Sex Hormone Binding Globulin as a Reliable Indicator of Hormone Dependence in Human Breast Cancer YOICHI MURAYAMA, JOJI UTSUNOMIYA, ISAMU TAKAHASHI,* MASATSUGU KITAMURA,* TAKESHI TOMINAGA*
One hundred nine pre- and postmenopausal mammary carcinoma cases were studied to elucidate the role of sex hormone binding globulin (SHBG) in the hormone dependence of human breast cancer. Our findings indicate that there is a significant negative correlation between SHBG binding capacity and plasma testosterone concentration. All patients with high SHBG titers were found to be cytosol estrogen receptor (CER) positive and the plasma SHBG binding capacity of CER positive was significantly higher than that of CER negative patients. We also found that the level of pretherapy plasma SHBG concentration is a reliable indicator in predicting the efficacy of hormone therapy. Our findings also confirm that, for a tumor to be hormone dependent, high plasma SHBG concentration and estrogen receptors must be present simultaneously. The present pretherapy determination of SHBG titers is easier and more reliable than previous methods for determining the hormone dependence of human breast tumors. S EX HORMONE BINDING GLOBULIN
(SHBG) is the
specific binding protein which has been suggested to regulate the biological activity of sex steroids in the blood.5 In normal subjects, SHBG is increased by an increase in endogenous estrogen5'6'27 and decreased by an increase in endogenous testosterone.'5'12-20 The change in SHBG during the menstrual cycle is much smaller than the radioimmunoassay-determined change of plasma estradiol or plasma testosterone.27 In breast cancer patients, the administration of exogenous sex steroids effects a change in the binding capacity of SHBG6 and after oophorectomy or adrenalectomy, SHBG decreases to the level of one-half or onethird of that before hormone therapy. McGuire, et al.14 reported that the presence of receptors in breast tumors is one of the reliable indicators * Tokyo Metropolitan Komagome Hospital, Tokyo, Japan. Reprint requests: Yoichi Murayama, The Second Department of Surgery, Tokyo Medical and Dental University, Tokyo 113, Japan. Supported in part by a research grant (51-7) from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan. Presented in part at the XII International Cancer Congress. Submitted for publication: November 14, 1978.
From the Second Department of Surgery, Tokyo Medical and Dental University, Tokyo, Japan
for their hormone dependence. However, not all receptor-positive tumors respond to endocrine therapy and only 60-65% of those tumors regress. Experimental and clinical studies revealed a change in the estrogen receptor level which was associated with a change in the levels of endogenous steroid hormones such as estrogen and progesterone.2'4'8'9"5'7 These findings suggest that the hormone dependence of breast tumors is subject to two factors-the presence of receptors and the endogenous hormone environment which reacts to the receptor system. We hypothesized that a specific endogenous hormone environment which shows an increase in SHBG initiates and promotes estrogen receptor synthesis in human breast tumors and that hormone-dependent breast cancer evidences an increase in SHBG and estrogen receptors. To test this hypothesis, we examined the relationship between SHBG binding capacity and endogenous testosterone concentration, analyzed the relationship between SHBG binding capacity and the presence of cytosol estrogen receptors (CER) and determined the reliability of SHBG binding capacity prior to hormone therapy as an indicator of the hormone dependence of the breast tumor. Materials and Methods
Subjects The 109 patients were divided into three groups. None of the patients or controls were pregnant, had endocrine disease, or were receiving medication, and all had normal liver and thyroid functions. With the exception of three patients in Group C, none of the patients had undergone prior hormone therapy. In Group A (24 patients), the relationship between plasma testosterone concentration and SHBG binding
0003-4932/79/0800/0133 $00.80 X J. B. Lippincott Company
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capacity was assayed simultaneously. This group consisted of 13 pre- and 11 postmenopausal primary breast cancer patients ranging in age from 25 to 74 years (average 47). In Group B (35 patients), the relationship between plasma SHBG binding capacity and CER was examined. This group consisted of 13 pre- and 22 postmenopausal primary breast cancer patients ranging in age from 29 to 73 years (average 52). There were no distant metastases. In Group C (50 patients), we assayed plasma SHBG concentration prior to therapy in order to determine whether pretherapy SHBG titers represent a reliable indicator for predicting the efficacy of hormone therapy. This group consisted of 17 pre- and 33 postmenopausal advanced breast cancer patients ranging in age from 33 to 72 years (average 54). These patients presented with metastatic breast cancer. Main metastatic lesions were found in soft tissues such as skin and lymph nodes of six pre- and 12 postmenopausal patients, in bone tissue of five pre- and ten postmenopausal patients, and in the lungs, pleura, liver, or brain of six pre- and 11 postmenopausal patients.
