Ewa M. Małunowicza Maria Ginalska-Malinowskab Tomasz E. Romerb Anna Ruszczyńska-Wolskab Małgorzata Durac
Departments of
a Laboratory Diagnostics, b Endocrinology and Pathology, Child Health Center, Warsaw, Poland
Key Words
Adrenocortical tumors Children Urinary steroid profiles Histopathological evaluation Treatment
Heterogeneity of Urinary Steroid Profiles in Children with Adrenocortical Tumors
Abstract
The excretory patterns of urinary steroids determined by capillary gas chromatography in 11 children (aged 0.8-16.5 years) with adrenocortical tumors were established. In 8 patients the predominant clinical feature was virilization, in 3 others, Cushing’s syndrome. In 5 patients (3 carcinoma, 2 adenoma) very high excretion of 3ß-hydroxy-5-ene steroids was observed. In 2 others (adenomas) only moderately elevated excretion of 11ß-hydroxyandrosterone was found. In 1 patient (adenoma) pregnanediol dominated in the steroid profile, accompanied by moderately elevated 3ß-hydroxy-5-ene steroids. Out of 3 Cushingoid patients (1 carcino- ma, 2 adenomas), 1 presented an atypical urinary steroid pattern for hypercortisolemia, with- out 5a-reductase and 11ß-hydroxysteroid dehydrogenase deficiencies. Neither the urinary steroid pattern nor tumor size alone were reliable indicators of tumor malignancy, as evaluat- ed by a pathological examination and subsequent metastasis-free survival.
| Abbreviation | Trivial or systematic name of steroid |
|---|---|
| AN | androsterone |
| ET | etiocholanolone |
| DHA | dehydroepiandrosterone |
| 45-AND | androst-5-ene-38,17ß-diol |
| 11-OHAN | 11ß-hydroxyandrosterone |
| 11-OAN | 11-oxo-androsterone |
| 11-OET | 11-oxo-etiocholanolone |
| 11-OHET | 11ß-hydroxyetiocholanolone |
| 16a-OHDHA | 16a-hydroxydehydroepiandrosterone |
| PD | pregnanediol |
| PT | pregnanetriol |
| 45-PD | pregn-5-ene-3฿,20a-diol |
| Δ5-ΑΝΤ(16α) | androst-5-ene-36,16a,17ß-triol |
| THS | tetrahydro-11-deoxycortisol |
| 45-16OHPN | 16a-hydroxypregnenolone |
| 45-PT(17) | pregn-5-ene-38,17a,20a-triol |
| THE | tetrahydrocortisone |
| 45-PT(16) | pregn-5-ene-38,16a,20a-triol |
| THB, a-THB | tetrahydrocorticosterone, allo-tetrahydrocorticosterone |
| THF, a-THF | tetrahydrocortisol, allo-tetrahydrocortisol |
| a-CTN, B-CTN | cortolone |
| a CT, ß CT | cortol |
| fF | free cortisol |
| 6a-OHF | 6B-hydroxycortisol |
| 20g-DHF | 20a-dihydrocortisol |
Introduction
Adrenocortical tumors are uncommon in children. Al- though nonsecreting tumors have been reported, the ma- jority of children manifest clinical symptoms of hormone excess such as hypercortisolemia, virilization, feminiza- tion, hyperaldosteronism or combinations of these fea- tures [1-8]. Both types of adrenocortical tumors (carcino- mas and adenomas) produce a wide variety of steroid hor- mones. This is a consequence of multiple enzyme defi- ciencies in tumor tissues. The tumor cells are capable of synthesizing a higher proportion of steroid hormone pre- cursors [9-12].
In the search for diagnostic methods and biochemical markers differentiating adrenocortical carcinoma and ad- enoma, various chemical methods have been used to ana- lyze the steroids from peripheral or adrenal venous blood as well as from the urine.
