A Case of Pediatric Virilizing Adrenocortical Tumor Resulting in Hypothalamic-pituitary Activation and Central Precocious Puberty Following Surgical Removal
YOKO MIYOSHI*, TAKAHARU OUE ** , MITSUGU OOWARI ** , HIDEKI SOH ** , MAKIKO TACHIBANA*,
SADAMI KIMURA*, YUKI KIYOHARA*, HIROYUKI YAMADA*, KAZUHIKO BESSYO*, SOTARO MUSHIAKE*,
KEIKO HOMMA *** , TOMONOBU HASEGAWA#, HIRONOBU SASANO# AND KEIICHI OZONO*
* Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
** Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
*** Central Clinical Laboratories, Keio University Hospital, Tokyo 160-8582, Japan
” Department of Pediatrics, Keio University School of Medicine, Tokyo 160-8582, Japan
Department of Pathology, Tohoku University School of Medicine, Sendai 980-8575, Japan
Abstract. We present a 6-year-old boy with a virilizing adrenocortical tumor who initially presented with peripheral preco- cious puberty. Development of facial acne, pubic hair and a growth spurt were noted at the age of five. A low-pitched voice as well as maturation of external genitalia was noted at the age of six. Both serum and urinary levels of adrenal androgens were elevated. Abdominal computed tomography revealed a large right suprarenal mass and he underwent surgical resection without any complications. The histological diagnosis was adrenocortical carcinoma according to the criteria of Weiss. Following surgical removal of the androgen-producing tumor, the patient subsequently developed hypothalamic-pituitary ac- tivation and demonstrated central precocious puberty. He was treated with a gonadotropin-releasing hormone agonist in order to delay further pubertal progression. Clinical follow-up of potential secondary effects of excess hormone secretion after re- moval is important in some pediatric patients with virilizing adrenocortical tumor.
Key words: Adrenocortical tumor, Adrenocortical carcinoma, Virilization, Precocious puberty
(Endocrine Journal 56: 975-982, 2009)
ADRENOCORTICAL CARCINOMA (ACC) is a rare neoplasm, especially in children, and the annu- al incidence has been calculated as 0.3 cases per mil- lion [1]. The molecular pathogenesis of ACC is not well understood, although it is strongly associated with germline mutation of tumor suppressor gene p53 on chromosome 17p13 [2]. ACC is often associated with the Li-Fraumeni and Beckwith-Wiedemann syn- dromes. Inactivating mutations of p53 tumor suppres- sor gene and alterations of the 11p15 locus leading to insulin-like growth factor (IGF)-II overexpression
are frequently present in these syndromes, respec- tively [3]. These pediatric adrenocortical tumors fre- quently secrete hormones, and nonfunctional tumors are relatively rare. The great majority of children with ACC presents with symptoms such as virilization [4]. Complete resection is the only curative therapy for successful treatment of ACC. The role of chemother- apy and mitotane (o, p’-DDD) in the management of childhood ACC has not been fully established. Local invasion or tumor extension into the inferior vena cava, as well as the presence of lymph nodes or distant metastases (lung and liver), are frequently detected in advanced pediatric ACC. Any virilizing conditions, when resolved, could theoretically trigger true preco- cious puberty, presumably because of maturation of the hypothalamic-pituitary axis; however, to the best of our knowledge only a few studies have reported such cases
Correspondence to: Keiichi OZONO, M.D., Ph.D., Department of Pediatrics, Osaka University Graduate School of Medicine, Suita City, Yamadaoka 2-2, Osaka, 565-0871, Japan.
E-mail: keioz@ped.med.osaka-u.ac.jp
in detail. Here we present a boy with a virilizing ACC, who subsequently developed hypothalamic-pituitary activation and central precocious puberty following tu- mor removal.
