, the lesions from lymph node and bone also were positive although the ER value from various sites may not be exactly the same. The one patient with a discordant ER value had ER of 31.6 fmoles from her primary breast lesion. Fifty-four months later the ER value obtained from a bone lesion was 3.5 fmoles but a negative ER value was found in a metastatic hepatic nodule. In Table 2 she was counted three times for the site of biopsy: primary, bone, viscera. The rest of the patients were counted once based on the first site of biopsy. Disease Free Interval
Ann.
Surg. * January
1976
tient had ER assay prior, during and 4 months after DES therapy. The ER values were 3.3, 0 and 11.5 fmoles/mg protein respectively. The other two patients each had ER assays while on DES and repeat assay after DES treatment was discontinued. One patient's ER level rose from 0 to 2.2 and the other went from 4.8 to 61 fmoles/mg protein after discontinuation of DES for 4 and 7 months respectively.
Survival Data
Forty-eight patients had expired as of December 15, 1974; 21 were positive for ER and 27 were negative.
Table 4 demonstrates the total duration of disease from
onset of cancer to death in each category. All but two
died of metastatic breast cancer; both had ER in their tumor. One died of toxicity from chemotherapy and the other died from pulmonary embolism 2 weeks after androgen therapy was initiated. Nine patients with negative ER had rapidly progressive disease which compelled the managing physicians to bypass endocrine treatment and initiate chemotherapy as the first systemic treatment for disseminated cancer. The difference in survival between ER positive and ER negative patients is statistically significant in favor of the ER positive group.
Seventeen patients are still free of recurrent cancer. Twenty-six patients presented with inoperable breast Comment cancer; 16 were ER negative patients. Therefore, in a Lower ER level in the premenopausal group is probably total of 43 patients the DFI cannot be determined. In due to partial saturation of the receptor sites by endogthe remaining 90 patients DFI could be calculated but no significant difference was observed between the positive and negative ER groups as illustrated in Table 3. 100POSITIVE ER NEGATIVE ER
90-Q
Dominant Site of Metastasis
80A total of 116 patients had recurrent or inoperable breast cancer. Fig. 1 shows a preponderance for bone 70metastasis in the positive ER group while in the negative ER group visceral metastasis appears dominant. The difference is highly significant with the P-value of 0.0001. PERCENT 60-
Patients with Multiple ER Assay Of 12 patients with multiple biopsies, 11 had concordant ER value regardless of the site of the biopsy. Three were negative for ER and 8 were ER positive. There were 6 patients who were ER positive subsequently rebiopsied after a period of 6 to 54 months. All were found to have decreasing values of ER except for one who displayed an increase in ER 11 months after bilateral adrenalectomy as shown in Table 5. None of these 6 patients received estrogen or androgen during the 6 months preceding the second biopsy. There were three patients who had ER assay performed while on Diethylstilbestrol (DES) 15 mg/day. One pa-
OF PATIENTS
49
0
4030an
J
16
SOFT TISSUE
r
SONE
VISCERA FIG. 1. Comparison of dominant site of metastasis between ER positive and ER negative group. More bone metastasis occurs in ER positive
and more visceral metastasis in ER negative groups. Fisher test for P = 0.001 for bone vs viscera and P = 0.01 for soft tissue vs viscera.
Vol. 183 . No. I
NATURAL COURSE OF BREAST CANCER
estrogen. The study of ER values in three patients while on and off therapy with DES seemed to be in agreement with this hypothesis, although the dose of DES used was much higher than the physiological amount of estrogens present in normal premenopausal women. It has been suggested that the critical level of ER in postmenopausal women to be considered as positive should be higher than premenopausal patients. On the other hand, several investigators6 reported good correlation of response to endocrine therapy in patients with borderline ER value. The interpretation of an ER value in a patient with disseminated cancer who is receiving or recently received estrogen therapy must be interpreted with caution because of the possibility of a false negative ER value. The binding between estrogen and receptor protein is strong. This tight binding appears to result from a slow rate of dissociation.8 How much time is required for the receptor sites to be freed up again after discontinuation of estrogen treatment is not known. The three patients who had re-biopsies for ER assays after interruption of DES for periods of 4 to 7 months showed increased ER levels. It is quite conceivable that less than 4 months is required for the receptor sites to become unsaturated. The fact that 11 out of 12 patients who had multiple ER determinations which were concordant would suggest that in most cases single biopsy of an accessible lesion is adequate for ER study. However when a change in endocrine therapy is contemplated, a repeat biopsy of an accessible lesion, when available, should be performed. This is to insure the presence of hormonal dependent cells in the tumor prior to institution of a new form of endocrine therapy. This is particularly important when major surgery such as bilateral adrenalectomy or hypophysectomy are involved. The fact that 5 of6 patients in this study showed a progressive decrease in ER value as the duration of metastatic disease increased, as shown in Table 5, appears to support the hypothesis that a patient whose tumor contains ER actually has both hormonal dependent cells and independent cells but in different proportions. After receiving endocrine manipulation, the hormonal dependent cells are destroyed allowing the hormonal independent cells to proliferate. The alternative explanation is the continuous existence of hormonal dependent cells, but ability to produce ER is lost after endocrine treatment. On the other hand, a patient with no ER in the tumor probably has few or none of hormonal dependent cells in their tumors. The clinical course of disseminated breast cancer, in general, correlates with the sites of metastasis and DFI. Patients with a long DFI or metastatic involvement of skin, lymph nodes, or bone without visceral metastasis are found to have a longer survival compared to those with visceral metastasis.1 Our ER study seems to coincide with the above clinical observations, with the exception of the prognostic role of DFI for which there was no
87
TABLE 4. Duration of Disease
enous
Positive ER 21
Mean* Median
56.7 54 1 5 15 3
Chemotherapy only
Endocrine therapy only Endocrine and chemotherapy Inoperable case at initial diagnosis *
cases
Negative ER 27
cases
29.6 months 23 months 9 3 15 8
P < 0.01 by Student's t-test.
