In vitro estrogen-binding by human breast carcinomas B.G. Mobbs,
ph
d; I.E. Johnson,
b sc
Patients whose breast carcinomas possess only low concentrations of a receptor molecule that binds estrogens with high affinity are unlikely to respond to
hormonal manipulative therapy when the disease recurs. The estrogen-binding capacity of 106 breast carcinomas was measured by an in vitro method and was expressed per milligram wet weight and in some cases related to the concentration of deoxyribonucleic acid (DNA) of the tumours. The ability of tumours to bind 3H-estradiol ranged from 0 to 1.3 fm/mg in pre- and perimenopausal women, and from 0 to 16.8 fm/mg in postmenopausal women. Menopausal status or serum concentrations of endogenous estrogen, or both, should therefore be considered when tumours are classified into low and high estrogen-binding capacity. It is not necessary to carry out Scatchard analysis for every tumour, and expressing estradiol binding on the basis of DNA concentration may be preferable to
expressing it
on a
wet-weight basis.
Les malades dont le cancer mammaire ne contient que de faibles concentrations de la molecule receptrice ayant une forte affinite de fixation pour les estrogenes ne reagiront probablement pas a une hormonotherapie si la maladie recidive. La capacite estrogenofixatrice de 106 cancers mammaires a ete mesuree par une methode in vitro et a ete exprimee par miHigramme de tissue humide et, en certains cas, reliee a la concentration d'acide desoxyribonucleique (DNA) des tumeurs. La capacite des tumeurs de fixer I'estradiol marque au tritium (3H) a varie de 0 a 1.3 fm/mg chez les femmes premenopausees ou perimenopausees, et de 0 a 16.8 fm/mg chez les femmes postmenopausees. II importe done de tenir compte du statut de la femme au point de vue menopausique ou des concentrations seriques d'estrogene endogene, ou des deux facteurs, quand les tumeurs sont classees dans les categories de capacite estrogenofixatrice faible ou forte. II n'est pas necessaire de pratiquer l'analyse de Scatchard pour toutes les tumeurs, et il est preferable d'exprimer La capacite fixatrice de I'estradiol d'apres la concentration des tumeurs en DNA qu'apres la base de tissu humide.
From the department of surgery, Banting Institute, University of Toronto, and Women's College Hospital, Toronto of surgery, Banting Reprint requests to: Dr. B.G. Mobbs,ONDepartment M5G 1L5 Institute, 100 College St., Toronto,
216 CMA JOURNAL/FEBRUARY 7, 1976/VOL. 114
There is
now
little doubt that in vitro measurement of
high-affinity binding of estradiol to specific protein macromolecules in the cytoplasm of human breast carcinomas can be of value in aiding the clinician to predict the response of recurrent disease to endocrine ablation or hormonal therapy. Evaluation of the data of 13 groups of investigators from eight countries showed that patients with breast carcinomas of low estrogen-binding capacity are unlikely to respond to hormonal manipulation.1 Treatment by endocrine ablative surgery or additive hormonal therapy is therefore not indicated in these patients. As yet there have been no data published from such an investigation in Canada. We now report results from an ongoing study of the estrogen-binding capacity of breast carcinomas from patients at Women's College Hospital, Toronto. Materials and methods
Estrogen-binding capacity was investigated in 106 breast deposits, mainly from lymph nodes. One primary tumour was from a 64-year-old man. Samples of two benign tu¬ mours (one specimen of fibrocystic disease and one fibroadenoma) and samples of nontumorous breast tissue from three patients were also examined. Tumour samples (usually 0.1 to 0.5 g) were placed in an iced container as soon as possible after removal and trans¬ ferred to the laboratory. Each sample was trimmed of fat and extraneous tissue and a portion was fixed for histologic examination. From this point on, minor modifications in technique have been made over the period of the study, but there is no indication that the modifications have changed appreciably the results obtained. Our current meth¬ od is described below, with the differing details of the earlier method in parentheses. The samples are frozen and stored in a liquid nitrogen container (formerly at .17 °C) until assay. The frozen tissue is pulverized in a stainless steel pulverizer (Thermovac Industries Corp., St. Copiague, New York), cooled with an liquid nitrogen and the resulting powder transferred to are iced homogenizer vessel. All subsequent procedures carried out at 0 to 4°C. Tissue is homogenized in TRIS 1.5 mM ethylenhydrochloride buffer (pH 8.0) containing concentration of ediaminotetraacetate (EDTA), the final 100 Homogenizamg/ml. being approximately homogenate carcinomas, including 97 primary tumours and 9 metastatic
