ORIGINAL PAPER
Walter Bonfig . Iris Bittmann . Susanne Bechtold Birgit Kammer . Vera Noelle . Sissi Arleth Klemens Raile . Hans-Peter Schwarz
Virilising adrenocortical tumours in children
Received: 7 January 2003 / Revised: 19 March 2003 / Accepted: 20 March 2003/ Published online: 14 June 2003 @ Springer-Verlag 2003
Abstract Adrenocortical tumours (ACT) are a rare but important cause of virilisation in infancy and childhood. Four cases of virilising ACT are presented. Two girls (age 0.9 years and 3.9 years) and two boys (age 6.2 years and 6.4 years) had symptoms and signs of virilisation before the age of 6 years. Diagnosis of a virilising adrenal tumour was confirmed by laboratory tests, diagnostic imaging and histology. However, one female patient was misdiagnosed and treated for 3 months as atypical congenital adrenal hyperplasia. Ultrasonogra- phy of the adrenal region could not visualise the tumour in three out of four cases. The most sensitive method of diagnostic imaging was MRI. In all cases, treatment consisted of complete surgical resection of the adrenal tumour by open abdominal surgery. Immunohisto- chemistry was performed in all patients and in two pa- tients there was an overexpression of p53, indicating p53 mutation and in three cases the ki67 proliferation index was greater than 5%. The classification of ACT in childhood is extremely difficult. Histology scores adap- ted from adrenal tumours in adults and molecular markers are under investigation, but there is still not enough clinical experience since ACT are so rare. Conclusion: Iong-term follow-up is mandatory not only because of the uncertainty in classification of adreno- cortical tumours, but also for observation of growth and pubertal development.
Keywords Adrenocortical tumour . Children . Virilisation
W. Bonfig ☒ · S. Bechtold · B. Kammer · V. Noelle
S. Arleth · K. Raile . H .- P. Schwarz Department of Paediatric Endocrinology and Radiology, University Children’s Hospital Munich, Lindwurmstrasse 4, 80337, Munich, Germany E-mail: wbonfig@web.de
Tel .: +49-89-51602811 Fax: +49-89-51604784
I. Bittmann
Department of Pathology, Ludwig-Maximilians-University, Munich, Germany
Abbreviations ACT adrenocortical tumour . ACTH adrenocorticotrophic hormone . DHEAS dihydroepiandrosterone sulphate · 17-OHP 17-hydroxyprogesterone
Introduction
Adrenocortical tumours (ACT) are rare in children. The worldwide annual incidence ranges from 0.3 to 0.38 per million children below the age of 15 years [25] with 65% of them occurring in children younger than 5 years of age. The incidence of ACT seems to be higher in young girls with a female/male ratio of 2:1, whereas in ado- lescents, the female/male ratio is 1:1 [5]. Virilisation is the most common presentation, followed by hypercor- tisolism and hyperaldosteronism. In contrast, there is a predominance of non-hormone secreting tumours in adults. The classification of ACT is difficult. Histo- pathological classification may not be reliable for sepa- rating benign from malignant tumours. Significant criteria predicting malignant behaviour include clinical evidence of weight loss and histologically broad fibrous bands, diffuse growth pattern, and vascular invasion, although no single criterion is reliable. Recently, molecular markers have been investigated as a diag- nostic aid [2, 3, 6, 9, 17, 18, 19, 21, 26,31]. Abnormal hormone concentrations correlate well with clinical symptoms. Diagnosis is supported by analysis of serum and urinary steroid hormones and physically confirmed by diagnostic imaging with ultrasound, CT scan or MRI. Four cases of virilising adrenocortical adenoma are presented here.