Case Report
Clinical course prior to admission
The patient was a 6-year-old Japanese boy. His past and family medical history were unremarkable. None of his family members had endocrine tumors, and their age with initiation of puberty was normal. A neo- natal screening test for congenital adrenal hyperplasia, which measures 17a-hydroxy progesterone (17OHP) levels on filter paper, was negative. The patient devel- oped facial acne, pubic hair, and a growth spurt at the age of five. Maturation of external genitalia and a low- pitched voice were noted at the age of six. At the age of six years and six months, he was diagnosed with pre- cocious puberty at a local hospital, and was admitted to Osaka University Hospital for further evaluation of precocious puberty.
Physical status
His height was 127.5 cm (+2.2 SD) and body weight was 30.4 kg (+15.2 % for ideal weight) on ad- mission. He had a muscular body, and percentage of body fat was +9.1%. Height gain was accelerat- ed from five years old on the growth chart. He had a low-pitched voice, facial acne and hirsutism. His Tanner stages were G3 and PH3, and each of his tes- tis was 4 mL in volume. Erection was also frequent- ly observed during physical examination and at home. Axillary hair was not present. No other abnormalities, such as hypertension, Cushingnoid symptoms or hemi- hypertrophy, were detected. No signs of Beckwith- Wiedemann syndrome, including macroglossia and ear creases, were identified on physical examination.
Laboratory studies
Serum and urinary laboratory findings are shown in Table 1. Total cholesterol was extremely low (63 mg/dL), and alkaline phosphatase (ALP) (1976 U/ L) and IGF-1 (749 ng/ml) were high for his age. Follicle-stimulating hormone (FSH) and luteiniz-
| Na | 137 mEq/L | TSH | 0.95 | μU/mL |
| K | 4.2 mEq/L | FT4 | 1.0 | ng/dL |
| Cl | 105 mEq/L | FT3 | 3.8 | pg/mL |
| BUN | 15 mg/dL | ACTH | 19 | pg/mL |
| Cr | 0.5 mg/dL | cortisol | 4.1 | µg/dL |
| Ca | 9.6 mg/dL | DHEAS | 3096 | ug/dL |
| IP | 5.4 mg/dL | 17OH-P | 30.4 | ng/ml |
| ALP | 1737 U/L | FSH | <0.2 | mIU/mL |
| AST | 21 U/L | LH | <0.2 | mIU/mL |
| ALT | 12 U/L | testosterone | 2.18 | ng/ml |
| LDH | 283 U/L | estradiol | 79 | pg/mL |
| T-chol | 60 mg/dL | GH | 12.48 | ng/mL |
| TG | 29 mg/dL | IGF-I | 749 | ng/ml |
| HDL-C | 31 mg/dL | PRA | 2.1 | ng/ml |
| LDL-C | 21 mg/dL | PAC | 262 | pg/mL |
| TP | 6.8 g/dL | Ad | 0.03 | ng/ml |
| Alb | 4.0 g/dL | NA | 0.2 | ng/ml |
ing hormone (LH) were lower than measurable limit (<0.2 mIU/mL). Serum dehydroepiandrosterone sul- fate (DHEAS) was extremely elevated to 3096 µg/dL. The levels of adrenal hormones were also elevated: 17-OHP (30.4 ng/ml), testosterone (T) (2.18 ng/ml), and urinary 17-ketosteroid (U-17KS) (314 mg/day). Serum morning cortisol level was not high at 4.1 ug/ dl, and ACTH was not suppressed (19 pg/mL); how- ever, daily urinary free cortisol excretion (95 µg/m2 body surface area/day) and urinary 17-hydroxycorti- costeroid (U-17OHCS) (3.5 mg/m2 body surface area/ day) were mildly elevated. HCG, HCG-ß, and AFP were lower than the measurable limit. Serum studies, including renin (PRA), aldosterone (PAC), and neu- ron-specific enolase (NSE), yielded normal-for-age re- sults. Urine vanilmandelic acid, homovanilmandelic acid and serum cathecholamine levels were also with- in normal ranges. Spot urine samples were analyzed for urinary steroid metabolites using gas chromatog- raphy/mass spectrometry as previously described [5]. The values of metabolites of pregnenolone, 17OH pregnenolone, and DHEA were abnormally high (Figure 1, Table 2). These hormonal data were inter- preted as neoplastic excretion of massive45-steroids (substrates of 3ß-hydroxysteroid dehydrogenase).