ice-cooled vessel with a Potterhomogenizer, with 15-second bursts of 790
tion is carried out in
Elvehem-type
an
rpm
separated by 45-second cooling periods. The homo-
genate is centrifuged at 1000 x g for 10 minutes. Any fat present forms a layer over the supernatant and can be
removed. An aliquot for estimation of deoxyribonucleic acid (DNA)2 is taken either from the homogenate before centrifugation or from the pellet afterwards. The super¬ natant is recentrifuged at 98 000 x g (formerly 48 000 x g) for 1 hour, then the supernatant (cytosol) is diluted 1:1 with buffer and used for assay. Replicate aliquots are in¬ cubated with 1.0 x 109 M 3H-estradiol (specific activity, 85 to 105 Ci/mmol (formerly 40 to 50 Ci/mmol) (New England Nuclear, Boston). Unlabelled estradiol is added to some aliquots at a concentration of 4 x 10~7 M. Blanks containing 1.0 ml buffer instead of cytosol are run concurrently. Incubation is carried out at 4°C overnight (for¬ merly at 25 °C for 30 minutes). Separation of bound from unbound steroid with dextran-coated charcoal and extrac¬ tion of bound steroid are carried out as previously described.3 The difference in the amount of radioactivity in the absence and in the presence of unlabelled estradiol is considered to be due to binding by the high-affinity estrogen receptor. This is expressed as femtomoles (10'15 moles) of 3H-estradiol bound per milligram of tissue homogenized or per microgram of DNA in the homogenate. Scatchard plot analysis4'5 was carried out on 17 carcino¬ ma specimens, with a range of 3H-estradiol concentrations from 1.2 x 1010 to 2.0 x 109 M. Low-affinity binding was determined by adding unlabelled estradiol to give a concen¬ tration 400 times that of the labelled hormone. The disso¬ ciation constant (Kd) of the estradiol receptor complex was calculated from the slope of the curve, and the con¬ centration of binding sites by extrapolation of the curve to the
Results For all the carcinomas investigated, estrogen-binding from 0 to 1.3 fm/mg in premenopausal women and in women within 5 years of the menopause, and from 0 to 16.8 fm/mg in postmenopausal women (Fig. 1). The values for estrogen-binding in the secondary de¬ posits fell within the range for the primary tumours. The value for the carcinoma from the male patient was 1.3 fm/mg. None of the nontumourous breast specimens bound detectable amounts of 3H-estradiol, and both the benign specimens had low binding capacity: 0.11 fm/mg for the specimen of fibrocystic disease and 0.12 fm/mg for the
capacity ranged
fibroadenoma. Scatchard plot analysis yielded Kds ranging from 9.1 x 10"9 to 3.4 x 10n M, and the majority were of the order of 1010 M, demonstrating high-affinity binding be¬ tween the 3H-estradiol and its receptors. One of these plots is illustrated in Fig. 2. The concentration of estradiol bound by receptors, as determined from the Scatchard analysis, was plotted against the concentration of estradiol bound at the "single-dose" incubation concentration of 1.0 x 10'9 M for the same tumours (Fig. 3). The two methods gave equivalent values except for tumours of very high binding capacity, in which not all the sites were saturated at this concentration. Thus, for tumours with a receptor concen¬ tration of estradiol greater than 3 fm/mg, incubation at 1.0 x 10~9 M 3H-estradiol may result in underestimation of the estrogen-binding capacity.
abscissa.
. (16.8)
5.5-
Premenopausal o Perimenopausal a
5.0"
Postmenopausal . Male
d*
4.5-
i
4.0n= 4.85 fm/mg tissue Kd= 5.21xl(r10M
3.53.0-
2.52.01.51.0-
0.5-
Ck
*
"
°
o°°
*
.