Case reports
The clinical data of the patients are given in Table 1. Patient 1 is a boy who was referred in February 2000 at the age of 6.2 years for evaluation of gynaecomastia which had begun 5 months previ- ously. On physical examination he was found to have gynaeco- mastia, pubic hair, a large penis measuring 8 cm and a deep voice.
| Patient | Age at first signs (years) | Sex | Age at diagnosis (years) | Presenting signs | Tumour weight | (g)/site | Histology | Immunohistochemistry | |
|---|---|---|---|---|---|---|---|---|---|
| p53 positive cells (%) | ki67 index (%) | ||||||||
| 1 | 5.7 | M | 6.2 | Gynaecomastia, pubic hair (P3), large penis (8 cm), deep voice, growth acceleration | 3 | Left | 2-15 mitoses, partially pleomorphic nuclei, no necrosis | 76.0 | 6.0 |
| 2 | 2.7 | F | 3.9 | Pubic hair (P3), clitoromegaly, acne, partially adult body odour, growth acceleration | 10 | Left | Up to 10 mitoses, pleomorphic nuclei, no necrosis | 54.5 | 9.0 |
| 3 | 5.4 | M | 6.4 | large penis (9.5 cm), acne, growth acceleration | 52 | Right | Single mitoses, no necrosis | 5.0 | 2.0 |
| 4 | 0.8 | F | 0.9 | Pubic hair (P2), clitoromegaly, growth acceleration, muscular aspect | 14 | Right | Up to 9 mitoses, partially pleomorphic nuclei, small necrosis | 12.0 | 17.0 |
Retrospectively, a growth spurt was diagnosed (growth velocity 11 cm/year). At diagnosis, bone age was 8.5 years. There was no acne, no axillary hair and no striae. Testicular size and structure were normal for age. Diagnosis was confirmed by laboratory data (Table 2), MRI and CT scan (Table 3). A left adrenalectomy was performed and a 3 g brown tumour was removed. Histological examination of the tumour showed marked nuclear pleomorphism and hyperchromasia along with increased mitotic activity without tumour necrosis or vascular invasion. No metastases were found. Post-operative blood samples showed a normal steroid hormone profile. The patient was last seen in January 2003 and there was no evidence of recurrence.
Patient 2 is a girl who was first seen in September 1997 at the age of 3.4 years with a 10-month history of pubic hair and clitoromegaly. On physical examination she was found to have pubic hair, a clitoris measuring 2 cm, acne and an adult body odour. Retrospectively, a growth spurt was diagnosed (growth velocity 11.5 cm/year). Bone age in September 1997 was 5 years (with a chronological age of 3.4 years). She was diagnosed as having atypical non-salt-wasting con- genital adrenal hyperplasia and was treated with hydrocortisone (12.5 mg/day) for 3 months. Since elevated 17-hydroxyprogesterone (17-OHP), androstenedione and urinary steroids were not sup- pressed, treatment was discontinued in February 1998 and the diagnosis was reassessed. Laboratory data from March 1998 are shown in Table 2. MRI showed an adrenal tumour on the left side. During surgery, a 10 g yellow-brown tumour was removed. Histo- logical examination of the tumour revealed areas of marked nuclear pleomorphism and hyperchromasia with single, sometimes atypical, mitotic figures. No tumour necrosis or vascular invasion was seen. No metastases were found. Post-operative blood samples showed normal steroid hormone profiles. In September 2000, the girl was diagnosed with a lumbar paravertebral rhabdomyosarcoma on the left side. The tumour was removed and the patient received radiation therapy afterwards.
Patient 3 is a boy who was 6.4 years old when he was referred in May 2000 for evaluation of acne that had started 1 year previously. On physical examination, he was found to have severe acne, a large penis measuring 9.5 cm and a growth spurt (growth velocity 12 cm/ year). At diagnosis, the bone age was 9.5 years. There was no axillary hair and no striae. Testicular size and structure were nor- mal for age. Diagnosis was confirmed by laboratory data (Table 2), ultrasound (Fig. 1) and MRI (Table 3). Surgical excision disclosed a 52 g brown tumour. Histological examination showed a tumour composed of cells with predominantly eosinophilic compact cyto- plasm arranged in short cords with only small foci of atypical nuclei. Only a few mitotic figures were detected. An adrenal cortical adenoma was diagnosed. Post-operative blood samples showed normal steroid hormone profiles. The patient was last seen in September 2002 and there was no evidence of recurrence.