Radiological studies
His bone age was equivalent to a 11.6-year-old Japanese boy. Both abdominal computed tomogra- phy (CT) and ultrasonogram (US) demonstrated a large
Pregnenolone-Ms
17OHpregnenolone-Ms
DHEA-Ms
estrogen
Progesterone-M
17OHprogesterone-Ms
Androstenedione-Ms
DOC-M
11-deoxycortisol-M
corticosterone-Ms
cortisol-Ms
aldosterone-M
410%ile
10-50% ile
50-90% ile
90% ile<
The values of metabolites [Ms] of pregnenolone, 17OH pregnenolone, and DHEA were abnormally high range for control boys with 6-8 year of age.
B
A
B
right suprarenal homogeneous mass measuring 55 x 41 mm and atrophic left adrenal gland (Figure 2). CT en- hancement was cancelled because of an asthma attack. The tumor measured 40 Hounsfield units (HU) on un- enhanced CT. The brain, lung and liver were free from metastases in MRI and tholaco- abdominal CT, respec- tively. Androgen secreting adrenocortical tumor was clinically suspected.
Surgery and pathological findings of adrenocortical tumor
Laparoscopy-assisted tumor resection was per- formed in Osaka University Hospital. Macroscopically,
| patient | 90%tile of control boys with 6-8 years of age | |
|---|---|---|
| pregnenolone-Ms | 13.4 | 0.1 |
| 17OHpregnenolone-Ms | 32.1 | 0.1 |
| DHEA-Ms | 597.8 | 0.3 |
| progesterone-M | 1.8 | 0.2 |
| 17OHprogesterone-Ms | 5.5 | 0.3 |
| androstenedione-Ms | 23.3 | 0.5 |
| DOC-M | 0.02 | 0.02 |
| corticosterone-Ms | 1.2 | 1.2 |
| aldosterone-M | 0.03 | 0.07 |
| 11-deoxycortisol-M | 0.2 | 0.2 |
| cortisol-Ms | 6.6 | 11 |
M: metabolite
(mg/g creatinine)
there was no foci of metastasis to regional lymph nodes during the operation. A round solid tumor was excised en bloc with the right adrenal gland. The tumor was encapsulated, soft, measured 5.5 x 4.5 x 3.0cm and weighed 63 g. The cut surface appeared yellow-red- dish with areas of focal hemorrhage (Figure 3). A thin layer of non-neoplastic adrenocortical tissue was mac- roscopically identified around the tumor. The staging
of adrenocortical tumors in children (modified from Sandrini et al.) was stage I, and the TNM staging was T2N0M0, stage II [3, 6].
Representative light microscopic findings of the re- sected specimen are shown in Figure 4. The tumor comprised cells with eosinophilic cytoplasm and het- erogenic nuclei with minimum pleomorphism and was arranged in an alveolar pattern (Figure 4A, 4B). Mitotic activity was low and capsular or vascular inva- sion was not identified. Immunohistochemical staining of steroid-producing enzymes were consistent with dis- organized steroidogenesis, which is characteristic of ACC [7]. P450scc and P450c17a were expressed in almost all tumor cells. P450c11ß and P450c21 were also expressed in some tumor cells, whereas 3ß-HSD was not expressed. These immunohistochemical find- ings were compatible with serum and urinary steroid profiles. The labeling index of Ki 67/ MIB1 staining was 8 % (Figure 4C) [8, 9]. Histological diagnosis was ACC with low-grade malignancy according to the criteria of Weiss: atypical mitosis, atypical cytoplasm, atypical architecture, nuclear atypia; and Weiss crite- ria 4 out of 9.
