Estrogen Receptor Assay of Carcinomas of the Breast by a
Simplified Dextran-Charcoal Method
JOHN S. MEYER, M.D., SUE C. STEVENS, PH.D., WILMA L. WHITE, B.S., AND BARBARA HIXON, B.S.
Meyer, John S., Stevens, Sue C , White, Wilma L., and Hixon,
Barbara: Estrogen receptor assay of carcinomas of the breast
by a simplified dextran-charcoal method. Am J Clin Pathol 70:
655-664, 1978. Carcinomas of the breast from 352 women were
assayed for binding of tritiated estradiol by tumor cytosol with
dextran-charcoal adsorption, saturation analysis, and twopoint Scatchard plots; the level of saturable binding defined a
cytosol as positive or negative for estrogen receptor. Valid
assays were obtained on specimens as small as 120 mg. Assays of
replicate samples of a cytosol were more reproducible than
assays of replicate samples of the tumor itself. Occasional
disparity of results between a primary mammary carcinoma
and its axillary metastases could be related to differences in
tumor cellularity. Saturable binding consistent with the
presence of estrogen receptor was found in 59% of 305 primary
carcinomas and in 57% of 47 metastatic or recurrent
carcinomas. There was a significant negative correlation between the patient's age and saturable estrogen binding in the
tumor. Serum estradiol levels of less than 250 pg/ml appeared to
have a negligible effect on estrogen receptor content. A small
subgroup of high-binding carcinomas had high dissociation
constants, but the significance of this observation is not clear.
(Key words: Breast; Carcinoma; Receptor; Hormone; Estrogen.)
THE FIRST PUBLISHED report of hormonal therapy
of mammary carcinoma, by Beatson, appeared in 1896.'
Interest in this form of therapy has been sustained since
a report by Huggins and Bergenstal in 1952 of the
efficacy of adrenalectomy in management of advanced
carcinoma of the breast. 8 Experience indicates that
nearly 50% of premenopausal patients benefit from
oophorectomy, but only 20-25% of postmenopausal
patients respond favorably to adrenalectomy or hypophysectomy. 18 The work of Folca and co-workers,
who demonstrated greater binding of radioactive hexestrol by responsive than by nonresponsive tumors,
pointed toward development of a practical method for
prediction of response. 5 Following description of
estrogen receptor protein in the rat uterus by Toft and
Gorski, 29 Jensen and co-workers reported detection of
estrogen receptor in human mammary carcinomas. 10
Received July 8, 1977; received revised manuscript August 15,
1977; accepted for publication August 15, 1977.
Financial aid was provided by the Miriam United Order of True
Sisters.
Address reprint requests to Dr. Meyer: Department of Pathology
and Laboratory Medicine, The Jewish Hospital of St. Louis, 216
South Kingshighway, P.O. Box 14109, St. Louis, Missouri 63178.
The Jewish Hospital of St. Louis and the Department of
Pathology and Division of Biostatistics, Washington
University School of Medicine, St. Louis, Missouri
Subsequently, the correlation between estrogen receptor content of carcinomas of the breast and their
responsiveness to steroid hormonal therapy has been
established in a number of laboratories. 319 The
methods used most often for assay of estrogen receptor
have employed: (1) dextran-charcoal to remove
unbound tritiated estradiol-17/3 ( 3 H-E 2 ), supplemented
by Scatchard plot analysis, and test for saturability of
3
H-E 2 binding sites by preincubation of cytosol with unlabeled estrogen, or (2) sucrose gradient ultracentrifugation of cytosol following incubation with 3 H-E 2 and
fractionation of the gradient to determine the sedimentation coefficient of binding proteins, supplemented
with saturation analysis. These two methods give good
agreement in classifying cytosols as positive or negative for estrogen receptor. 21 The advantages of the dextran-charcoal method are that it requires relatively
small amounts of cytosol and does not require long-term
ultracentrifugal sedimentation. Sucrose gradient ultracentrifugation measures sedimentation coefficient,
which can differentiate between estrogen receptor and
binders of lower affinity. Positive identification of estrogen receptor by the dextran-charcoal method requires determination of the dissociation constant by
multipoint Scatchard plots, and the identification should
be confirmed by demonstration of specificity of steroid
binding. 29 Such an exhaustive analysis would preclude
routine clinical application and would require larger
volumes of tissue than often are available. Therefore,
we have used a simplified dextran-charcoal assay. In
the absence of rigorous identification of estrogen receptor, we use the term "saturable binding" (of
estradiol) in its place. Our purposes in analyzing our
data include estimation of the reproducibility of the
assay, comparison of results from biopsy of the primary
lesion, the residual tumor in the mastectomy specimen,
and the metastatic tumor in axillary lymph nodes, and
study of the relationships between the age of the patient
and circulating estradiol levels and saturable estrogenbinding of the tumor.