.
ph
d; I.E. Johnson,
b sc
Patients whose breast carcinomas possess only low concentrations of a receptor molecule that binds estrogens with high affinity are unlikely to respond to
hormonal manipulative therapy when the disease recurs. The estrogen-binding capacity of 106 breast carcinomas was measured by an in vitro method and was expressed per milligram wet weight and in some cases related to the concentration of deoxyribonucleic acid (DNA) of the tumours. The ability of tumours to bind 3H-estradiol ranged from 0 to 1.3 fm/mg in pre- and perimenopausal women, and from 0 to 16.8 fm/mg in postmenopausal women. Menopausal status or serum concentrations of endogenous estrogen, or both, should therefore be considered when tumours are classified into low and high estrogen-binding capacity. It is not necessary to carry out Scatchard analysis for every tumour, and expressing estradiol binding on the basis of DNA concentration may be preferable to
expressing it
on a
wet-weight basis.
Les malades dont le cancer mammaire ne contient que de faibles concentrations de la molecule receptrice ayant une forte affinite de fixation pour les estrogenes ne reagiront probablement pas a une hormonotherapie si la maladie recidive. La capacite estrogenofixatrice de 106 cancers mammaires a ete mesuree par une methode in vitro et a ete exprimee par miHigramme de tissue humide et, en certains cas, reliee a la concentration d'acide desoxyribonucleique (DNA) des tumeurs. La capacite des tumeurs de fixer I'estradiol marque au tritium (3H) a varie de 0 a 1.3 fm/mg chez les femmes premenopausees ou perimenopausees, et de 0 a 16.8 fm/mg chez les femmes postmenopausees. II importe done de tenir compte du statut de la femme au point de vue menopausique ou des concentrations seriques d'estrogene endogene, ou des deux facteurs, quand les tumeurs sont classees dans les categories de capacite estrogenofixatrice faible ou forte. II n'est pas necessaire de pratiquer l'analyse de Scatchard pour toutes les tumeurs, et il est preferable d'exprimer La capacite fixatrice de I'estradiol d'apres la concentration des tumeurs en DNA qu'apres la base de tissu humide.
From the department of surgery, Banting Institute, University of Toronto, and Women's College Hospital, Toronto of surgery, Banting Reprint requests to: Dr. B.G. Mobbs,ONDepartment M5G 1L5 Institute, 100 College St., Toronto,
216 CMA JOURNAL/FEBRUARY 7, 1976/VOL. 114
There is
now
little doubt that in vitro measurement of
high-affinity binding of estradiol to specific protein macromolecules in the cytoplasm of human breast carcinomas can be of value in aiding the clinician to predict the response of recurrent disease to endocrine ablation or hormonal therapy. Evaluation of the data of 13 groups of investigators from eight countries showed that patients with breast carcinomas of low estrogen-binding capacity are unlikely to respond to hormonal manipulation.1 Treatment by endocrine ablative surgery or additive hormonal therapy is therefore not indicated in these patients. As yet there have been no data published from such an investigation in Canada. We now report results from an ongoing study of the estrogen-binding capacity of breast carcinomas from patients at Women's College Hospital, Toronto. Materials and methods
Estrogen-binding capacity was investigated in 106 breast deposits, mainly from lymph nodes. One primary tumour was from a 64-year-old man. Samples of two benign tu¬ mours (one specimen of fibrocystic disease and one fibroadenoma) and samples of nontumorous breast tissue from three patients were also examined. Tumour samples (usually 0.1 to 0.5 g) were placed in an iced container as soon as possible after removal and trans¬ ferred to the laboratory. Each sample was trimmed of fat and extraneous tissue and a portion was fixed for histologic examination. From this point on, minor modifications in technique have been made over the period of the study, but there is no indication that the modifications have changed appreciably the results obtained. Our current meth¬ od is described below, with the differing details of the earlier method in parentheses. The samples are frozen and stored in a liquid nitrogen container (formerly at .17 °C) until assay. The frozen tissue is pulverized in a stainless steel pulverizer (Thermovac Industries Corp., St. Copiague, New York), cooled with an liquid nitrogen and the resulting powder transferred to are iced homogenizer vessel. All subsequent procedures carried out at 0 to 4°C. Tissue is homogenized in TRIS 1.5 mM ethylenhydrochloride buffer (pH 8.0) containing concentration of ediaminotetraacetate (EDTA), the final 100 Homogenizamg/ml. being approximately homogenate carcinomas, including 97 primary tumours and 9 metastatic
