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[CANCER RESEARCH 42, 4812-4814,
0008-5472/82/0042-OOOOS02.00
November 1982]
Cytoplasmic and Nuclear Estrogen and Progesterone Receptors in Male
Breast Cancer1
Rosemary J. Pegoraro,2 Dharamraj Nirmul, and Septimus M. Joubert
Departments of Chemical Pathology ¡P..J. P. , S. M. JJ and Surgery [D. N.¡,University of Natal, P. O. Box 17039, Congella 4013, Durban, South Africa
ABSTRACT
Cytoplasmic estrogen receptors were detected in 12 of 13
male breast cancer tumors. There was no significant correlation
of receptor levels with the age of the patient, size and histológ
ica! grading of the tumor, and stage and nodal involvement of
the disease. Nuclear estrogen receptors were found in eight of
10 tumors and six of nine patients had tumors positive for
Cytoplasmic progesterone receptors, two of which were also
found to contain nuclear progesterone receptors. The presence
of Cytoplasmic progesterone receptors, in addition to cytoplasmic and nuclear estrogen receptors, may be indicative of truly
hormone-dependent tumors in male breast cancer. Treatment
offered to patients included surgery, X-ray therapy, chemo
therapy, and orchiectomy, but as yet, no general conclusions
of the efficacy of the therapeutic regime can be drawn.
INTRODUCTION
Estrogen receptors have been studied extensively in neo
plasms of the female breast, and their measurement is now
firmly established as an aid in predicting response to endocrine
therapy (19) and as a guide to prognosis (5, 16). In the course
of an ongoing study of estrogen and progesterone receptors in
female breast cancer, malignant tumor tissue from 13 male
patients has been examined. Wherever possible, both CERs3
and NERs have been measured, since it is the nuclear bound
receptor that is believed to be necessary for the expression of
estrogen-mediated protein synthesis (19). It has been shown
in rat uteri that uterine responses and growth following estrogen
administration can be correlated to the number of estrogen
receptor complexes in the nuclei and to the duration of nuclear
binding (1), clearly illustrating the importance of measuring
nuclear receptors. Progesterone receptors have been mea
sured because their production is thought to be estrogen
dependent (13, 17) and their presence is predictive of an
improved response to endocrine therapy in females (4, 12).
In this paper, the steroid receptor status has been analyzed
in relation to the age of the patient, size and histological grading
of the tumor, and stage and nodal involvement of the disease,
and where available, the response to therapy is presented.
MATERIALS
AND
METHODS
Clinical Material. Breast tumor tissue was obtained either by biopsy
' This study was supported
by the South African Medical Research Council
through the Preclinical Diagnostic Chemistry Research Group.
1 To whom requests for reprints should be addressed.
3 The abbreviations used are: CER, Cytoplasmic estrogen
nuclear estrogen receptor; CPR, Cytoplasmic progesterone
clear progesterone receptors.
Received April 6, 1982: accepted August 10, 1982.
4812
receptor;
receptor;
NER.
NRP, nu
or at mastectomy from 13 men. Ten were black, 2 were Asian, and one
was white; their ages ranged from 25 to 77 years. In none of the
patients was there any clinical evidence of gynecomastia. Two of the
patients were classified as Stage II, 7 as Stage III, and 4 as Stage IV
according to the American Joint Committee Staging Manual Criteria.
Twelve of the tumors were histologically adenocarcinomas; one was a
squamous carcinoma. Tumor tissue was placed in 0.15 M NaCI on ice
and transported to the laboratory where it was trimmed of fat and
assayed immediately or stored in liquid nitrogen.
Homogenization of Tissue. Tumor tissue frozen in liquid nitrogen
was homogenized using a microdismembrator
(Braun, Melsungen,
West Germany) as recommended by the European Organisation for
Research on Treatment of Cancer Breast Cancer Co-operative Group
(7).
Chemicals and Reagents. 17/S-[2,4,6,7-3H]Estradiol (85 to 110 Ci/
mmol), [6,7-3H]ORG 2058 (40 to 60 Ci/mmol), and unlabeled ORG
2058 were obtained from the Radiochemical Centre, Amersham, United
Kingdom. All other chemicals used were Analar reagent grade.
Estrogen Receptor Assay. The assay method used has been de
scribed in detail previously (22) and therefore will be outlined only
briefly here. The homogenizing buffer used was 20 mw 4-(2-hydroxyethylM-piperazineethanesulfonic
acid, 1.5 mw EDTA, and 0.25 mw
dithiothreitol, pH 7.4. About 150 mg tumor tissue were pulverized and
homogenized in 3 ml buffer and then centrifuged at 700 x g for 10 min
to yield a crude cytosol and nuclear pellet. The nuclear pellet was
washed twice to remove trapped Cytoplasmic debris. In order to deter
mine whether nuclei were free of Cytoplasmic contamination, lactate
dehydrogenase activity was measured in 6 different nuclear suspen
sions and cytosols. In each case, the lactate dehydrogenase activity
detected in the nuclear suspension was negligible or insufficient to
account for the level of receptors measured. Portions of the tumor
supernatant and nuclear suspension were incubated separately at 4°
without shaking for 18 hr with 17/î-[3H]estradiol, serially diluted to give
final concentrations in the range 0.1 to 0.8 nw. Additional portions of
both fractions were incubated with 0.8 nw 17/H3H]estradiol
Plus a
100-fold excess of diethylstilbestrol.