A
C
Ki67
B
| Pre-operation Non Tx | 1 POD HC 150mg iv | 9 POD HC 15mg po | 14 POD HC 7.5mg po | 60 POD Non Tx | 240 POD LHRH analog | |
|---|---|---|---|---|---|---|
| T.chol (mg/dL) | 63 | 69 | ND | 158 | 150 | 160 |
| ALP (U/L) | 1976 | 1174 | ND | 833 | 954 | 663 |
| FSH (mIU/mL) | <0.2 | ND | ND | 0.8 | 4.6 | 0.1 |
| LH (mIU/mL) | <0.2 | ND | ND | 0.3 | 1.6 | 0.2 |
| testosterone (ng/mL) | 2.18 | <0.05 | <0.05 | <0.05 | 0.92 | <0.05 |
| estradiol (pg/mL) | 79 | 25 | <10 | <10 | <10 | <10 |
| ACTH (pg/mL) | 19 | 7 | 27 | 69 | 28 | 45 |
| cortisol (µg/dL) | 4.1 | 81.7 | 2.6 | 10.8 | 4.6 | 8.7 |
| DHEAS (µg/dL) | 3096 (max 4023) | 1199 | <20 | <20 | 24.8 | 21.4 |
| 17OHP (ng/ml) | 30.4 | ND | ND | 2 | 0.4 | 0.7 |
| renin (ng/ml) | 2.1 | ND | ND | ND | 4 | 2.6 |
| aldosterone (pg/mL) | 262 | ND | ND | ND | 97 | 160 |
| IGF-1 (ng/ml) | 749 | ND | ND | ND | 581 | 369 |
POD: post-operative days
Analysis of the p53 gene
DNA analysis of his peripheral mononuclear leu- kocytes for the p53 tumor suppressor gene was per- formed by the previously described method after ob- taining informed consent [10]. Neither Pro152Leu nor Arg158His was found in the p53 gene.
Clinical course after operation
He was treated with hydrocortisone replacement therapy before the surgery, and the dosage was gradu- ally tapered postoperatively to prevent adrenal insuf- ficiency due to atrophy of the left adrenal gland. The post-surgical course was uneventful, and he was dis- charged on postoperative day 14. Serum DHEAS, 17OHP, testosterone and estradiol rapidly decreased to the normal level (Table 3). Inversely serum cholester- ol level promptly normalized from 63 to 158 mg/dl on postoperative day 14. Because the tumor was well en- capsulated, completely resected on macroscopic obser- vation and no metastasis was evident on radiological findings, he received no additional treatment. A low- pitched voice and pubic hair had persisted at the same level after the discharge. Two months after the oper- ation, his mother once more noticed increased facial acne and erection. Each of his testis size was gradually increased to 8 mL in volume. His serum DHEAS and daily urinary free cortisol excretion (21.7 µg/ m2 body surface area/day) was not elevated, whereas testoster- one and FSH/ LH levels were elevated (FSH 4.6mIU/ mL, LH 1.6mIU/mL, T 0.92ng/mL) (Table 3). The
peak values of FSH and LH by LH-RH stimulation test were at pubertal levels (basal FSH 4.3mIU/mL, peak FSH 11.9mIU/mL, basal LH 1.3mIU/mL, peak LH 18.0mIU/mL), suggesting secondary central pre- cocious puberty. Treatment with a gonadotropin-re- leasing hormone (GnRH) agonist (Leuprolelin acetate, 60 µg/kg/4weeks) was initiated to delay further puber- tal progression three months after the operation. His pubertal level was gradually suppressed, and he re- mains disease free about two years after the operation.