0002-9173/78/1000/0655 $01.05 © American Society of Clinical Pathologists
655
656
MEYER ETAL.
Materials and Methods
Patient Population
Specimens of mammary carcinoma from 352 patients
were analyzed for estrogen receptor. Three hundred
five of the carcinomas were primary lesions, and 47
were either metastatic or locally recurrent. The specimens originated in 18 different hospitals, and eight hospitals submitted at least ten specimens each of primary
carcinomas of the breast. The mean age of patients with
primary carcinomas was 57.1 years, and the mean age
of patients with metastatic carcinomas was 53.1 years.
Sampling and Storage of Tissue
Specimens were obtained at the time of surgical
biopsy or mastectomy. Neoplastic tissue was trimmed
to exclude adjacent structures and necrotic areas. It
was then frozen by methods that varied with different
institutions and included -70 C with or without immersion in TED buffer (composition indicated below),
dry ice (-78.5 C) without buffer, liquid nitrogen
(-196 C) without buffer, and -20 C without buffer.
Specimens from outside institutions were transported
to our laboratory on dry ice on the day of or the day
following excision, and they were always received
frozen. They were then stored at -70 C for less than a
week prior to assay.
Method of Assay for Estrogen Binding
We used the dextran-charcoal method of Korenman,13 as adapted by Johnson, Nakamura, and Libby,12
with minor modifications. The essential features follow.
The tumor was minced with a scalpel during thawing,
and then was homogenized* in 5 ml cold TED buffer (tris[hydroxymethyl]-aminomethane, 10 mM, disodium ethylenediamine-tetraacetic acid, 1.5 mM, dithiothreitol, 1.0 mM, pH 7.4 at 4 C) by three ten-second
bursts at 800 rpm, separated by 30-second cooling intervals with the tubes immersed in ice. Cytosol was
prepared by centrifugation at 100,000 x g (mean) for
60 minutes at 4 C, and protein concentration was measured.16 The cytosol was diluted to a protein concentration of 2 mg/ml (80% of assays) or was used at a protein concentration between 0.66 and 2 mg/ml (20% of
assays), and results from duplicate samples were averaged for all phases of the assay. The 2 mg/ml and the
more dilute cytosols yielded similar rates of positive
assays. Assay tubes contained tritiated estradiol-17y3
(3H-E2), specific activity 93-115 Ci/mmolt 15 or 200
* Model S223 with S31 tubes and S21 pestles, Tri-R Instruments,
Inc., Rockville Centre, N.Y.
t New England Nuclear, Boston, Mass.
A.J.C.P. . October 1978
fmol in 50 (x\ TE buffer (TED without dithiothreitol)
and 200 jul cytosol. For saturation analysis, nonradioactive estradiol-17/3, 50 pmol (a 250-fold excess) in 25
fil TE buffer was added to one set of cytosols prior
to addition of 200 fmol 3H-E2 in 25 /x\ buffer. Blanks
contained either 15 or 200 fmol 3H-E2 with TED buffer
in place of cytosol. All tubes were incubated overnight
at 4 C, and then a 500 /al volume of dextran-charcoal
suspension was added to all tubes except two pairs,
which received 500 ixl distilled water. The latter were
used to derive the denominators for bound/free ratio
calculations. Standards contained 2 pmol 3H-E2/ml TE
buffer. The dextran-charcoal suspension consisted of
dextran, molecular weight 60,000-90,000$ 2.5 mg, and
activated charcoal,§ 250 mg in 100 ml TE buffer. All
tubes were shaken at 180 oscillations per minute at 4 C
for 30 minutes, and the charcoal was sedimented at
1,300 x g at 4 C. Five hundred-microliter samples of
supernatant were transferred to counting vials, and 10
ml scintillant" were added for scintillation counting.
Dextran-charcoal treatment consistently removed
more than 97% of the radioactivity from the blanks.
Calculations were performed according to the method
of Johnson and co-workers.12 A binding index was computed as the fraction of 3H-E2 binding after addition of
200 fmol 3H-E2 that was prevented by prior addition
of nonradioactive E2. For example, a binding index of
0.90 indicated that 90% of the binding was saturable.
A cytosol with binding of 100 fmol 3H-E2/mg protein
and a binding index of 0.90 was considered to have
saturable binding of 90 fmol/mg protein. Twopoint
Scatchard plots28 were drawn from data obtained after
addition of 15 fmol and 200 fmol 3H-E2 to cytosol as
described above. The dissociation constant (Kd) was
calculated as the reciprocal of the slope. Because dissociation constants computed from the Scatchard plots
did not vary consistently in accord with observed binding of 3H-E2, binding index, or saturable binding, and
were often lower when little or no binding was demonstrated than when both binding and binding index
were high, we did not use extrapolated values from
the. plots to estimate binding capacity.