Appropriate
blanks were included
in the cytoplasmic assay. Free and loosely bound ligand was removed
by 0.15% dextran-coated charcoal in the cytosols, while nuclear bound
radioactivity was separated on Whatman GF/C filters, using a Millipore
filtration unit. The data were plotted according to Scatchard (25) and
a straight line was calculated by the method of least squares. Receptors
were deemed present if the Kd was less than 7 x 10~'° M.
Progesterone Receptor Assay. Progesterone receptors were mea
sured in the same tumor homogenate as the estrogen receptors. All
buffers used were the same as those used for the assay of estrogen
receptors with the inclusion of 10% glycerol (v/v) (23). The assay
procedure and conditions were identical to those used for estrogen
receptors, except that the final concentrations of [3H]ORG 2058 were
in the range 0.1 to 2.4 nw. Additional portions from both fractions were
incubated with 2.4 nw [3H]ORG 2058 plus a 100-fold excess of
unlabeled ORG 2058. Criteria used to determine positivity were similar
to those applied to the assay of estrogen receptors except that the Kd
was expected to be in the range of 2 to 10 x 10~'° M (2).
Protein and DNA Assays. Cytosol protein concentration was mea
sured by the method of Lowry ef al. (18). The DNA content of the
nuclear fraction was determined by the method of Burton as modified
by Katzenellenbogen and Leake (14).
CANCER
RESEARCH
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VOL. 42
Steroid Receptors in Male Breast Cancer
RESULTS
Estrogen Receptors. Of the 13 patients whose tumors were
assayed for CERs, 12 (92%) were found to be positive with
results ranging from 36 to 389 fmol/mg protein (Table 1). Ten
of these tumors were examined for NERs, of which 8 were
positive. Of the 2 which were negative for NERs, one (Patient
5) was the squamous carcinoma and one was the tumor which
was negative for CERs. The 12 patients with CER-positive
tumors were all over the age of 48 years, while the only patient
whose tumor contained no estrogen receptors in either subcellular compartment was aged 25 years. There was, however,
no significant correlation of CER levels with age using the linear
regression analysis (r = 0.3013; p > 0.1). Similarly, no corre
lation could be found between CER values and the size of the
tumor (/•
= -0.4445; p > 0.1 ) (Table 2). Using the Kendall rank
correlation coefficient T, no correlation of any significance
could be observed between the CER levels and the stage of
the disease (r = -0.2883;
p > 0.1), nodal involvement (T =
0.0165; p > 0.5), or the histológica! grade of the tumor (r =
0.1233; p > 0.5) (Table 2).
Progesterone Receptors. Tumors from 9 of the patients in
this series were assayed for CPRs, which were found in 6
specimens ranging in value from 196 to 3231 fmol/mg protein
(Table 1). Only 2 of these specimens showed any evidence of
progesterone binding in the nuclei. Both of these tumors were
positive for CERs and NERs, as were the 4 other tumors with
CPRs only. Two specimens contained estrogen receptors in
both cytoplasm and nuclei, but no progesterone receptors,
while one tumor contained no receptors at all.
Assay Reproducibility. In order to demonstrate the reproducibility of these assays, 11 tumors were assayed on 2 or 3
occasions. The differences in quantitative results varied from
0.02% to 60% possibly due to tumor heterogeneity, but in no
instance did the receptor status change from assay to assay.
Clinical Response. Various treatment modalities were used
in the management of these patients. Primary treatment con
sisted of mastectomy and axillary node dissection, X-ray irra
diation to the chest wall, and draining lymph nodes and adju
vant chemotherapy. Recurrent or metastatic disease was man
aged by a variety of treatment regimens which included orchiectomy, chemotherapy, and X-ray therapy.
Only 3 patients with metastatic disease agreed to orchiectomy as part of the treatment regimen. Patient 2 had orchiectomy and combination chemotherapy for osseous métastases
and his disease showed stabilization for 3 months before he
was lost to follow-up. Patient 6, who developed osseous sec
ondaries 8 months after primary treatment with mastectomy
and X-ray therapy, was further treated by orchiectomy and
single-agent p.o. chemotherapy (cyclophosphamide).
He has
shown stabilization of his metastatic disease for 14 months.
Patient 12, who presented with an ulcerating breast tumor and
spinal métastases,was treated by orchiectomy and X-ray irra
diation to the spinal secondaries. The ulcerated breast lesion
healed following orchiectomy, and there was a 50% reduction
in the size of the breast tumor.
Details of treatment and response of the remaining patients
are not given as they did not receive hormonal therapy and
therefore are not relevant to this paper.