Determination of Plasma Testosterone Concentration (Group A) Plasma testosterone concentration was determined
by radioimmunoassay.13 Testosterone-3-oxime-BSA which was used as antigen was produced by the method of Erlanger.7 Testosterone- 1f,2,8-3H (SA: 47 Ci/mmol) was obtained from the Radiochemical Center (Tokyo). Emulsion was prepared from 1 mg testosterone-3oxime-BSA, 0.5 ml complete Freund's adjuvant and 0.5 ml normal saline. The antiserum was generated against testosterone-3-oxime-BSA, diluted according to the method of Furuyama,10 and cross-reacted with the following steroids. The per cents of cross-reactivity with 5a-dihydrotestosterone, epitestosterone and androst-4-ene-31, 17f3-diol were 118%, 47% and 26%, respectively. Separation and purification procedures were performed by the method of Wu26 and crossreaction was negligible. Separation of bound and free hormones was performed by 50% (NH4)2SO4. The intra- and interassay variances were 10.2% and 11%, respectively. The least detectable concentration was 20 pg. The recovery rate and water blank were 85.8 + 11%, and 7.46 + 2.8 pg, respectively. Determination of SHBG Binding Capacity (Group B) SHBG binding capacity was determined by agar-gel electrophoresis with dextran-coated charcoal treatment.19 One hundred microliters of serum and 1.5 nmol
Ann. Surg. * August 1979
of [3H] 17-,B-estradiol were incubated at 0-2° for two hours to obtain SHBG-17,1-estradiol complex. The free and loosely bound [3H] 17,3-estradiol was then removed by the addition of 3 ml of ice cold dextran-coated charcoal suspension. Separation and purification was performed by agar-gel electrophoresis at a low temperature24 and the radioactivity of each fraction was counted on a liquid scintillation counter (Packard). Determination of CER (Group B) CER was determined by the method of Wagner and
Jungblut.25 Two hundred fifty milligrams of frozen tissue stored at -80° was ground with an autopulverizer in liquid nitrogen and homogenized with a Teflon® glass homogenizer in ice cold 0.01 M Tris HCI buffer (pH 7.4) into a volume four times that of the original tissue volume. The homogenate was centrifuged in a cooled centrifugal butylate tube at 130,580 x g at 40 for 30 minutes to obtain cytosol fractions as the supernatant. Tritiated 17,8-estradiol (0.4 nmol) was incubated with 60 ,ul of cytosol at 0-2° for two hours to obtain 17,8-estradiol receptor complex. The free and loosely bound tritiated 17,8-estradiol was then removed by adding 0.5 ml ice
cold dextran-coated charcoal suspension. Separation and purification was performed by agar-gel electrophoresis at low temperature.24 After the agar-layer was fractionated into 3 mm widths, the radioactivity of each fraction was determined with a liquid scintillation counter (Packard).
Radioactivity Measurement Plasma and cytosol samples in 3 mm sections of agar-gel were dissolved overnight in 5 ml Soluene and counted in 10 ml Instagel in a liquid scintillation counter (Packard) with a counting efficiency of 35%. Hormone Therapy (Group C) In premenopausal metastatic breast cancer patients
(Group C), adrenalectomy, ovariotomy or androgen therapy were chosen. Adrenalectomy, including the resection of both ovaries, was performed in a onestep procedure via an upper abdominal incision. In three patients who had previously undergone ovariectomy for the treatment of advanced breast cancer, only the adrenal glands were removed. A maintenance dose of 50 mg cortisollday was administered to patients postoperatively. Androgen therapy consisted of 30 mg testosterone/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded.11 The treatment for postmenopausal patients consisted
SHBG AND HORMONE RESPONSIVENESS
Vol. 190 * No. 2
of adrenalectomy, androgen or estrogen therapy. If menstruation had ceased less than five years previously, androgen therapy (30 mg testosterone/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded") was instituted, if more than five years had elapsed since the patient's last menstrual period, estrogen therapy (15 mg diethylstilbestrol/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded") was chosen. All patients were closely observed for at least four weeks following initiation of the chosen treatment to determine its effectiveness.