On the basis of assay results from normal breast
and tissues not responsive to estrogen, we designated
specimens with saturable binding of less than 10 fmol/
mg protein negative for estrogen receptor. Specimens
with saturable binding in excess of 49 fmol/mg cytosol
protein were designated high-binding, and specimens
with intermediate values were designated low-binding.
These designations were defined with the realization
that the data do not indicate the existence of sharp
t Sigma Chemical Co., St. Louis, Mo.
§ Norit "A," Fisher Scientific Co., St. Louis, Mo.
" Biofluor, New England Nuclear.
BREAST CANCER ESTROGEN RECEPTOR
Vol. 70 . No. 4
657
Table I. Mean Differences, Standard Deviations, and Ranges of Absolute Differences of Duplicate
Measurements on Mammary Carcinomas Grouped According to Saturable Binding
<35 fmol/mg Protein or >35 fmol/mg Protein
Saturable Binding <35 fmol/mg Protein
Saturable Binding &35 fmol/mg Protein
n
Mean
Difference
SD
Range
n
Cytosol samples
Total binding*
Binding index
K„ x 10-'° M
Saturable binding*
10
10
10
10
0.62
0.067
0.93
0.91
0.58
0.065
0.98
0.37
0.09-1.65
0.01-0.21
0.00-2.6
0.02-1.38
10
10
10
10
Tumor samples
Saturable binding*
21
8.28
0.10-30.0
11
10.12
Mean
Difference
6.02
0.026
1.65
6.61
46.27
SD
Range
5.37
0.033
3.58
5.85
0.02-18.19
0.01-0.10
0.05-11.5
0.90-18.3
32.57
15-110
* fmol estradiol-17/3 bound per mg cytosol protein.
divisions between levels of estrogen-binding in carcinomas of the breast, but with recognition of the
need to define groups for the purpose of clinical decisions.
Serum Estradiol-17(3
Measurement
Blood was obtained from many of the patients who
had mammary carcinomas, in particular, those less than
60 years old, intraoperatively or the next morning.
Total (bound + free) estradiol-17/? was measured in
serum by radioimmunoassay after extraction with
diethyl ether and separation of estradiol from estrone
and estriol on a Sephadex LH-20 column. Antiserum
prepared in sheep was used to bind estradiol-17/3.
Reagents and procedural details were obtained from
New England Nuclear Corporation. 25
tein, precision was greater, i.e., standard deviations
were smaller, than when saturable binding was 35
fmol/mg protein or more, for total binding, K„ and
saturable binding but not for the binding index (Table 1).
Analysis of duplicate tissue specimens from 32 carcinomas of the breast also showed greater precision
for saturable binding when the values were less than 35
fmol/mg protein. For both categories, saturable binding
was more variable for the pairs of tissue specimens
than for the pairs of cytosol specimens (Table 1). This
occurred despite the fact that values for tissue samples
were derived from analysis of their cytosols in duplicate.
The additional variability between tumor samples in
comparison with cytosol samples can be attributed to
differences in cellularity and perhaps in cellular content of estrogen receptor from one site to another in
the tumor.
Processing of Data
Binding and saturable binding of :IH-E2 by cytosol,
expressed as fmol/mg cytosol protein, were rounded to
the nearest integer. The values for total binding, saturable binding, and K(, were log-normally distributed,
and a log10 transformation was made for those variables. For purposes of taking logarithms, binding values
of zero were arbitrarily reclassified as 0.1. In order
to assess the variation in assay values of more than
one sample from a given mammary carcinoma cytosol,
the differences within pairs were analyzed for randomly
chosen cytosols, ten with saturable binding less than
35 fmol protein and ten with bindings of 35 fmol/mg
protein or more. The mean difference between the
paired values and the standard deviation of the mean
difference (SD) were calculated.
Results
Reproducibility
of the Assay
Analysis of duplicate cytosol assays showed that
when saturable binding was less than 35 fmol/mg pro-
Effect of Re free zing of Cytosol
Consistent losses in saturable binding, which were
attributable to loss of binding of 3 H-E 2 rather than to
decrease in binding index, occurred when cytosol was
frozen and stored for seven days at - 7 0 C. The
loss approximated 30%. Losses were not evident when
separate samples of the same tissue were analyzed at
an interval of two weeks with storage at - 7 0 C (Fig. 1).
Mammary and Estrogen-nonresponsive
Involved by Carcinoma
Tissues Not
The results of assays of 25 specimens are summarized in Table 2. The only specimen that exceeded
10 fmol/mg protein saturable binding of :i H-E 2 was a
highly cellular intraductal papilloma from a 60-year-old
woman. These observations support the use of 10
fmol/mg protein 3 H-E 2 binding as a dividing point to
separate mammary carcinomas designated estrogen receptor-positive from those designated estrogen receptor-negative.