difference between positive and negative ER groups. Patients with ER negative tumors more frequently have visceral involvement, whereas those with ER positive have predilection for bone involvement. The survival was significantly shorter in the ER negative group. Because the inoperable breast cancer in the ER negative group outnumbered those in the ER positive at a ratio of 8 to 3, one may raise the question that the poorer prognosis in the ER negative group could have been the result of excessive patient procrastination in seeking medical treatment. However, in review of the medical records in those 11 cases who presented with inoperable breast carcinoma, the median delay in getting medical attention was 5 months (range 1-12) in the ER negative group as contrasted with 12 months (range 1-13) in the positive ER group. Moreover, 5 out of 8 patients in the negative ER group, at the time of initial diagnosis, had visceral metastases in addition to breast lesions. However, only one out of 3 in the positive ER group had visceral involvement at the time of diagnosis of breast cancer. These findings tend to support the hypothesis that the biological behavior rather than procrastination by the patients is the reason for shorter survival in ER negative patients. We feel that ER study should be done in every patient with breast cancer regardless of operability. A significant number of patients who undergo potentially curative mastectomy will eventually have recurrent cancer. If cancer recurs in bones or viscera, frequently the biopsy for ER assay cannot be done either because of technical TABLE 5. ER Level in Patients with Multiple Sequential Assay
Name
1st and 2nd ER level fmoles
E.F. W.B. V.T. H.B. G.G. G.T.
31.6 80.4 32 41.3 14.6 14.2
3.5 8.3 0.9 5.4 6.5 80.7
Treatment between 2 ER
Duration between 2 ER (in mos.)
Adr., chemo.* Adr. Adr. Adr. Adr. Adr.
* Adr. = bilateral surgical adrenalectomy. t L.N. = lymph node.
54 7 6 8 7 11
M M M M M M
Sites of biopsy
Primary, bone Skin, skin Skin, skin L.N., L.N.t L.N., L.N. L.N., L.N.
Ann. Surg. * Januzu-% 1976
SINGHAKOWINTA AND OTHERS
88
or ethical reasons. The only tissue available for ER assay in these individuals would be the primary lesions. It is proposed that ER study not only can guide the physician in selecting the appropriate type of therapy for disseminated cancer but in our opinion it can provide additional information regarding prognosis and probable future sites of metastasis.
Addendum Since the submission of this article, 8 more patients whose tumors contained ER were subjected to repeat biopsies for ER after termination of endocrine therapy (and in some cases after both endocrine and chemotherapy). All 8 patients showed decrease in ER value in the second ER assays. Not all patients responded to endocrine therapy.
Acknowledgment The authors wish to thank Mrs. Cynthia Van De Walle for her statistical assistance in this study.
References 1. Cutler, S. J.. Asire. A. J. and Taylor. S. G.: Classification of Patients with Disseminated Ciancer of the Breast. Cancer. 24: 861-869. 1969. 2. Godefroi. V. C. and Brooks. S. C.: Impr-oved Gel-Filtration Method for Analysis of Estrogen Receptor Binding. Anal. Biochem.. 51:335-344. 1973. 3. Jensen. E. V. and DeSombre. E. R.: Estrogen-Receptor- Interaction. Science, 182:126-134. 1973. 4. Jensen. E. V.. Nemato. M.. Brecher. P. I. and DeSombre. E. R.: The Biochemistry of Steroid Hormone Action. Edited by R. M. S. Smillie. London. Academic Press. 1971. 5. Lowry. 0. H.. Rosebrough. N. J.. Farr. A. L. and Randall. R. J.: Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.. 193:265-275. 1951. 6. McGuire. W. L.. Carbone. P. P. and Vollmer, E. P.: Estrogen Receptors in Human Breast Cancer. New York. Raven Press. 1975. 7. Robison. G. A.. Butcher. R. W. and Sutherland. E. W.: Cyclic AMP. New York. Academic Press. 1971. 8. Truong. H.. and Baulien. E. E.: Interaction of Uterus Cytosol Receptor with Estradiol. Equilibrium and Kinetic Studies. Biochim. Biophys. Acta. 237:167-172. 1971.