140l 0
120-
SHBG P moles/ml
100* 0
80o r=
60 -
40 -
0
20
0
Results Relationship Between SHBG and Plasma Testosterone Concentration (Group A) A significant negative correlation was noted between plasma testosterone concentration and SHBG binding capacity in the premenopausal patients of Group A (Fig. lA). In the postmenopausal patients, an inverse correlation between SHBG binding capacity and plasma testosterone concentration was noted. However, this correlation was not significant (Fig. iB).
SHBG
Yz-55x+85
100
r
=
-0.78
13 P' 0.01
n= 80 0
0
0
0.6
I
0
0.6
0.8
I
1.0
1.4
1.2
1.6
FIG. lb. Relationship between plasma testosterone concentration and SHBG binding in postmenopausal breast cancer patients.
Relationship Between SHBG and CER (Group B) In premenopausal CER-positive and negative patients, the SHBG plasma concentrations were 59 + 12 (mean + SD) p moles/ml of plasma and 28 + 7 (mean + SD) p moles/ml of plasma, respectively. The concentration difference was significant (p < 0.05) (Fig. 2). The plasma SHBG concentration in the premenopausal controls was 52 + 11 (mean ± SD) p moles/ml of plasma and concentration difference between the controls and premenopausal CER-negative patients was significant (p < 0.05). There was no significant difference between the controls and premenopausal CER-positive patients. Plasma SHBG concentration in the postmenopausal controls, CER-positive and negative patients was 35 76 (mean SD) p moles/ml of plasma, 71 34 (mean SD) p moles/ml of plasma, and 34 8 (mean + SD), respectively. There was a significant difference between postmenopausal CER-positive and negative (p
One hundred nine pre- and postmenopausal mammary carcinoma cases were studied to elucidate the role of sex hormone binding globulin (SHBG) in the hormone dependence of human breast cancer. Our findings indicate that there is a significant negative correlation between SHBG binding capacity and plasma testosterone concentration. All patients with high SHBG titers were found to be cytosol estrogen receptor (CER) positive and the plasma SHBG binding capacity of CER positive was significantly higher than that of CER negative patients. We also found that the level of pretherapy plasma SHBG concentration is a reliable indicator in predicting the efficacy of hormone therapy. Our findings also confirm that, for a tumor to be hormone dependent, high plasma SHBG concentration and estrogen receptors must be present simultaneously. The present pretherapy determination of SHBG titers is easier and more reliable than previous methods for determining the hormone dependence of human breast tumors. S EX HORMONE BINDING GLOBULIN
(SHBG) is the
specific binding protein which has been suggested to regulate the biological activity of sex steroids in the blood.5 In normal subjects, SHBG is increased by an increase in endogenous estrogen5'6'27 and decreased by an increase in endogenous testosterone.'5'12-20 The change in SHBG during the menstrual cycle is much smaller than the radioimmunoassay-determined change of plasma estradiol or plasma testosterone.27 In breast cancer patients, the administration of exogenous sex steroids effects a change in the binding capacity of SHBG6 and after oophorectomy or adrenalectomy, SHBG decreases to the level of one-half or onethird of that before hormone therapy. McGuire, et al.14 reported that the presence of receptors in breast tumors is one of the reliable indicators * Tokyo Metropolitan Komagome Hospital, Tokyo, Japan. Reprint requests: Yoichi Murayama, The Second Department of Surgery, Tokyo Medical and Dental University, Tokyo 113, Japan. Supported in part by a research grant (51-7) from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan. Presented in part at the XII International Cancer Congress. Submitted for publication: November 14, 1978.