658
MEYER ETAL.
300
tient's age (r = -0.331, P < 0.00001). When two outlying values of serum E 2 (571 pg/ml and 3,600 pg/ml,
the latter associated with pregnancy) were eliminated,
serum E 2 showed only slight correlation with log saturable binding (r = -0.107, P = 0.05). The partial correlation between log saturable binding and serum E 2 ,
removing the effect of age, was -0.010. Therefore,
the apparent correlation was actually due to the correlation of both serum E 2 and log saturable binding
with age. However, none of the six carcinomas associated with serum E 2 in excess of 250 pg/ml showed
saturable 3 H-E 2 binding in excess of 49 fmol/mg cytosol
protein, whereas binding in excess of 49 fmol/mg protein was found in 67 of the 221 specimens associated
with lower serum E 2 levels. While serum E 2 levels
less than 250 pg/ml appear to have little effect on the
estrogen receptor content of tumors, the data are consistent with the possibility that higher E 2 levels may
be associated with decreased binding of 3 H-E 2 .
Cytosol
Tissue
5 250
<b
• * *
5
g> 200
I
5? 150
I
.e:
,»
100
S 50-
14
Days stored
A.J.C.P. • October 1978
at
-70 C
FIG. I. Effect of refreezing and storage of mammary carcinoma
cytosol on saturable binding in comparison with the effect of continued storage of tissue without thawing and refreezing. Two pieces
of each of four tumors were assayed. A cytosol was prepared from
the first piece after less than seven days of storage at -70 C. The
cytosol was assayed immediately, was then stored at -70 C, and was
reassayed seven days later. Fourteen days after preparation of cytosol from the first piece of tumor, the second piece was removed
from -70 C storage, and a cytosol was prepared from it. The
cytosol was assayed immediately and again after storage at -70 C
for seven days. Refreezing and storage of cytosols was associated
with loss of saturable binding in seven of eight instances, whereas
prolongation of storage of whole tumor did not cause loss of
saturable binding in freshly prepared cytosols.
3
H-E2 Binding of Carcinomas of the Breast
The results of saturable 3 H-E 2 binding assays of 305
primary carcinomas of the breast and 47 carcinomas
that were metastatic or recurrent following mastectomy
are presented in Table 3. The geometric mean of the
primary carcinomas was slightly greater than that of
the metastatic carcinomas, but the difference was not
significant when tested by analysis of variance {P
> 0.38). The proportions of primary and metastatic or
recurrent carcinomas in each of the three arbitrarily defined classifications of binding were not significantly
different.
Log saturable binding was correlated with the age of
the patient for both primary (r = 0.315, P < 0.00001)
and metastatic sites (r = 0.221, P < 0.004) (Fig. 2). The
rather low, although highly significant, correlation coefficients reflect the wide ranges of saturable binding
values found in all age groups.
Serum estradiol-17/3 (E2) was measured at the time
of excision of 231 mammary carcinoma specimens
(Fig. 3). Serum E 2 correlated negatively with the pa-
Specimens from both the primary carcinoma in the
breast and metastatic carcinoma in axillary lymph
nodes were analyzed for estrogen binding in 26 instances. Saturable binding in primary and metastatic
tumors was similar in most instances (Fig. 4). The geometric mean was 9.23 fmol/mg protein for the primary
lesions and 12.7 for the metastases. A t test for paired
data showed no significant difference (P > 0.20). However, differences observed in four instances deserve
comment, because in each the axillary metastases were
high-binding, whereas the primary lesions were negative
for estrogen receptor (two instances) or low-binding
(two instances) (Table 4). Two tumors were infiltrating
lobular carcinomas with low cellularity in the primary
lesion, and the others were infiltrating ductular carcinomas. Microsections of the metastasis were available from three of the cases and showed higher cellularity. Therefore, the higher saturable binding values
of the metastatic lesions might be explained by their
higher cellularity with resultant lesser dilution of carcinoma cytosol protein by extraneous protein.
Table 2. Saturable 3 H-E 2 Binding of Tissues Other
Than Mammary Carcinoma
Tissue
Normal breast
Pregnant breast
Benign proliferative mammary
lesions
Gynecomastia
Estrogen-nonresponsive
tissues
Number
of
Cases
Saturable 3H-E2
Binding
fmol/mg Protein
Median
Range
1
1
11
1
0-85
13
0-9
BREAST CANCER ESTROGEN RECEPTOR
Vol. 7 0 . No. 4
Two or occasionally three samples of the same tumor
were assayed in 32 instances, and the results showed
closer correspondence without the occasional wide
variations that were sometimes observed when primary
lesions were compared with metastatic lesions (Fig. 4).