ice-cooled vessel with a Potterhomogenizer, with 15-second bursts of 790
tion is carried out in
Elvehem-type
an
rpm
separated by 45-second cooling periods. The homo-
genate is centrifuged at 1000 x g for 10 minutes. Any fat present forms a layer over the supernatant and can be
removed. An aliquot for estimation of deoxyribonucleic acid (DNA)2 is taken either from the homogenate before centrifugation or from the pellet afterwards. The super¬ natant is recentrifuged at 98 000 x g (formerly 48 000 x g) for 1 hour, then the supernatant (cytosol) is diluted 1:1 with buffer and used for assay. Replicate aliquots are in¬ cubated with 1.0 x 109 M 3H-estradiol (specific activity, 85 to 105 Ci/mmol (formerly 40 to 50 Ci/mmol) (New England Nuclear, Boston). Unlabelled estradiol is added to some aliquots at a concentration of 4 x 10~7 M. Blanks containing 1.0 ml buffer instead of cytosol are run concurrently. Incubation is carried out at 4°C overnight (for¬ merly at 25 °C for 30 minutes). Separation of bound from unbound steroid with dextran-coated charcoal and extrac¬ tion of bound steroid are carried out as previously described.3 The difference in the amount of radioactivity in the absence and in the presence of unlabelled estradiol is considered to be due to binding by the high-affinity estrogen receptor. This is expressed as femtomoles (10'15 moles) of 3H-estradiol bound per milligram of tissue homogenized or per microgram of DNA in the homogenate. Scatchard plot analysis4'5 was carried out on 17 carcino¬ ma specimens, with a range of 3H-estradiol concentrations from 1.2 x 1010 to 2.0 x 109 M. Low-affinity binding was determined by adding unlabelled estradiol to give a concen¬ tration 400 times that of the labelled hormone. The disso¬ ciation constant (Kd) of the estradiol receptor complex was calculated from the slope of the curve, and the con¬ centration of binding sites by extrapolation of the curve to the
Results For all the carcinomas investigated, estrogen-binding from 0 to 1.3 fm/mg in premenopausal women and in women within 5 years of the menopause, and from 0 to 16.8 fm/mg in postmenopausal women (Fig. 1). The values for estrogen-binding in the secondary de¬ posits fell within the range for the primary tumours. The value for the carcinoma from the male patient was 1.3 fm/mg. None of the nontumourous breast specimens bound detectable amounts of 3H-estradiol, and both the benign specimens had low binding capacity: 0.11 fm/mg for the specimen of fibrocystic disease and 0.12 fm/mg for the
capacity ranged
fibroadenoma. Scatchard plot analysis yielded Kds ranging from 9.1 x 10"9 to 3.4 x 10n M, and the majority were of the order of 1010 M, demonstrating high-affinity binding be¬ tween the 3H-estradiol and its receptors. One of these plots is illustrated in Fig. 2. The concentration of estradiol bound by receptors, as determined from the Scatchard analysis, was plotted against the concentration of estradiol bound at the "single-dose" incubation concentration of 1.0 x 10'9 M for the same tumours (Fig. 3). The two methods gave equivalent values except for tumours of very high binding capacity, in which not all the sites were saturated at this concentration. Thus, for tumours with a receptor concen¬ tration of estradiol greater than 3 fm/mg, incubation at 1.0 x 10~9 M 3H-estradiol may result in underestimation of the estrogen-binding capacity.
abscissa.
. (16.8)
5.5-
Premenopausal o Perimenopausal a
5.0"
Postmenopausal . Male
d*
4.5-
i
4.0n= 4.85 fm/mg tissue Kd= 5.21xl(r10M
3.53.0-
2.52.01.51.0-
0.5-
Ck
*
"
°
o°°
*
.
.