From the Second Department of Surgery, Tokyo Medical and Dental University, Tokyo, Japan
for their hormone dependence. However, not all receptor-positive tumors respond to endocrine therapy and only 60-65% of those tumors regress. Experimental and clinical studies revealed a change in the estrogen receptor level which was associated with a change in the levels of endogenous steroid hormones such as estrogen and progesterone.2'4'8'9"5'7 These findings suggest that the hormone dependence of breast tumors is subject to two factors-the presence of receptors and the endogenous hormone environment which reacts to the receptor system. We hypothesized that a specific endogenous hormone environment which shows an increase in SHBG initiates and promotes estrogen receptor synthesis in human breast tumors and that hormone-dependent breast cancer evidences an increase in SHBG and estrogen receptors. To test this hypothesis, we examined the relationship between SHBG binding capacity and endogenous testosterone concentration, analyzed the relationship between SHBG binding capacity and the presence of cytosol estrogen receptors (CER) and determined the reliability of SHBG binding capacity prior to hormone therapy as an indicator of the hormone dependence of the breast tumor. Materials and Methods
Subjects The 109 patients were divided into three groups. None of the patients or controls were pregnant, had endocrine disease, or were receiving medication, and all had normal liver and thyroid functions. With the exception of three patients in Group C, none of the patients had undergone prior hormone therapy. In Group A (24 patients), the relationship between plasma testosterone concentration and SHBG binding
0003-4932/79/0800/0133 $00.80 X J. B. Lippincott Company
133
134
MURAYAMA AND OTHERS
capacity was assayed simultaneously. This group consisted of 13 pre- and 11 postmenopausal primary breast cancer patients ranging in age from 25 to 74 years (average 47). In Group B (35 patients), the relationship between plasma SHBG binding capacity and CER was examined. This group consisted of 13 pre- and 22 postmenopausal primary breast cancer patients ranging in age from 29 to 73 years (average 52). There were no distant metastases. In Group C (50 patients), we assayed plasma SHBG concentration prior to therapy in order to determine whether pretherapy SHBG titers represent a reliable indicator for predicting the efficacy of hormone therapy. This group consisted of 17 pre- and 33 postmenopausal advanced breast cancer patients ranging in age from 33 to 72 years (average 54). These patients presented with metastatic breast cancer. Main metastatic lesions were found in soft tissues such as skin and lymph nodes of six pre- and 12 postmenopausal patients, in bone tissue of five pre- and ten postmenopausal patients, and in the lungs, pleura, liver, or brain of six pre- and 11 postmenopausal patients.
Determination of Plasma Testosterone Concentration (Group A) Plasma testosterone concentration was determined
by radioimmunoassay.13 Testosterone-3-oxime-BSA which was used as antigen was produced by the method of Erlanger.7 Testosterone- 1f,2,8-3H (SA: 47 Ci/mmol) was obtained from the Radiochemical Center (Tokyo). Emulsion was prepared from 1 mg testosterone-3oxime-BSA, 0.5 ml complete Freund's adjuvant and 0.5 ml normal saline. The antiserum was generated against testosterone-3-oxime-BSA, diluted according to the method of Furuyama,10 and cross-reacted with the following steroids. The per cents of cross-reactivity with 5a-dihydrotestosterone, epitestosterone and androst-4-ene-31, 17f3-diol were 118%, 47% and 26%, respectively. Separation and purification procedures were performed by the method of Wu26 and crossreaction was negligible. Separation of bound and free hormones was performed by 50% (NH4)2SO4. The intra- and interassay variances were 10.2% and 11%, respectively. The least detectable concentration was 20 pg. The recovery rate and water blank were 85.8 + 11%, and 7.46 + 2.8 pg, respectively. Determination of SHBG Binding Capacity (Group B) SHBG binding capacity was determined by agar-gel electrophoresis with dextran-coated charcoal treatment.19 One hundred microliters of serum and 1.5 nmol
Ann. Surg. * August 1979
of [3H] 17-,B-estradiol were incubated at 0-2° for two hours to obtain SHBG-17,1-estradiol complex. The free and loosely bound [3H] 17,3-estradiol was then removed by the addition of 3 ml of ice cold dextran-coated charcoal suspension. Separation and purification was performed by agar-gel electrophoresis at a low temperature24 and the radioactivity of each fraction was counted on a liquid scintillation counter (Packard). Determination of CER (Group B) CER was determined by the method of Wagner and
Jungblut.25 Two hundred fifty milligrams of frozen tissue stored at -80° was ground with an autopulverizer in liquid nitrogen and homogenized with a Teflon® glass homogenizer in ice cold 0.01 M Tris HCI buffer (pH 7.4) into a volume four times that of the original tissue volume. The homogenate was centrifuged in a cooled centrifugal butylate tube at 130,580 x g at 40 for 30 minutes to obtain cytosol fractions as the supernatant. Tritiated 17,8-estradiol (0.4 nmol) was incubated with 60 ,ul of cytosol at 0-2° for two hours to obtain 17,8-estradiol receptor complex. The free and loosely bound tritiated 17,8-estradiol was then removed by adding 0.5 ml ice
cold dextran-coated charcoal suspension. Separation and purification was performed by agar-gel electrophoresis at low temperature.24 After the agar-layer was fractionated into 3 mm widths, the radioactivity of each fraction was determined with a liquid scintillation counter (Packard).
