The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 649-652
Copyright © 2001 by The Endocrine Society
Aromatase P450 Expression in a Feminizing Adrenal Adenoma Presenting as Isosexual Precocious Puberty
Chanika Phornphutkul,
Tomoharu Okubo,
Kebin Wu,
Zeev Harel,
Thomas F. Tracy, Jr.,
Halit Pinar,
Shiuan Chen,
Philip A. Gruppuso and
Gregory Goodwin
Departments of Pediatrics (C.P., Z.H., P.A.G., G.G.) and Surgery
(T.F.T.), Brown University and Rhode Island Hospital, Providence, Rhode
Island 02903; Department of Pathology, Brown University and Women and
Infants Hospital (H.P.), Providence, Rhode Island 02905; and Division
of Immunology, City of Hope/Beckman Research Institute (T.O., K.W.,
S.C.), Duarte, California 91010
Address all correspondence and requests for reprints to: Gregory Goodwin, M.D., Department of Pediatrics, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail:
gregory_goodwin_md{at}brown.edu
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Abstract
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A 7-yr-old girl presented with isosexual precocious puberty secondary
to a feminizing adrenal adenoma. The adrenal tumor was found to express
aromatase messenger ribonucleic acid. Enzyme kinetic studies revealed a
high level of aromatase activity in the adrenal tumor, with a
Km of 45 nmol/L and a maximum velocity of 25.6 pmol/mg·h.
Aromatase activity was approximately 500-fold higher in the tumor than
in adjacent normal adrenal tissue. Although histopathological
examination of the tumor was most consistent with a benign adenoma, the
aromatase transcripts present in the tumor corresponded to those
previously associated with malignant as well as benign tumors. We
consider the pattern of aromatase expression sufficient to warrant
continued follow-up for tumor recurrence. Our case demonstrates that
isosexual precocious puberty secondary to a feminizing adrenal tumor
can be due to estrogen synthesis from the tumor itself rather than
peripheral aromatization as had been previously theorized.
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Introduction
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FEMINIZING ADRENAL tumors are a rare cause
of isosexual precocious puberty in females (1). The
mechanism of estrogen production in feminizing adrenal tumors has been
proposed to be due to either peripheral aromatization of androgens
(2) or estrogen production from the tumor itself
(3). Increased aromatase messenger ribonucleic acid (mRNA)
expression and activity have been demonstrated in a feminizing
adrenocortical carcinoma from an adult male presenting with
gynecomastia (4), presumably accounting for estrogen
production by the tumor. We encountered a 7-yr-old female who presented
with isosexual precocious puberty and adrenal adenoma. The adenoma was
shown to possess high aromatase P450 activity and to express exon PII-
and I.3-containing aromatase mRNA transcripts. To our knowledge, this
is the first report of increased aromatase (CYP19) gene expression in
an adrenal adenoma resulting in isosexual precocious puberty in a
child.
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Case Report
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A 7 9/12-yr-old Caucasian female presented with a 1-yr history of
pubic hair development and a 1-month history of breast development.
Physical examination revealed Tanner II pubic hair and Tanner II
breasts. There was no virilization. Her bone age was advanced at 10 yr.
Initial hormonal studies were notable for serum estradiol of 21 pg/mL
(77 pmol/L), LH below 0.1 IU/L, and FSH below 0.3 IU/L. Six months
later, her breast development had progressed to Tanner III. Her
interval growth velocity was in the pubertal range at 13.6 cm/yr. Her
predicted final adult height was 155 cm, based on Bayley and Pinneau
height prediction tables (5), whereas her calculated
midparental height was 158 cm.
Menarche occurred at 8 9/12 yr, and by 9 yr of age the patient had
developed dysfunctional uterine bleeding. She was treated with high
dose ethinyl estradiol/norgestril, which resulted in
cessation of menses. Menorrhagia and menstrual cramping occurred when
treatment was discontinued, prompting a pelvic ultrasound. The
ultrasound revealed a 6-cm, well circumscribed right adrenal mass.
Laboratory studies at this time (Table 1
)
showed elevations of serum estrone and dehydroepiandrosterone sulfate.
Twenty-four-hour urine studies (Table 2)
showed increased 17ketosteroids, estrone, estradiol, and estriol
relative to adult female normal values. Urinary
17-hydroxycorticosteroid and free cortisol levelswere normal. However,
overnight low dose (1 mg) and high dose (2 mg, three times daily, for 3
days) dexamethasone did not suppress her serum cortisol [morning serum
cortisol concentrations after low and high dose dexamethasone
treatments were 7.4 µg/dL (204.2 nmol/L) and 7.4 µg/dL (204.2
nmol/L), respectively]. Chest, abdominal, and pelvic computed
tomography scans did not show any evidence of tumor metastasis.
The patient underwent a complete right adrenalectomy without
complications. The weight of the well encapsulated adrenal mass was
66 g. Pathological examination was consistent with adrenal
adenoma. There were few mitotic cells, no atypical mitotic figures, and
no evidence of vascular invasion. Tumor tissue and adjacent normal
adrenal tissue were frozen in liquid nitrogen and stored at -70 C.