In 21 instances results of assay of the surgical biopsy
could be compared with results from residual tumor
in the mastectomy specimen. The geometric mean of
the biopsies was 8.42 fmol/mg protein, and the geometric mean of the mastectomy residuals was 11.9
fmol/mg protein. By paired t test the difference was
not significant. However, when six pairs in which either
or both assay values were less than or equal to 1.0
were omitted, the respective geometric means were
22.9 and 33.0, and the difference was significant (P
< 0.05). The mastectomy specimen usually afforded a
better opportunity for selection of tumor with the gross
appearance of high cellularity and viability than did
the biopsy specimen, and this may account for the
higher values observed on assays of tumors from
mastectomies.
The geometric mean saturable bindings for primary
mammary carcinomas from eight hospitals that submitted at least ten specimens were computed, and analysis of variance showed no significant difference among
the hospitals (P = 0.19). Therefore, the different methods of freezing, which included -20 C, -70 C, dry
ice, and liquid nitrogen at different hospitals, did not
appear to affect the results of estrogen receptor assay.
The assay generated three pieces of data that have
been used to judge the presence or absence of estrogen
receptor: the total binding as fmol/mg protein after incubation with a large dose of E2 (200 fmol/0.4 mg
cytosol protein),21 the binding index, and the dissociation constant.12 Of these three variables, only the log
total binding and the binding index correlated highly
(r = 0.723, P < 0.00001) (Fig. 5). Log Kd showed a low
but significant negative correlation with log saturable
binding (r = -0.197, P < 0.0001), and log Kd showed
a somewhat higher negative correlation with binding
index (r = -0.449, P < 0.00001). A scatter plot of dissociation constants against saturable binding suggested
a tendency for high dissociation constants to occur with
659
400
• Primary
200
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recurrence
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1 -j.8—o-,:—4. 9§| b—|» l » •'•*] -4 • 1-j—°—r—
20
30
40
50
60
70
80
Age, years
90
FIG. 2. Saturable estradiol-17/3 binding of 352 carcinomas plotted
on a log scale against the age of the patient. Axillary metastases from
radical mastectomy specimens are not included in the plot. Values of
zero are plotted as 1 (r = 0.315, p < 0.00001 for primary tumors;
r = 0.221, p < 0.0004 for metastases).
low and to a lesser extent with high saturable bindings, but not with saturable bindings between 35 and
110 fmol/mg protein (Fig. 6). Dissociation constants
exceeded 10~9 M in eight of 114 specimens with saturable
binding in excess of 50 fmol/mg protein, and in these
cases binding indices always exceeded 0.9. Dissociation constants exceeded 10-9 in two of 72 specimens
with saturable binding between 15 and 50, and in 35
of 159 specimens with saturable binding less than 15. In
the latter 159 instances binding indices usually were
less than 0.9.
Specimen weights ranged from 0.12 to 2.24 g, but
in only seven instances were they less than 0.25 g,
Table 3. Estrogen Receptor in Mammary Carcinomas
3
H-E 2 Saturable Binding
fmol/mg Cytosol Protein
Primary
Metastatic
or recurrent
Classification
Range
Negative
(<10
fmol/mg P)
Low-binding
(10-49
fmol/mg P)
High-binding
(>50
fmol/mg P)
54.1
0-399
125 (41%)
81 (27%)
99 (32%)
44.6
0-285
20 (43%)
15 (32%)
12 (25%)
No.
Geometric
Mean
95%
Confidence
Interval
Arithmetic
Mean
305
13.6
10.8-17.0
47
10.3
5.6-18.9
660
MEYER ETAL.
A.J.C.P. • October 1978
400
• •• •
100- • • 1
|
so-!;.- . "
!
binding fM/mg
IFV ••' ' . '
1 ••
. .
••••
•1
•
I
•
1*1t
13 "
•l"l
FIG. 3. Saturable binding of estradiol-17/3 on a
log scale and serum estradiol-17/3 concentration
at the time of biopsy or excision of 230 mammary carcinomas. An additional patient who
was pregnant and whose carcinoma showed no
saturable estradiol binding in the presence of
a serum estradiol level of 3,600 pg/ml is omitted.
Saturable binding values of zero are plotted as
0.1 (r = -0.164, P = 0.006); when the patient
with serum E2 = 571 is omitted, r = -0.107,
P = 0.054).
•
•
•
••
•
•
•• • * •
.1 •
•
••
s
0.1 -
1
c)
• •
i
r
i
50
100
150
Serum
— I —
200 250
— I —
300
estradiol -17fi,
— I —
350 400
600
500
pg/ml
and in only four instances were they more than 1.75 g.