Gas chromatography of urinary steroid metabolites can be of value in the diagnosis and management of patients with adrenocortical tumors as has been con-
Received: November 29, 1994 Accepted after revision: April 11, 1995
Ewa M. Małunowicz, PhD
Department of Laboratory Diagnostics
Al. Dzieci Polskich 20
Child Health Center 02-736 Warsaw (Poland)
@ 1995 S. Karger AG, Basel 0301-0163/95/0444-0182 $8.00/0
Downloaded by:
| Case | Age years | Symptoms | 17-KS umol/1 | Outcome after operation | Type of steroid pattern |
|---|---|---|---|---|---|
| 1 | 0.8 | V+C | 103.8 | alive and well 5.1 years | |
| 2 | 1.3 | V+C | 79.6 | alive and well 10 years | |
| 3 | 5.0 | V | 346.0 | alive and well 10 years | |
| 4 | 7.0 | V | 224.0 | alive and well 22 months (under treatment with op'-DDD) | I |
| 5 | 14.0 | V | 1304.0 | alive and well 25 months (treated 1 year with op'-DDD) | |
| 6 | 3.3 | V | 17.3 | alive and well 5.2 years | |
| 7 | 6.0 | V | 17.3 | alive and well 9 years | II |
| 8 | 2.6 | V | 20.4 | alive and well 6.5 years | III |
| 9 | 3.0 | C+V | 44.3 | died after 11 months (multiple metastases: lymph nodes, liver, lungs) | |
| 10 | 13.5 | C+V | 93.0 | alive and well 23 months (treated 1 year with op'-DDD) | IV |
| 11 | 16.5 | C | 51.5 | alive and well 4.5 years |
Reference values: girls 1-7 years (n=10), <6.9; girls 14-18 years (n = 43), 12.8-52.0. V = Virilization; C = Cushing’s syndrome.
firmed by several authors [13-17]. The aim of this investi- gation was to study the excretory pattern of urinary ste- roids by capillary gas chromatography as an alternative to single hormone measurements in the serum of children with adrenocortical tumors. Attention was focused on diagnosis, prediction of clinical course in correlation with histopathological data and follow-up of patients in the context of possible tumor recurrence.
Patients
Eleven girls with adrenocortical tumors who were admitted to the Child Health Center, Warsaw, Poland, between 1984 and 1992 were included in this study. Their ages ranged from 10 months to 16.5 years. The clinical histories of the patients are summarized in table 1.
At surgery, 8 tumors were found to be encapsulated which per- mitted complete resection, but in two patients (9 and 10) the tumor was soft and broke apart during surgery. Infiltration of tumor cells to the adrenal vein was observed in patient 4 and metastases to lymph nodes were found in patient 9. Histopathologically, 4 children were found to have carcinomas and 7 adenomas. The morphological char- acteristics of the tumors are presented in table 2.
| Case | Tumor size, cmª | Primary diagnosis | No.b Weiss's criteria |
|---|---|---|---|
| 1 | 9 | Ca | 8 no venous invasion |
| 2 | 8 | Ca | 9 all |
| 3 | 6 | Ad | 3 only sinus invasion, diffuse architecture and less than 25% clear cells |
| 4 | 9 | Ca | 7 no high mitotic rate, no atypical mitosis |
| 5 | 12 | Ad | 4 no venous or capsule invasion, no high mitotic rate, no atypical mitoses, no high nuclear grade |
| 6 | 3 | Ad | 4 no venous or capsule invasion, no high mitotic rate, no atypical mitoses |
| 7 | 7 | Ad | 2 only high nuclear grade and less than 25% clear cells |
| 8 | 4 | Ad | 1 less than 25% clear cells only |
| 9 | 5 | Ca | 9 all |
| 10 | 5 | Ad | 4 no venous or capsule invasion or sinusoidal invasion, no high mitotic rate, no atypical mitoses |
| 11 | 3 | Ad | 1 less than 25% clear cells only |
a Evaluated by a pathologist.
b ≤2 = Nonmetastasizing/nonrecurring tumor; 3 = border zone; ≤4-9 = metastasizing/recurring tumors.