Discussion
ACC is an extremely rare neoplasm in childhood, with an incidence of around 0.3 cases per million, rep- resenting 0.2% of all childhood tumors [1]. Females clearly predominate, and bimodal age distribution is well known, with the first peak occurring before 5 years old, and the second in the fourth to fifth decade [11]. Approximately 90% of children with ACC have clini- cal evidence of adrenocortical dysfunction. Virilization, alone or in combination with signs of overproduction of other adrenocortical hormones, is the most common en- docrinological presentation [12]. Isolated Cushing’s syndrome, which is the major symptom of adult ACC, is extremely rare in pediatric patients with ACC [12, 13]. Our male patient was associated with signs of iso- sexual precocious puberty. He had pubic hair, matu- ration of external genitalia, a low-pitched voice, acne, hirsutism, and growth acceleration. Hormonal findings were characterized as an increment in androgens, es-
pecially serum DHEAS, T and urinary 17-KS. These serum and urinary steroid hormone profiles were con- sistent with the immuno- histochemical findings of ad- renal tumor. He had severe hypocholesterolemia, and after resection of the tumor, his serum cholesterol lev- el dramatically rose to normal, suggesting a role of this tumor in his marked hypocholesterolemia. The mech- anism for this association remains unclear, however, Nakagawa et al. had suggested that increased LDL re- ceptor activity and unrestricted uptake of LDL by the adrenal tumor [14]. Further study would be needed for the clarification of the mechanism.
Two months after the operation, his serum DHEAS levels had decreased to the normal range, although the physical signs of precocious puberty were exagger- ated. The peaks of FSH and LH by LH-RH stimula- tion test were at pubertal levels, suggesting secondary central precocious puberty. One of the most com- mon mechanisms of progressive precocious puber- ty is the early activation of pulsatile GnRH secretion (central or gonadotropin- dependent precocious pu- berty), which may be caused by hypothalamic hamar- toma, but the etiology in the great majority of report- ed cases has remained unknown [15]. Several causes of peripheral or gonadotropin-independent puberty have been recognized, including gonadal and adrenal tumors and tumors producing human chorionic go- nadotropin, a constitutive active mutation in the go- nadotropic pathway, and exposure to exogenous sex steroids. Peripheral precocious puberty may result in activation of pulsatile GnRH secretion and central pre- cocious puberty. In addition, any virilizing conditions, when resolved, could trigger true precocious puberty, presumably because of maturation of the hypothalam ic- pituitary axis. This may occur after removal of an androgen-producing adrenal tumor, as reported in this study. It may also occur after the initiation of gluco- corticoid treatment for long-untreated vililizing con- genital adrenal hyperplasia, and after androgen thera- py for various disorders. GnRH agonists are indicated in progressive central precocious puberty, and it was really effective in our male patient. These agents ex- ert their effects by providing continuous stimulation of the pituitary gonadotrophs, leading to desensitization and decreases in the release of LH and, to a lesser ex- tent, FSH [15].
Because of the heterogeneity and rarity of ACC in a pediatric population, prognostic factors have not been established [16]. A high mitotic rate, tumor necrosis,
atypical mitotic figures, high Ki67 staining, and evi- dence of mutated p53 have been associated with ad- vanced ACC and poor prognosis [17, 18]. In child- hood ACC, clinical staging (modified from Sandrini et al.) is proposed according to tumor size and clini- cal as well as surgical parameters [6]. Patients with stages I and II have the best chance of a cure, where- as patients with stages III and IV have a poor progno- sis. Our case was classified as stage I (tumor totally excised with negative margins. The tumor weighed < 200g, there was no evidence of metastasis, and ab- normal hormone levels returned to normal after sur- gery). According to the Union International Contre Cancer (UICC) staging system published by the WHO in 2004 [19], stages I and II are defined as localized tumors, ≤5 cm and >5 cm, respectively; therefore, this case would be classified as UICC stage II (tumor was 5.5×4.5×3.0cm). In both staging systems, stage I and II tumors are recommended to be treated only with surgery. The benefits and risks of chemothera- py for ACC in children, including mitotane or other chemotherapeutic agents, are still controversial [20]. Mitotane has been introduced as a specific adrenocor- ticolytic therapeutic agent, but only 2-30% of patients responded to the treatment and the treatment itself was associated with marked side effects. We there- fore did not perform adjuvant chemotherapy. A matter of debate is the use of laparoscopic adrenalectomy for ACC. Since its introduction in 1992, minimal inva- sive laparoscopic adrenalectomy has become the treat- ment of choice for adrenal tumors with a small diame- ter because of lower complication rates, less operative blood loss, less postoperative pain and a shorter hos- pital stay [21]. We initially tried laparoscopic adrena- lectomy, but difficulty in stemming bleeding prompted us to select an additional abdominal approach. There were no problems related to the operation.