One month after surgery, the patient’s serum dehydroepiandrosterone
sulfate and estrone levels were normal, as was urinary
17-ketosteroid excretion. An overnight low dose dexamethasone
suppression test showed normal suppression (morning cortisol, <0.2
µg/dL; <5.5 nmol/L). Follow-up adrenal computed tomography scans at
7 months and 1 yr after surgery revealed no residual or recurrent
tumor. The patient resumed menses at the age of 10 8/12 yr. Her final
height is 150.9 cm.
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Materials and Methods
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Aromatase activity assay
Aromatase activity was determined by
[3H]H2O release assay
using homogenates prepared as described by Kadohoma et al.
(6).
[1ß-N-3H]Androst-4-ene-3,17-dione was used as
substrate. Enzyme kinetic analyses were performed at six different
substrate concentrations (5–200 nmol/L) of
[1ß-3H]androst-4-ene-3,17-dione.
Analysis at 100 nmol/L substrate was performed using NCI-H295 cell
extract to validate our assay results.
RT-PCR Southern blot analysis
Exon I primer-specific RT-PCR has been shown to be a useful tool
for examining the alternative use of aromatase gene exon I promoters in
aromatase-expressing tissues (7, 8). We synthesized seven
oligonucleotides (7, 8), including the 1a, 1b, 1c, 15,
N69, 1d, and 2a sequences described by Harada (7), Toda
et al. (9), and Shozu et al.
(10). These primers have sequences derived from exons I.1,
I.3, I.4, I.5, I.6, pII, and II, respectively. A reverse primer (2d)
with a sequence derived from exon II and situated downstream from 2a
was also used. The membranes were hybridized with an exon II-specific
oligonucleotide probe that corresponds to the middle of exon II
(5'-ATGGTTTTGGAAATGCTGAA-3'). A schematic representation of the
relative locations of five exons I in the human aromatase gene and the
positions of the primers used in our primer-specific RT-PCR analysis
are shown in Fig. 1.
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Figure 1. A schematic representation of the relative
locations of six exons I and promoters in the human aromatase gene. The
oligonucleotides used for amplification are shown as 1a, 1b, 1c, 1d,
2a, N69, 15, and 2d. Although the position of PI.5 is not known, it is
contiguous with exon II in the processed mRNA (dashed
line).
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Total RNA was isolated from the tumor and normal adrenal tissue as
described by Chomczynski (11). RT-PCR was performed at
nonsaturating conditions using the Titan One Tube RT-PCR System
(Roche, Indianapolis, IN) (8).
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Results
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In vitro aromatase activity in the tumor tissue was
substrate concentration-dependent. The Km and
maximum velocity (Vmax) values derived from a
double reciprocal plot (Fig. 2) were 45
nmol/L and 25.6 pmol/mg·h, respectively. The Km
value was comparable to that determined for human placenta aromatase
expressed in CHO cells (31 nmol/L) (6). The
Vmax value for aromatase in our adrenal tumor
homogenate was only one fourth of that measured in a microsomal
preparation from a feminizing adrenocortical tumor in a 29-yr-old man
(4). However, because the amount of tumor tissue and
normal adrenal tissue was limited, we performed the enzyme assay using
an unfractionated tissue homogenate rather than a microsomal
preparation. The specific activity would presumably be significantly
higher if the assay were performed using a microsomal preparation. Of
note, activity in normal adrenal tissue adjacent to the tumor was very
low, approximately 0.2% of that seen with the tumor tissue. The low
activity in normal adrenal tissue prevented an accurate determination
of kinetic parameters. We also performed a comparative study using the
NCI-H295 human adrenocortical carcinoma cell line, which is known to
express aromatase (12, 13). Aromatase activity in the
NCI-H295 cell lysates, measured at a 100 nmol/L substrate
concentration, was determined to be 0.28 pmol/mg·h. After incubating
the cells with 8-bromo-cAMP for 24 h, aromatase activity increased
to 0.52 pmol/mg·h. These results were similar to those obtained by
Sanderson et al. (14), supporting the validity
of our assay conditions. Of note, the aromatase specific activity in
the NCI-H295 cells was much lower than that in homogenates of our
patient’s adrenal tumor measured at the same 100 nmol/L substrate
concentration (
20 pmol/mg·h).
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Figure 2. Kinetic analysis of aromatase activity in
unfractionated adrenal tumor homogenate as measured by
[3H]H2O release assay. Data obtained using
six substrate concentrations are shown as a double reciprocal plot.
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Human aromatase gene exon I-specific primer RT-PCR analysis revealed
that the aromatase P450 mRNA species in the adrenal tumor were mainly
exon PII derived, suggesting that aromatase expression in this tumor
was primarily driven by promoter II. Unspliced promoter I.3-driven
transcripts (I.3 A) were also detected in the adrenal tumor (Fig. 3). The level of aromatase mRNA in normal
adrenal tissue was undetectable by RT-PCR.