Total protein content of the cytosol and cytosol protein per gram tumor wet weight were calculated for the
last 254 specimens assayed. Protein extraction from the
smaller specimens tended to be more complete because
the standard 30-second time of homogenization allowed their more complete disruption. The mean
cytosol protein per gram of tumor assayed was 15.6
mg for 50 consecutive specimens, and the range was
5.1 to 35.6 mg. Saturable binding did not correlate with
specimen weight (r = 0.010, P - 0.42). Thus we have
evidence that small specimens can give valid results.
Log saturable binding showed a low order of negative
correlation with cytosol protein concentration per gram
400
!•
• Primary carcinoma, biopsy
200
•t
o Primary carcinoma, mastectomy
* Axillary metastasis
ill
5 IOO
I
• a
fc. 50 H
^
30
£
20 H
FIG. 4. Saturable estradiol-17/3 binding on a log
scale in multiple samplings of primary mammary
carcinomas and axillary lymph nodal metastases.
Each vertical array represents results from a single
patient. Saturable binding values of zero are
plotted as 1 to permit use of a logarithmic scale.
"S
10 -
1
5 3 **»i9
.IsS^oSAtSSft • ».o..
Vol. 70 • No. 4
661
BREAST CANCER ESTROGEN RECEPTOR
tuu-
100-
I
I
.
-
••••
•
50-
-
• tl.l
.1 I M . i *
l0
FIG. 5. Total estradiol-17/3 binding of 352 primary
and metastatic or locally recurrent mammary carcinoma cytosols on a log scale plotted against
binding index (r = 0.723, P < 0.00001).
•
• :j.:»..v
•"
"...
• •
1
v
*
.
" I '
.. | .
.i
.
• •••:.
•
5-1
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• • •• •••
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-
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•
. . ..| •
1 -
•
1
1
0.0
1
0.25
0.50
Binding
tissue homogenized (r = -0.111, P < 0.038). This finding suggests that the extent of cellularity of the carcinomas may be inversely related to the level of estrogen receptor of the carcinoma.
Discussion
The dextran-charcoal, gel filtration, sucrose gradient, and electrophoretic assays have proven to have
predictive value for response of carcinomas of the
breast to hormonal therapy to the extent that approximately 55% of estrogen receptor-positive tumors have
responded to additive or ablative therapy and only approximately 8% of estrogen receptor-negative tumors
have responded.19
The simplified dextran-charcoal assay for estrogen
receptor is attractive because it can be performed on
small volumes of tumor, and the simplicity of the
0.75
i
1.00
1.25
index
technic permits analysis of specimens in batches.
We have found that the proportion of positive assays
is not affected by specimen size in the approximate
range of 0.12-1.5 g, and Johnson and Nakamura" have
reported successful assay of specimens as small as 50
mg. This is a decided advantage because of the small
size of many cutaneous recurrences in the chest wall
following mastectomy. A 5-mm spherical nodule of carcinoma has a mass of approximately 65 mg. Lesser
volumes of tissue would appear to be inadequate for
assay. Unfortunately, even the simplified dextrancharcoal method would not be sensitive enough for
assay of needle biopsies of bone or liver, for a core
of tissue 1.4 mm in diameter and 20 mm long, such
as may be obtained by percutaneous needle biopsy of
the liver,23 has a mass of only 31 mg. Because many
recurrences of carcinoma of the breast involve only in-
Table 4. Low-binding Primary Carcinomas and High-binding Axillary Metastases
Primary Lesion
SPNo.
76-2257
77-755
76-6505
76-7664
Histologic Type
Infiltrating
Infiltrating
Infiltrating
Infiltrating
lobular
lobular
ductal
ductal
Saturable
Binding
fmol/mg Protein
2, 8t
8
17
11, 35t
Metastatic Lesion
Cellularity*
Saturable
Binding
fmol/mg Protein
Cellularity*
0.1
0.1
0.4
0.2
55
75
108, 184$
282
0.7
0.8
0.5
* Cellularity was measured as the product of the fraction of the tumor examined in microsections under scanning magnification that consisted of neoplastic tissue multiplied by the
fraction of 100 points (ocular grid) that fell over neoplastic cells at x 100 magnification in the most cellular area. No section of the axillary metastasis from the first case was available.
t Results of assays of two separate samples of primary carcinoma.
X Results of assays of two different axillary metastases.
662
A.J.C.P. • October 1978
MEYER ETAL.
115-r
100
75
FIG. 6. Dissociation constants of 352 primary and
metastatic or locally recurrent mammary carcinoma
cytosols for binding of estradiol-17/3 plotted against
saturable binding, the latter on a log scale (r
= 0.002, P = 0.48). (For log K« correlated with log
saturable binding, r = -0.197, P < 0.0001.)