| 3ß-Hydroxy-5-ene steroids | Patient | 1 Patient 2 Patient 3 | Patient | 4 Patient 5 | Reference values | ||
|---|---|---|---|---|---|---|---|
| girls 1-7 years (n = 10) | girls 14-18 years (n = 20) (range) | ||||||
| DHA | 19.4 | 22.0 | 110.7 | 96.5 | 572.0 | <0.08 | 0.3-1.9 |
| 45-AND | 3.8 | 13.2 | 18.4 | 10.0 | 105.9 | ND | <0.3 |
| 16a-OHDHA | 4.6 | 74.3 | 27.7 | 13.2 | 105.3 | ND | 0.2-1.37 |
| Δ5-ΑΝΤ (16β) | - | - | 21.8 | 4.0 | 25.6 | ND | not evaluated |
| Δ5-ΑΝΤ(16α) | 7.5 | 21.5 | 16.2 | 6.3 | 11.1 | ND | 0.52-2.19 |
| 45-PD | 2.5 | 4.3 | 6.2 | 2.9 | 14.5 | <0.06 | 0.17-1.3 |
| 45-16OHPN | - | - | 1.8 | 1.9 | 2.4 | ND | ND |
| Δ5-PT(17) | <0.1 | <0.1 | 1.2 | 10.4 | 13.1 | <0.06 | 0.39-1.05 |
Materials and Methods
Steroids, conjugated and then liberated by enzymatic hydrolysis (ß-glucuronidase/arylsulfatase from Helix pomatia; Serva), were ex- tracted from urine with WatersTM Sep-pak C-18 cartridges (Millipore Co., USA) and prepared for gas-chromatographic analysis as delin- eated by Shackleton [18]. Gas chromatography was carried out on a 25 m × 0.32 mm i.d., film 0.25 um OV-1 glass capillary column, housed in a Hewlett-Packard 5890 II gas chromatograph with split 1:20 injection. Stigmasterol (Sigma Chemical Co., USA) was used as the internal standard.
When doubts about peak identification occurred, GC/MS was performed (Hewlett-Packard 5970 MSD).
Total urinary 17-oxosteroids (17-KS) were determined according to the procedure of Kraushaar et al. [19]. Tumors were evaluated according to standard methods [20] and, additionally, according to the nine criteria of Weiss [21] by the same histopathologist (M.D.). These criteria were: (1) nuclear grade; (2) mitotic rate; (3) atypical mitoses; (4) character of cytoplasm; (5) architecture of tumor cells; (6) necrosis; (7) invasion of venous structure; (8) invasion of sinusoi- dal structures; (9) invasion of capsule tumor.
Results
An assessment of urinary steroid patterns in children with adrenocortical tumors confirmed their heterogene- ity. Four types of excretion patterns were identified. Rep- resentative steroid profiles for each pattern are shown in figure 1.
Pattern I (patients 1-5): High excretion of the metabo- lites of 3ß-hydroxy-5-ene steroids. DHA and its 16- hydroxylated derivatives such as 16a-DHA and androst- 5-ene-30,16a,17ß-triol predominated. In spite of the mild Cushingoid appearance in our youngest patients (1 and 2), their cortisol metabolite (THE, THF, allo-THF) patterns
| Name of steroid | Patient 6 Patient | 7 Patient | 8 Reference values girls 1-7 years (n= 10) | |
|---|---|---|---|---|
| AN | 3.5 | 2.1 | 6.7 | <0.5 |
| ET | 0.77 | 0.86 | 3.4 | <0.5 |
| DHA | 0.30 | 0.18 | 1.4 | <0.08 |
| 11-OHAN | 4.5 | 5.6 | 2.4 | <0.8 |
| 16a-OHDHA | - | - | 3.3 | ND |
| Δ5-ΑΝΤ(16α) | 1.24 | 2.6 | 1.8 | ND |
| 45-PT(17) | - | - | 1.4 | <0.06 |
| 45(16) | - | - | 6.1 | ND |
| PD | 1.4 | 9.3 | <0.37 | |
| PT | 2.8 | 2.9 | <0.457 | |
were normal (data not shown). Only excretion of 11ß- hydroxyandrosterone was elevated. The excessive amounts of steroids excreted by all of the individuals in this group over a 24-hour period are illustrated in table 3.