Patient 4 is a girl who was referred in August 2001 at the age of 11 months for evaluation of pubic hair and clitoromegaly that had been noticed 1 month before. On physical examination she had pubic hair, a muscular aspect and an enlarged clitoris measuring 1.5 cm. At diagnosis the bone age was 1 year. There was no acne, no axillary hair and no striae. Diagnosis was confirmed by laboratory data (Table 2) and MRI (Table 3) (Fig. 2). A right adrenalectomy was performed
and a 14 g yellow tumour was excised. Histological examination of the tumour showed marked nuclear abnormalities with pleomor- phism and hyperchromasia and increased mitotic count, sometimes with atypical mitotic figures (Fig. 3). Small foci of necrosis were found but no vascular invasion. No metastases were found. Post- operative blood samples showed normal steroid hormone profiles. The patient was last seen in October 2002 and there was no evidence of recurrence of the tumour.
Methods
Between 1997 and 2001, four cases of adrenal tumour were ob- served, representing all cases of this rare childhood tumour seen at our hospital from 1990 to 2002. Each patient’s clinical record was reviewed. Bone age was assessed from hand and wrist radiographs
| Patient | 1 Patient | 2 Patient | 3 Patient 4 | Normal (1-6 years) | |
|---|---|---|---|---|---|
| Serum | |||||
| DHEAS (µg/dl) | 343 | 721 | 4800 | 779 | <5-30 |
| Testosterone (ng/dl) | 462 | 204 | 63 | 571 | <20 |
| Androstenedione (ng/dl) | 54 | 499 | 226 | 463 | <10-45 |
| Cortisol basal (ng/dl) | 6 | 8 | 16 | 11 | 5-20 |
| Cortisol after ACTH (ng/dl) | 18 | 22 | 18 | 37 | >20 |
| 17-OHP basal (ng/dl) | 148 | 1081 | 2093 | 1482 | <10-100 |
| 17-OHP after ACTH (ng/dl) | 248 | 1446 | 1890 | 1465 | A<260 |
| Oestradiol (pg/ml) | 33 | 26 | ND | ND | <5 |
| LH basal (mU/ml) | <0.5 | <0.5 | <0.5 | 0.6 | <0.5-1.2 |
| FSH basal (mU/ml) | <0.5 | <0.5 | <0.5 | <0.5 | <0.5-1.9 |
| Insulin-like growth factor 1 (ng/ml) | 314 | 383 | 210 | 531 | 25-185 |
| Urine | |||||
| Pregnanetriol (ug/day) | 243 | 1383 | 1593 | 62 | 200-500 |
| Pregnanetriolone (µg/day) | 87 | 182 | 352 | <20 | <20 |
| Pregnanediol (ug/day) | 120 | 432 | 778 | 26 | <20 |
| Pregnenetriol (ug/day) | 88 | 690 | 1578 | 34 | <20 |
| Tetrahydro-11-deoxycortisol (ug/day) | 277 | 188 | 74 | ND | <20 |
| Tetrahydrocortisone (ug/day) | 313 | 944 | 1452 | 46 | < 500 |
| 5-alpha-tetrahydrocortisone (ug/day) | 137 | 546 | ND | 40 | <300 |
| 5-beta-tetrahydrocorticosterone (µg/day) | 94 | 355 | 482 | <20 | <20 |
| Patient | Ultrasonography | MRI | CT |
|---|---|---|---|
| 1 | Left adrenal gland not visible | Tumour on top of left kidney pole 1.5x2.4 cm | Tumour left adrenal cortex with small calcification 2×2 cm Not performed |
| 2 | No tumour found | Tumour left adrenal cortex 2.5×3.5×1.3 cm | |
| 3 | Tumour right adrenal cortex | Tumour right adrenal cortex 5.5×4 cm | Not performed |
| 4 | No tumour found | Tumour right adrenal cortex 2.8×2.7×3.1 cm | Not performed |
×
×
DISTANCE = 48.5mm DISTANCE = 38.2mm
by the method of Greulich and Pyle [8]. Growth velocity was cal- culated and acceleration of growth was defined as a growth velocity greater than the 95th percentile for age and as crossing of two or more height percentiles [20]. Urinary steroids were measured by gas chromatography [12]. Serum steroid hormones were determined by
fluorescence immunoassay (dehydroepiandrosterone sulphate (DHEAS), cortisol, LH, FSH), radioimmunoassay (androstenedi- one, 17-OHP, oestradiol) and ELISA (testosterone, insulin-like growth factor 1). Adrenocorticotrophic hormone (ACTH) stimu- lation tests were performed by administration of 0.25 mg tetraco- sactide (Synacthen) and measurement of 17-OHP and cortisol at baseline and 60 min after injection. Immunohistochemistry was performed on paraffin-embedded tissue with antibodies against p53 (dilution 1:50, Dakopatts, Germany) and ki67 (dilution 1:30, Da- kopatts, Germany) after microwave pretreatment according to standard procedures.