Radioactivity Measurement Plasma and cytosol samples in 3 mm sections of agar-gel were dissolved overnight in 5 ml Soluene and counted in 10 ml Instagel in a liquid scintillation counter (Packard) with a counting efficiency of 35%. Hormone Therapy (Group C) In premenopausal metastatic breast cancer patients
(Group C), adrenalectomy, ovariotomy or androgen therapy were chosen. Adrenalectomy, including the resection of both ovaries, was performed in a onestep procedure via an upper abdominal incision. In three patients who had previously undergone ovariectomy for the treatment of advanced breast cancer, only the adrenal glands were removed. A maintenance dose of 50 mg cortisollday was administered to patients postoperatively. Androgen therapy consisted of 30 mg testosterone/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded.11 The treatment for postmenopausal patients consisted
SHBG AND HORMONE RESPONSIVENESS
Vol. 190 * No. 2
of adrenalectomy, androgen or estrogen therapy. If menstruation had ceased less than five years previously, androgen therapy (30 mg testosterone/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded") was instituted, if more than five years had elapsed since the patient's last menstrual period, estrogen therapy (15 mg diethylstilbestrol/day for at least 30 days or until new lesions appeared or existing lesions increased by at least 25% above their smallest size recorded") was chosen. All patients were closely observed for at least four weeks following initiation of the chosen treatment to determine its effectiveness.
140l 0
120-
SHBG P moles/ml
100* 0
80o r=
60 -
40 -
0
20
0
Results Relationship Between SHBG and Plasma Testosterone Concentration (Group A) A significant negative correlation was noted between plasma testosterone concentration and SHBG binding capacity in the premenopausal patients of Group A (Fig. lA). In the postmenopausal patients, an inverse correlation between SHBG binding capacity and plasma testosterone concentration was noted. However, this correlation was not significant (Fig. iB).
SHBG
Yz-55x+85
100
r
=
-0.78
13 P' 0.01
n= 80 0
0
0
0.6
I
0
0.6
0.8
I
1.0
1.4
1.2
1.6
FIG. lb. Relationship between plasma testosterone concentration and SHBG binding in postmenopausal breast cancer patients.
Relationship Between SHBG and CER (Group B) In premenopausal CER-positive and negative patients, the SHBG plasma concentrations were 59 + 12 (mean + SD) p moles/ml of plasma and 28 + 7 (mean + SD) p moles/ml of plasma, respectively. The concentration difference was significant (p < 0.05) (Fig. 2). The plasma SHBG concentration in the premenopausal controls was 52 + 11 (mean ± SD) p moles/ml of plasma and concentration difference between the controls and premenopausal CER-negative patients was significant (p < 0.05). There was no significant difference between the controls and premenopausal CER-positive patients. Plasma SHBG concentration in the postmenopausal controls, CER-positive and negative patients was 35 76 (mean SD) p moles/ml of plasma, 71 34 (mean SD) p moles/ml of plasma, and 34 8 (mean + SD), respectively. There was a significant difference between postmenopausal CER-positive and negative (p