Pattern II (patients 6 and 7): A metabolite of 11ß- hydroxyandrostendione (e.g. 11ß-hydroxyandrosterone) is the major androgen in the profile. Moderately increased excretion of androst-5-ene-30,16a,17ß-triol was also found (table 4).
Pattern III (patient 8): Progesterone and pregnenolone metabolites dominate in the steroid profile (e.g. pregnane- diol, pregn-5-ene-30,16a,20a-triol). 45- and 44-androgens were moderately elevated (table 4).
II
1
=
13
AN
DHA
ET
AN
11 - OET
11 - 0AN
DHA
A -AND
11 - OHAN
1&C-ONDNA
PT
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N -PD
ANTIS AI
A -ANTII&O
THE
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- THB
THE
45-P7117)
THF
·- THF
THF
PCTN + PCT
CCTN
CCTN
TNF
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PCTN + PCT
1.5.
I.S.
IV
III
IF
IF
AN
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ET
AN
DHA
11-0AN
11 - OET
11 - OHET
11 - OHAN
11 - OHAN
164 - OHDNA
PT
PT
PD
45.PD 45- ANT(18CC)
THS
0
5 -PT(17)
THE
THE
5 A -PT(18)
THF
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L- CTN
-CTN
P-CTN +B-CT
PCTN + PCT
L-CT
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L.S.
36P - OHF 20&-DHF
pattern II = patient 6; pattern III = patient 8; pattern IV = patient 10.
Fig. 1. Representative GC profiles of steroids in urine of girls with adrenocortical tumors. Pattern I = Patient 4;
Pattern IV (patients 9-11): High levels of excreted cor- tisol metabolites (THE, THF, allo-THF, fF, 6ß-OHF) and 11-deoxycortisol metabolites (THS) were characteristic for this pattern. Moderately elevated levels of 45-andro- gen metabolites were also found (table 5).
In all of the children except one (patient 9), the urinary steroid profile normalized after surgery. In patient 9, urine sample examination 1 month after surgery revealed
a high level of adrenal steroids, when compared to that before operation. Metastases to the liver were found.
Repeated determination of steroids after 1, 3 and 6 months then annually showed a normal urinary pattern in some patients or steroid suppression in patients treated with o,p’-DDD and/or hydrocortisone.
| Name of steroid | Patient 9 Patient 10 Patient | 11 Reference values (mean ± SD) | ||
|---|---|---|---|---|
| AN | 1.4 | 11.2 | 1.7 | 7.3±2.8 |
| ET | 2.8 | 18.2 | 14.1 | 6.3±3.1 |
| 11-OHAN | 7.0 | 28.7 | 11.0 | 3.45±1.1 |
| THS | 6.2 | 16.6 | 7.7 | <0.14 |
| THE | 7.7 | 38.6 | 7.7 | 6.85±1.9 |
| THF | 16.0 | 24.6 | 38.2 | 3.4±1.1 |
| a-THF | 7.6 | 24.0 | 1.3 | 2.89±0.9 |
| fF | 1.53 | 1.3 | 1.8 | <0.55 |
| 6ß-OHF | 1.18 | 4.1 | 4.7 | <0.52 |
| 20a-DHF | 1.9 | 3.0 | 2.4 | <0.55 |
| Ratio | ||||
| ET/AN | 2.0 | 1.62 | 8.29 | 0.88±0.32 |
| THE/THF | 0.48 | 1.57 | 0.2 | 2.07±0.44 |
| THF/a-THF | 2.1 | 1.02 | 29.4 | 1.24±0.35 |
Discussion
A urinary steroid profile by capillary gas chromatogra- phy identifies up to 40 steroid metabolites that appear along the biosynthesis pathway of cortisol and aldoste- rone. Urinary steroid profiles have revealed a variety of enzyme defects involving steroid biosynthesis in vivo in the adrenocortical tumors of our patients.