«>
O
5040 J .
30
.<0
ii
20 -l' !
u^..ii.) I...Y_J\,:
200
25 50
100
Saturable binding, fM/mg
300
400
protein
ternal structures and major surgery is necessary to obtain large biopsies, it is desirable to obtain data routinely from the primary lesions. Block and co-workers2
found that results of estrogen receptor assay of carcinoma of the breast usually remained consistent when
repeat samples were taken at intervals of as long as 80
months, and suggested that results from the primary
tumor be used to plan therapy of recurrences.
Small specimens, and some larger but poorly cellular
specimens, yield relatively little cytosol protein. The
rates of positive assays, 68% and 56%, respectively,
for cytosols with less than 2.0 mg protein/ml and for
cytosols with 2.0 mg protein/ml when assayed, were not
significantly different (x 2 = 1.386, df = \,P> 0.2). Although our data provide no evidence that moderate decreases in cytosol protein concentration below 2 mg/ml
interfered with detection of estrogen receptor, ideally
all specimens should be assayed at the same cytosol
protein concentration to avoid differences in the efficiency of separation of bound from unbound 3H-E2
by dextran-charcoal. To avoid low protein concentrations in cytosols, small samples can be homogenized
in reduced volumes of buffer, and a filler can be used
during ultracentrifugation to prevent collapse of tubes.11
In the simplified dextran-charcoal assay, E2 binding
is measured after incubation of cytosol with a substantial (0.2 pmol) dose of 3H-E2. We have examined
several high-binding cytosols with multiple-point
Scatchard plots; and in agreement with Johnson and
Nakamura,11 we have found that the 0.015 and 0.02
pmol :!H-E2 points fall on the straight-line segment of
the plot. The use of 0.015 pmol and 0.2 pmol quan-
tities of 3H-E2 permits an estimate of the Kd to be
made, in addition to providing an approximation of
the binding capacity of the cytosol, and dissociation
constants computed from two-point and multiple-point
Scatchard plots performed on the same cytosol usually
agreed within a ratio of 2.11 Binding measured after
addition of 0.2 pmol 3H-E2 underestimated the binding capacity obtained from Scatchard plot extrapolation by no more than 10 fmol/mg protein when the
binding capacity was less than 50 fmol/mg protein.
Binding capacities obtained by extrapolation from
Scatchard plots of high-binding cytosols were underestimated by much greater amounts. But once a cytosol
is established as high-binding, the precise measurement of the binding capacity contributes no further
clinically useful information.
Given the agreement between duplicate cytosol assays in Table 1, when our practice of averaging duplicate cytosol results is followed, we would estimate
the precision of the assay to be within ± 25% for total
binding, binding index, and saturable binding, except
for lesser precision of measurement of saturable binding in low-binding cytosols. Precision of measurement
of the Kd is less, particularly in high-binding cytosols.
The variability of saturable binding measurements on
duplicate tissue samples of carcinomas of the breast
was far greater than can be explained by variability
among cytosol samples. Nonhomogeneous cellularity
of the tumor can be offered as an explanation for the
variability, and the higher cellularity of certain higherbinding axillary metastases in comparison with that
of lower-binding primary tumors supports this explana-
Vol. 70 • No. 4
BREAST CANCER ESTROGEN RECEPTOR
tion. We suggest that inferences of alteration in estrogen receptor content of tumors should not be drawn
without careful comparison of the cellularity of the
tumors in question, and that all assays should be accompanied by histologic assessment of a representative portion of the specimen. This can be accomplished
by removing and fixing a thin slice through the center
of the specimen prior to freezing it for estrogen receptor assay.
Our comparisons between saturable bindings of
tumor from breast biopsy and mastectomy specimens
indicate that the receptor activity remains constant
during a period of devascularization between detachment of the breast from the chest wall and completion
of the axillary dissection. We estimate that the duration of devascularization prior to detachment of the
specimen is approximately 30 minutes, during which
the tumor cools toward room temperature. Provided
that the specimens are obtained promptly from the
operating room, assays of estrogen receptor may be
done with confidence on tumor from mastectomy specimens, and the axillary metastases should routinely be
assayed because of occasional instances when their
greater cellularity may enable significant estrogen binding to be detected in them and not in the primary lesion.
Tissue can be stored at - 7 0 C for as long as six months
without loss of receptor," but thawing and refreezing
must be avoided, and cytosoi cannot be frozen and
stored without loss of activity.