DISCUSSION
In common with observations made throughout the world,
the incidence of male breast cancer in South Africa is low. This
fact considerably impedes the study of steroid receptors in this
disease, and in response to an appeal by Everson et al. (8),
results obtained in our laboratory from 13 male patients over
a 6-year period have been presented. The high prevalence of
CER-positive cancers is in agreement with the literature where
74 of 89 tumors are reported to contain CERs (6, 8-10, 24).
Ten of our patients were black, and although numbers pre
sented here are small, the indication is that there is no variation
in the occurrence of receptors among black and white popuTable 2
CEP concentrations and disease characteristics
in
volve
tee stageIIIIVIIIIIIIIIIIIIIIIIVIIIIVIVIIINodal
ment231b1b31b1b01b1blb3lb
(cm)7x7Multiple
size
Patient12345678910111213CERs(fmol/mgprotein)2183892161099413433618236010349239Tu
lesions5
x56.5x6Ulcération3.5x31
1.53
.Sx
x44x55
x66
X4.51
11.51
1.5 x
x 1WHOhisto-logicalgradeIIIIIIIIIIII1IIIIIII11IIIIIIAmericanJointComm
Table 1
Estradici and progesterone receptors in patients with male breast cancer
CERsPatient1
(yr)59
protein218
M)4.32
fmol/mg
M)Not DNA
10"'°
pro
teinNot
DNANot
done
done
done
doneNot
Not
BlackBlackBlackBlackAsianAsianWhiteBlackBlackBlackBlackBlackfmol/mg
4.014.106.200.860.960.270.661.061.880.990.58NERsK„(x
Not
doneNot
Not
doneNot
2345678910111213Age
506448565352635025755677RaceBlack
3892161099413433618236010349239K„(x10*'°
done1250011521340876102301288116712204.392.051.012.553.592.902.690.89CPRsfmol/mg
done624Not
done0Not
done5166181960032310519Ka(x1.971.173.502.172.450.59NPRsfmol/mg
done12480000665600Ka(X4.413.34
NOVEMBER
1982
4813
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R. J. Pegoraro et al.
lations. This observation has already been made in females
with breast carcinoma (22).
In female breast cancer, it is well established that the re
sponse rate to hormonal therapy among CER-positive patients
is approximately 50 to 60% (2, 4,13). There have been several
reports demonstrating an improved prediction rate, as high as
77%, in the response of patients whose tumors contained
NERs or CPRs in addition to CERs (2, 4, 15). Friedman ef al.
(9) have suggested that, since male breast cancer patients
respond to orchiectomy and most have tumors with high CER
levels, it would be of greater clinical value to identify those
patients who are unlikely to respond to endocrine therapy, so
that an alternative form of treatment could be initiated promptly.
In Table 1, however, it can be seen that, of the 9 tumors
assayed for both estrogen and progesterone receptors, 6
(67%) contained an apparently functional estrogen receptor
system; i.e., the CERs appeared to have retained the ability to
translocate into the nucleus, resulting in the synthesis of the
CPR protein. This figure agrees closely with the 68% objective
regression among 41 castrated males reported by Trêves (27).
In addition, Neifeld et al. (20) reported an objective 62.5%
response rate in their 8 patients and their review of the literature
showed a response rate of 50 to 70%. It may be that only
tumors with CERs, NERs, and CPRs are truly hormone de
pendent, while patients whose tumors contain an impaired
receptor mechanism may benefit more from an alternative
therapy regimen. The significance of the presence of NRPs is,
as yet, uncertain.
As only one of our patients (Patient 12) had orchiectomy as
the only form of systemic therapy, with partial response, gen
eral conclusions regarding the response to hormonal treatment
in relation to receptor status cannot be made. It was interesting
to note, however, that Patient 6, who had a full receptor
complement in his tumor, has shown stabilization of his rib and
skull métastasesfor at least 14 months, following orchiectomy
and p.o. cyclophosphamide.
The single specimen of squamous carcinoma of the breast
was included for interest. Whether such a tumor could be
hormone dependent is undetermined, but low levels of cytoplasmic estrogen receptor have been reported in skin (11 ) and
estrogen receptors have been measured in squamous carci
noma of the cervix (26).
The lack of correlation of CER level with age did not support
the findings of Everson ef al. (8) but was in accordance with
those of Friedman et al. (9), as was the absence of any
correlation between CER status and the stage of the disease
or the histological grade of the tumor.
A recent review revealed an objective response rate of 48%
to treatment with tamoxifen in 31 patients, while another 5
patients exhibited disease stabilization (21 ). The steroid recep
tor status of these patients was not known. Considering the
psychological effects of orchiectomy and the reluctance of
several patients in this study to undergo castration, we would
support Becher ef al. (3) in the use of tamoxifen as initial
endocrine therapy in advanced male breast cancer.
ACKNOWLEDGMENTS
We are indebted to the Department of Pathology. University of Natal, for the
histological diagnosis and grading of the tumors.
4814
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CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on April 30, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Cytoplasmic and Nuclear Estrogen and Progesterone Receptors
in Male Breast Cancer
Rosemary J. Pegoraro, Dharamraj Nirmul and Septimus M. Joubert
Cancer Res 1982;42:4812-4814.
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