p53 positivity has been associated with ACC in children, but no mutation of the p53 tumor suppres- sor gene in codon 152 and 158 was found in our case. Measurement of HU on unenhanced CT is of great value to differentiate malignant from benign adrenal lesions. Using a threshold value of 10 HU, sensitivity and specificity for characterization an adrenal lesion as a benign adenoma in unenhanced CT were 71% and 98%, respectively, in a meta-analysis of 10 studies [22]. The present case was 40 HU, which suggest ad- renal carcinoma.
Differentiation between benign and malignant adre-
nal lesions is primarily based on macroscopic features (tumor weight, hemorrhage, breached or intact tumor capsule) and a light microscopic diagnostic score or the criterion of Weiss [23, 24]. The criterion of Weiss has been most widely used, and has become the gold standard of histological diagnosis of adrenocortical carcinoma, as seen in its incorporation into the recent WHO tumor classification [19]; however, distinguish- ing an adrenal cortical adenoma from adrenal corti- cal carcinoma on the basis of histological findings is sometimes difficult, especially in children. Several studies have demonstrated the value of Ki67 staining in differentiating benign from malignant lesions [8, 9]. In addition, Ki67 expression may be of prognostic rel- evance as high expression (>10%) has been associated with poor survival. Histopathologically, the present case would be classified as an adrenocortical carcino- ma, based on the 4 out of 9 criteria by Weiss, and 8 % Ki67 labeling index. Wieneke JA et al. had proposed that nine features associated with an increased prob- ability of a malignant clinical behavior included tumor weight (>400 g), tumor size (>10.5 cm), vena cava in- vasion, capsular and/or vascular invasion, extension into periadrenal soft tissue, confluent necrosis, se- vere nuclear atypia, >15 mitotic figures/20 high power fields, and the presence of atypical mitotic figures [25]. Our patient met three of these criteria above, including
confluent necrosis, severe nuclear atypia, and the pres- ence of atypical mitotic figures, and he is considered to fall into the intermediate outcome category.
In functioning tumors, hormonal markers should be measured every 3 months after removal for early detection of tumor recurrence. Because surgical re- moval of a local relapse or metastases is a valid thera- peutic option, early detection of recurrent tumor is im- portant; however, in most patients, imaging is more sensitive for monitoring tumor recurrence. Therefore the patient should be additionally followed every 3 months by CT (abdomen plus chest), or abdominal US and chest X-ray alternatively, during the first 2 years. Our patient remains disease free after about two years of close observation, however, patients with ACC re- main at moderate risk for relapse even after 2 years. Regular restaging should go on for at least 5 years, whereas imaging intervals may increase [13].
We present a 6-year-old boy with a virilizing adre- nocortical tumor, who demonstrated peripheral or go- nadotropin-independent precocious puberty. Surgical resection of the tumor triggered hypothalamic-pituitary activation and central precocious puberty. We suggest that after removal of an androgen-producing adreno- cortical tumor, it is important to follow up for potential secondary effects of excess hormone secretion.
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