Results
All four patients were younger than 6 years of age when clinical signs first appeared. Virilisation was present in all, with pubic hair and enlargement of the clitoris or penis. Other clinical signs were growth acceleration, deepening of the voice, adult body odour, acne, gynae- comastia and a muscular aspect. Urinary steroid profiles showed an irregular pattern in all four patients (Table 2). In collected blood samples, DHEAS, andro- stenedione, 17-OHP and testosterone were elevated (Table 2) and 17-OHP did not respond abnormally to ACTH stimulation, except in patient 2, who was initially
A
B
misdiagnosed as having congenital adrenal hyperplasia. A tumour was detected by ultrasonography in one case only, whereas the presence of an ACT was confirmed by MRI in all patients. Definite diagnosis was made be- tween 0.1 to 1.2 years after the appearance of the first
clinical signs. All four patients underwent abdominal surgery. Tumour weight ranged from 3 to 52 g. In one case only (patient 3), the diagnosis of a benign adrenal adenoma was made because of the histopathological appearance of the tumour. In the remaining three cases, nuclear abnormalities with pleomorphism along with atypical mitotic figures were seen, not allowing mor- phological distinction between adrenal cortical adenoma and carcinoma. Vascular invasion and metastasis were not found in any patient. The results of the immuno- histochemical evaluation are summarised in Table 1. In patients 1 and 2, there were more than 30% of cells positive for nuclear p53, indicating a somatic p53 mutation. The ki67 proliferation index was greater than 10% in patient 4, reflecting the fast progression of vi- rilisation and tumour growth in this patient. In patients 1 and 2, the ki67 index was above 5%. Only in patient 3 a low immunohistochemical expression of p53 and ki 67 was found.