Urinary steroid pattern I is characteristic for most common virilising adrenal tumors. This pattern indi- cates impairment of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity in tumor adrenal tissue resulting in high urinary excretion of DHA metabolites (e.g. 16a-hydroxy DHA, androst-5-ene-3,17ß-diol, androst- 5-ene-30,16a,17ß-triol) and pregnenolone (pregn-5-ene- 30,20ß-diol) or 17a-hydroxypregnenolone (pregn-5-ene- 30,17a,20a-triol). This type of steroidogenesis in adrenal tumors with wide individual variations has been con- firmed by urinary steroid profiling in children and adults [13-17] and by studies on the steroid content of the adre- nal venous effluent [22].
Very high levels of urinary 17-ketosteroids (17-KS) were observed in all our patients with steroid pattern I; Honour et al. [14] made similar observations in their series.
Although pattern I of urinary steroids suggests an adre- nal cortex tumor, it alone is not sufficient for a biochemi-
cal diagnosis, especially in older children who need fur- ther testing (dexamethasone suppression test). This type of urinary steroid profile is common in pubertal girls and women with nontumor symptoms of androgenization (hirsutism, acne) and, in some patients, can reflect CAH due to nonclassical 36-HSD deficiency. This possibility should be investigated by additional serum steroid deter- minations after the ACTH stimulation test [23-26].
However, when excessive excretion of 30-hydroxy- 5-ene-steroids is accompanied by elevated levels of other cortisol precursors, e.g. tetrahydro-11-deoxycorti- sol (THS), this reflects ‘mixed’ enzyme deficiencies, and the urinary steroid profile alone is highly suggestive of an adrenal tumor [15, 17].
Benign or malignant adrenocortical tumors are often difficult to differentiate by pathology evaluation or tumor size [27-30]. Some authors attempted to find preopera- tive discrimination between this two types of tumors by determining urinary and serum steroids. They consider increased excretion of 3ß-hydroxy-5-ene-steroids and/or THS (as well as serum 11-deoxycortisol) or decreased plasma corticosterone/11-deoxycorticosterone ratio as biochemical markers for malignancy [15, 17, 31-33].
All our patients with urinary steroid pattern I and tumor size from 6 to 12 cm were candidates for adrenal carcinoma. Among 5 patients, histopathological (conven- tional and according to Weiss’s criteria) signs of malig- nancy were found in only 3. Tumors from two other patients (3 and 5) were conventionally evaluated as ade- noma but fulfilled Weiss’s histological criteria on 3 and 4, classifying them as borderline in the first case and poten- tially metastasizing in the second. The 10-year metastasis- free survival of patient 3 seems to indicate that the adre- nocortical tumor was benign.
The clinical symptoms in patients with type II and III steroid profiles with marginally elevated urinary 17-KS suggested nonclassical forms of CAH. The results of urine steroid profiling which were not typical for CAH pointed to an adrenal tumor, which was then confirmed by CT scan.
Similar steroid profiles in children with characteristic 11ß-hydroxyandrosterone elevation (pattern II) were pre- sented by Shackleton’s group [13, 18] and Honour et al. [14]. However, patient 8 (pattern III) represents the unique steroid profile for adrenal tumor with evidence of excessive progesterone production (pregnanediol domi- nates among the metabolites). The urinary steroid profile of this patient suggests a combination of 3ß-HSD, 21a- hydroxylase and 17a-hydroxylase deficiencies in tumor tissue.
Patients 6 and 7 with 3- and 7-cm tumors, respectively, were diagnosed with adrenal adenoma although the small- er tumor was qualified as potentially metastasizing ac- cording to Weiss’s criteria. The 5-year follow-up after sur- gery shows no signs of reccurrence. In the report by Hon- our et al. [14], similar biochemical data as in our patients 6 and 7, were found in 5 children, and in one of them a 3.5-cm adrenal carcinoma was confirmed.
Of 3 patients with a type IV steroid pattern, presenting with Cushing’s symptoms, a typical urinary steroid pat- tern reflecting hypercortisolism was found in 2 (9 and 11), e.g. higher ratios of 50/5a steroid metabolites (ET/AN, THF/allo-THF) and lower ratio of THE/THF than in nor- mal controls. This indicated changes in the hepatic me- tabolism of cortisol and androstendione (5a-reductase and 11ß-hydroxysteroid dehydrogenase deficiencies). Similar results were shown by other authors [13, 15-18]. In the third patient (patient 10) normal ratios of hyperse- creted cortisol metabolites were observed, this reflects hypercortisolism without alteration of their hepatic me- tabolism (table 5).