Our results for prevalence of estrogen receptor in
carcinomas of the breast are similar to results reported from other laboratories. In eight reports of at
least 100 assays by various methods, the rates for
positivity ranged from 41 to 70%, with a mean of
58%.4.6.14.17.20,22.31 j n fOUr studies primary tumors and
metastases were listed separately; 41 to 72% (mean
57%) of the primary tumors were estrogen receptorpositive and 42 to 58% (mean 50%) of the metastatic
tumors were estrogen receptor-positive. 415 - 20 - 22 We
found saturable estrogen binding in excess of 10
fmol/mg cytosoi protein, which we consider consistent
with presence of estrogen receptor, in 58.8% of our
352 specimens of carcinoma of the breast, and in 59%
of the 305 primary carcinomas, and in 57% of the
metastatic carcinomas. Most studies have shown
slightly higher rates of estrogen receptor positivity in
primary carcinomas of the breast than in postoperative recurrences or metastases, but in our experience
the rates of estrogen receptor positivity were not
significantly different for the two groups, although the
data suggest that the postoperative recurrences or
metastases are less often high-binding than are the primary lesions. Block and co-workers 2 introduced the
separation of estrogen receptor-positive carcinomas of
663
the breast into low-binding and high-binding groups on
the basis of estrogen binding per g tumor wet weight,
and noted that the low-binding tumors did not respond
to hormonal therapy. Heuson and co-workers 7 reported
a significant relationship between estrogen receptor
content per mg cytosoi protein and response to hormonal therapy. We found a range of 5.1 to 35.6 g cytosoi protein per g tumor and a mean of 15.6 g in 50
consecutive tumors. At 15.6 g cytosoi protein per g
tumor, the figure of 750 fmol E2 binding per g tumor
by which Block and co-workers define the lower limit
of high-binding would correspond to 48 fmol E2
binding per mg cytosoi protein, which is close to the 50
fmol/mg cytosoi protein that we have used. We and
others 20 - 2231 have observed less estrogen binding in
tumors from younger (premenopausal) women than in
tumors from older (postmenopausal) women. The effect of high circulating estrogen levels on assayable
estrogen receptor content of the tumor cytosols may
play a role, but our data confirm the impression
that only levels of serum E2 in excess of 250 pg/ml are
likely to affect the estrogen binding of carcinomas. 17
We have recently reported evidence that the lower
estrogen binding found in tumors from younger women
may relate to lesser differentiation and faster growth
rates of those tumors in comparison with tumors of
older women. 24
The K(ts of estrogen receptor from uterus" and from
mammary carcinomas induced in rats by dimethylbenz(a)anthracene 27 are in the range of 0.1 to 10 x 10~10 M.
However, higher K d s have been found in some human
mammary carcinomas that appeared to contain estrogen receptor by the presence of high levels of estrogen-saturable binding.14-22 Thirty of our 111 highbinding mammary carcinomas showed dissociation
constants in excess of 10.0 x 10"'° M (mean 24.4,
range 10.7-92.8). Nine of the lesions with high K d s
were primary, and one was metastatic. Their institutional origins and methods of freezing were similar to
those of the other specimens. The mean age of these
patients was 61.7 years, which exceeds the 56.2-year
mean for the other patients, and the serum E2 concentrations, which were known for eight patients, were
less than 50 pg/ml in six patients. The binding indices
were consistently high (mean 0.98, range 0.96-1.00),
and the mean saturable E 2 binding was 249 fmol/mg
protein (range 175-399). The mean Kd for the 30 patients in question was similar to the 17 x 10"10 M that
characterizes serum testosterone-estrogen-binding
protein (TeBG). 26 Leclercq and co-workers found
dissociation constants in excess of 20 x 10 -10 M for
nine of 73 breast carcinomas that they judged positive
for estrogen receptor. 14 They considered several possible explanations, including contamination by TeBG,
MEYER
664
and appreciable quantities of albumin present in some
cytosols indicated contamination with plasma proteins.
However, neither we nor they have found significant
binding in samples of nontarget tissues or sera of
women of comparable age. The presence of TeBG in
mammary carcinoma cytosol has been demonstrated
by electrophoresis.30 Its concentration in serum increases in response to estrogens,26 but we interviewed
five of the patients who had high-binding, high-Kd
carcinomas with regard to estrogen therapy, with uniformly negative results. Because the significance of the
high K(l in certain high-binding mammary carcinomas
is not understood, prospective studies will be necessary
to determine whether tumors with these characteristics
will respond to hormonal therapy.
Acknowledgments.
Walter C. Bauer, M.D.. Thomas J. Cooper,
M.D., Carl G. Kardinal, M.D., Frederick T. Kraus, M.D., Carlos
Perez-Mesa, M.D., William H. Sheffield, M.D., and Eugene F.
Tucker, M.D., contributed specimens of mammary carcinoma.
Thomas C. Cantley, M.S., Rhoda Facher, B.S., Nancy Van
Dillen, A.B., Ann Ward, Joann Weisler, B.S.. and Amy E. White,
B.S., furnished technical assistance; and B. Ramanath Rao, Ph.D.,
gave advice.
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