Discussion
First described in 1865, ACT are rare in children [1]. Most tumours appear in children younger than 5 years of age. Worldwide annual incidence of childhood ACT ranges from 0.3 to 0.38 per million children below the age of 15 years [25]. For unknown reasons there is an increased incidence of ACT in Southern Brazil [6, 13, 22,24]. Clinical signs and symptoms of virilising ACT include pubic hair, hypertrophy of the clitoris or penis, acne, gynaecomastia, deepening of the voice, accelerated growth velocity and, in a few cases, a palpable abdom- inal tumour. Diagnosis is supported by measurement of both serum DHEAS, androstenedione, testosterone, 17- OHP and urinary steroids [4, 10, 14,17]. These hormones are also helpful as tumour markers for detection of recurrence in follow-up until puberty. The definitive diagnosis is best made by abdominal MRI. Although ultrasonography can image large adrenal masses, it is inferior to CT or MRI in detecting smaller lesions [7]. In particular, the left adrenal gland is difficult to visualise by ultrasonography. In our small series, three out of four adrenal tumours were not seen in sonogram (two on the left, one on the right side). MRI and CT are reported to be virtually equivalent for the detection of adrenal masses larger than 1.0-1.5 cm in diameter. For detection of small non-functioning adrenocortical ade- nomas seen in adults, so called incidentalomas, MRI is the method of choice. Necrosis, calcification and inva- sion of the renal vein or inferior vena cava are best de- tected by CT [7]. Assessing the malignant potential of adrenal tumours is difficult. Even established histo- pathological criteria and algorithms adapted from tu- mours in adults [11, 23, 28,] generally do not allow clear classification in children. Recently, molecular and ge- netic markers have been assessed for their potential to differentiate adrenocortical carcinomas from benign adenomas [2, 3 ,6, 9, 19, 21, 26]. Overexpression of
insulin-like growth factor-2, insulin-like growth factor- 1-receptor and high levels of insulin-like growth factor binding protein 2 have been detected in malignant ACT [27]. Somatic p53 mutations (on chromosome 17- 17p13.1) occur in approximately 30-50% of sporadic ACT [3]. Two of the tumours of this case report had more than 30% of nuclear p53 positive cells indicating a p53 mutation. Moreover, the onset of a second tumour (rhabdomyosarcoma) in patient 2 is suggestive of a genomic p53 mutation in this case. Mutation analysis is still in progress in this patient. Arola et al. [2] found the majority of ACT to be positive for expression of p53 (more than 10% of cells positive for p53) and for a proliferative activity ki67 index of more than 5%. Wa- chenfeld et al. [26] found that a ki67 index (expression of the proliferation-associated antigen ki67 detected by MIB1 antibody) above 5% is a sensitive and specific marker of ACT in adults. Three out of four patients in our study had a ki67 index greater than 5%. On the other hand, Cagle et al. [5] found that clinically benign ACT in children have significantly more mitosis, necrosis, broad fibrous bands and moderate to marked nuclear pleomorphism. In one of our four patients, a definite diagnosis of benign adrenal adenoma was made by histology. In the remaining three patients, no metastases were found and there was no recurrence of the tumour on follow-up, which could also be due to complete tumour resection. From our point of view, it is questionable whether to use ki67 index as marker of malignancy. The ki67 index is just an index of prolifer- ation and primarily reflects tumour tissue proliferation. Thus, we believe one has to be cautious when using the ki67 index to assess the potential of adrenal tumours in childhood. Loss of heterozygosity of 11q13 (menin gene locus) was more frequently found in adrenal carcinoma than in adrenal adenoma [26]. No benign adenomas with loss of both heterozygosity of MEN1-locus and the p53 gene have been described so far. Mutations of another gene, p57 (KIP2) on chromosome 11 (11p15), have been described in malignant sporadic ACT. Genetic syn- dromes may also be associated with ACT: in Li-Frau- meni syndrome and Beckwith-Wiedemann syndrome, ACT are not uncommon [21]. Tumour weight and size, signs of vascular invasion and metastases are morpho- logical signs of tumour carcinogenic potential. A weight of more than 100 g and a diameter greater than 5 cm are indicators of malignancy and infiltration of surrounding tissue is an obvious sign of carcinoma [11, 14,30]. Treatment of choice is radical resection of the complete tumour by open abdominal or laparoscopic surgery. Peritoneal metastatic spreading has been described after laparoscopic removal of a malignant ACT, so that open abdominal surgery is the method of choice in tumours of undetermined carcinogenic potential, which is the case in children [24]. Chemotherapy in the management of ACT is clearly a second choice, although long-term relapse- free survival after treatment with mitotane has been reported [15,16]. The role of radiation therapy in the management of childhood ACT has not been
established. Long-term outcome of adrenal adenoma is generally very good, in contrast to adrenocortical car- cinoma, especially if metastases are present. Early diagnosis is important and essential when virilisation and androgen secretion is present in childhood. Because of the rarity of ACT in children, the percentage of malignant tumours is not known. Although all of our patients seem to have been cured by surgery, long-term observation, also with regard to early pubertal devel- opment, is warranted.
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