In all our patients with clinical symptoms of Cushing’s syndrome we found increased excretion of THS, confirm- ing 11ß-hydroxylase deficiency in tumor tissues; this could be a marker of tumor malignancy. Of the 3 patients with this steroid pattern, a malignant tumor (size 5 cm) was unequivocally identified in only 1 (patient 9). The malignancy of one tumor (also 5 cm) was questionable (patient 10), it had no features of malignancy on conven- tional microscopic examination but fulfilled Weiss’s crite- ria on 4 (metastasizing tumor). After 1 year of treatment with op’-DDD and a subsequent 11 months’ follow-up without treatment, the patient is well with no signs of recurrence. Her urinary steroid profile is no longer patho- logical. The third patient (patient 11) was diagnosed with an adenoma (3 cm) and nonmetastasizing tumor accord- ing to Weiss’s criteria and is well after 4.5 years of follow- up.
The presented biochemical results, histopathological evaluation and size of adrenocortical tumors in children as well as the observed clinical course, confirmed that pre- diction of malignant behavior in this type of tumor is dif- ficult.
Treatment of adrenocortical carcinoma also represents a frustrating problem and is a matter of controversy [34, 35]. The toxicity of op’-DDD is one of the major limita- tions in the therapy with this drug, especially in pediatric patients. This was taken into account in the decision not to introduce op’-DDD treatment after radical surgery in our youngest patients with adrenal carcinoma (patients 1
and 2). Fortunately, 5.1- and 10-year metastasis-free sur- vival, respectively, is observed.
The possibility of introducing monitored therapy with op’-DDD, i.e. establishing individual doses to maintain the plasma level of the drug in the range of 14-25 µg/ml, prompted us to introduce improved op’-DDD treatment in 3 patients [36]. The decisions to begin treatment were based on the following: (1) in case 4 diagnosis of carcino- ma with infiltration of tumor cells to the adrenal vein; (2) in cases 5 and 10, the very fast clinical course of the disease, unclear histopathological diagnosis (4 morpho- logical characteristics of tumor by Weiss’s criteria) and findings of necrotic foci in the tumors in both cases; (3) additionally, in case 10, the soft consistency of the tumor, which caused it to break apart during surgery.
The individual doses of op’-DDD in monitored thera- py varied from 1 to 11 g/day.
The presented steroid patterns in our series of children with virilising and/or hypercortisolemic adrenocortical tumors do not cover all of the possible biochemical mani- festations of the tumor tissues, responsible for the above- mentioned clinical syndromes. Care should be taken in diagnosis, because the presence of excessive amounts of steroid precursors in the circulation can sometimes mimic the hormonal profile of CAH. Recently, a unique case of virilising adrenal adenoma in a 5-year-old girl, showing the urinary and plasma steroid characteristics for CAH due to 11ß-hydroxylase deficiency, was described. Addi- tional molecular studies have been performed to exclude CAH [37].
Conclusions
Urinary steroid profiling confirms the variability of steroidogenic properties in adrenocortical tumor tissue in vivo and can be used as an alternative to multiplied serum steroid determinations in the diagnosis and follow-up of patients in terms of possible recurrence of the tumor.
There is no single marker of adrenal cortex malignancy in children. Steroid pattern, tumor size and even histolog- ical evaluation, taken individually, can be misleading.
Acknowledgements
This work was presented in part at the Lawson Wilkins Pediatric Endocrine Society and European Society for Paediatric Endocrinolo- gy Fourth Joint Meeting, San Franscisco, USA, June 3-7, 1993 [1]. T.E.R. is a member of the ESPE.
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Małunowicz/Ginalska-Malinowska/Romer/ Ruszczyńska-Wolska/Dura
Urinary Steroid Profiles in Adrenocortical Tumors
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