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Figure 3. Chemiluminescent detection of adrenal tumor
RT-PCR products generated with exon I-specific RT-PCR followed by
Southern analysis. The sizes of the detected PCR products for exons
I.3, PII, and II are 333, 233, and 169 bp, respectively.
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Discussion
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Although feminization secondary to an adrenal tumor has previously
been attributed to peripheral conversion of adrenal androgen
(2), our case confirms that an adrenal adenoma can possess
the molecular pathways necessary for estrogen biosynthesis and that it
can cause isosexual precocious puberty in a female. This conclusion is
based on direct determination of aromatase activity in our patient’s
tumor, which was approximately 500 times higher than that in the
adjacent, normal adrenal tissue.
In humans, estrogen is derived from the conversion of androstenedione,
testosterone, and 16
-hydroxyandrostenedione to estrone, estradiol,
and estriol, respectively. Most of this conversion takes place in
ovaries and adipose tissue, where it is catalyzed by aromatase P450.
Normal adrenal tissue expresses very low level of aromatase P450. In
our patient, the major circulating estrogen was estrone. The serum
estrone presumably came from the aromatization of androstenedione by
the adrenal adenoma. We speculate that estradiol was probably
synthesized by conversion of estrone via peripheral
17ß-hydroxysteroid dehydrogenase type I (15), rather
than intraadrenal aromatization of testosterone, as the peripheral
testosterone level was unmeasurable. This interpretation is consistent
with findings described by Young et al. (4),
who demonstrated tumor estrone (but not estradiol) production by direct
catheterization of adrenal veins.
Analysis for aromatase mRNA expression in the tumor showed that Exon
PII-containing mRNA was the major form detected, although unspliced
promoter I.3 driven transcript (I.3 A) was also detected in the adrenal
tumor. To our knowledge, this is the first report of exon I.3 mRNA
expression in an adrenal adenoma. Exon PII-containing mRNA is normally
expressed in gonadal tissue (16) and exon I.3-containing
mRNA is normally expressed in adipose tissue (17). Normal
adult liver, endometrium, myometrium and adrenal tissues do not express
exon PII or I.3-containing mRNA (16). However, excessive
expression of exon pII and I.3-containing mRNAs has been detected in
various malignant and nonmalignant conditions, including endometriosis,
leiomyoma, hepatocellular carcinoma, endometrial cancer, breast cancer,
and adrenocortical cancer (16). Promoters I.3 and II are
both inducible by cAMP (17). Thus, cAMP-dependent
signaling is a potential mechanism for abnormal CYP-19 gene expression
in our patient’s adrenal tumor.
Several guidelines have been used to differentiate between adrenal
carcinoma and adrenal adenoma on clinical grounds. These include
weight, size of the tumor, and evidence of metastasis (18, 19). Tumors weighing more than 500 g or having diameter
greater than 6 cm are more likely to be carcinomas. Tissue histology
is, of course, more definitive. Findings in carcinomas included
abnormal mitoses, necrosis, vascular and capsular invasion, broad
fibrous bands, and cellular pleomorphism. All of these features were
absent in our patient. Therefore, we originally concluded that our
patient’s diagnosis was most consistent with an adenoma.
The urinary estrogen concentration in feminizing adrenal tumors has
been reported to be a useful tool for following these patients. Very
high concentrations indicate that the tumor is more likely to be a
carcinoma (20). Our patient’s urinary estrogen was
elevated in the range of some of the patients with adrenal carcinoma
reported by Wotiz et al. (20). Given the
increased expression of exon I.3 and II mRNA, both of which are
associated with multiple carcinomas (17), and the
extremely elevated urinary estrogen concentration, we have concluded
that ongoing surveillance for recurrence of our patient’s tumor is
indicated.
This case illustrates the importance of ongoing monitoring and
follow-up of patients with what appears to be simple or benign early
puberty. Based on the large cross-sectional study of 17,000 girls by
Herman-Giddens et al. (21), precocious puberty
has been defined as the onset of breast development before the age of 7
yr in white females (22). When considering that our
patient was 7 9/12 yr at presentation, and her predicted final adult
height fell within her genetic potential, a thorough evaluation for
pathological causes of precocious puberty was not undertaken. Her
clinical course, however, became more concerning when she developed
unremitting dysfunctional uterine bleeding associated with abdominal
cramping. This prompted further investigation.
In summary, the present case is the first demonstration of increased
aromatase P450 activity in a feminizing adrenal adenoma leading to
isosexual precocious puberty. The aromatase activity was probably due
to abnormal enhancement of the transcriptional activity of the promoter
I.3 and II aromatase gene, a finding previously associated with
malignancy. This association warrants careful longitudinal surveillance
despite an otherwise good prognosis based on clinical pathological
features most consistent with a benign adrenal adenoma.
Received June 22, 2000.
Revised October 16, 2000.
Accepted October 27, 2000.
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Abstract
Introduction
