Evidence of 11 Beta-Hydroxylase Deficiency in Childhood Adrenocortical Tumors
The Plasma Corticosterone/11-Deoxycorticosterone Ratio as a Possible Marker for Malignancy
HELMUTH G. DOERR, MD,* WOLFGANG G. SIPPELL, MD,t STENVERT L. S. DROP, MD, PHD,# FRANK BIDLINGMAIER, MD, PHD,§ AND DIETER KNORR, MD*
In search for a biochemical marker to differentiate between adrenocortical carcinoma (AC) and ade- noma (AA), plasma levels of the following steroids were studied preoperatively and postoperatively: 11-deoxycorticosterone (DOC), corticosterone (B), 11-deoxycortisol (S), and cortisol (F). Levels were measured by Sephadex LH-20 chromatography and specific radioimmunoassays. The subjects included eight children ages 2 years, 5 months to 9 years, 10 months. There were three girls and 5 boys with pseudoprecocious puberty due to adrenocortical tumors (histologically, four were AC and four, AA). The preoperative showed that DOC and S levels were elevated in all patients, F levels were elevated in four of eight children when compared with age-matched controls, whereas B was normal. Postoperatively, all levels returned to normal. The ratios of B/DOC and F/S as an index of adrenal 11 beta-hydroxylase activity were calculated. The preoperative ratios of B/DOC were markedly decreased in all patients with AC compared to controls (7.7,4.1,5.9,1.9 versus 23.5, median), but normal in three of four patients with AA (16.2, 29.6, 16.1). The F/S ratios were significantly lower in AC and AA when compared with controls. The data indicate a deficiency in 11 beta-hydroxylation in cases of adrenocortical tumors. Despite a still limited number of patients, the decreased B/DOC ratios may possibly indicate malig- nancy and could be helpful in distinguishing by biochemical means between benign and malignant adrenocortical tumors.
Cancer 60:1625-1629, 1987.
T HE EXPERIENCE WITH ADRENOCORTICAL tumors in childhood is limited because they are extremely rare.1-4 In children, carcinomas are three times more common than adenomas.5 Both types produce a wide variety of steroid hormones of which androgens are, by far, the most prevalent.6 The histologic evaluation of malignancy is very difficult;7 only the size was regarded as an important morphologic predictor of clinical be- havior.28 The biochemical differentiation by hormonal studies was, to date, unsuccessful.8 Even an attempt to
correlate malignancy with increased dehydroepiandros- terone sulfate (DHEAS) levels failed since high DHEAS levels were also observed in proven adenoma cases.30
The aim of this study was to examine the question of whether in children a specific steroid pattern of a 11- beta-hydroxylase deficiency is detectable as it has been described in some cases for adults.9-11 The object also was to check the possibility of a biochemical differentia- tion between adrenocortical carcinoma and adenoma.
Materials and Methods
Eight children with adrenocortical tumors admitted between 1978 and 1983 were included in the study (Table 1). The clinical data and course of Patients 2, 3, and 8 have been reported.12 Six patients showed an iso- sexual, pseudoprecocious puberty, and Patients 1 and 3 showed a heterosexual, pseudoprecocious puberty. All patients presented as markedly tall and with accelerated bone development. None had hypertension. Histologi- cally, there were four children with carcinoma and four with adenoma.
From the Departments of Pediatrics, University of Munich,* Uni- versity of Kiel, t the Department of Clinical Biochemistry, University of Bonn,§ FRG, and Sophia Children’s Hospital, University of Rotter- dam,# The Netherlands.
Supported by grants from the Deutsche Forschungsgemeinschaft (Do 273/1-2, Si 197/2-2).
The authors thank Mrs. Reinhild Joswig for her skillful technical assistance.
Address for reprints: Helmuth G. Doerr, MD, Department of Pedi- atrics, University of Munich, Medical School, Lindwurmstr. 4,D-8000 Muenchen 2, West Germany.
Accepted for publication April 7, 1987.
| Patient | Sex | Age | Symptoms | Pathologic diagnosis |
|---|---|---|---|---|
| 1 | F | 2 yrs, 5 mos | V + C | Adenoma |
| 2 | M | 4 yrs, 8 mos | V | Adenoma |
| 3 | F | 9 yrs | V | Adenoma |
| 4 | M | 9 yrs, 10 mos | V + C | Adenoma |
| 5 | M | 3 yrs, 4 mos | V | Carcinoma |
| 6 | M | 3 yrs, 7 mos | V | Carcinoma |
| 7 | M | 4 yrs, 2 mos | V | Carcinoma |
| 8 | F | 6 yrs | F | Carcinoma |
V = virilisation; C = cushing; F = feminisation.
Among various hormonal parameters within the labo- ratory investigations, plasma levels of 11-deoxycortico- sterone, corticosterone, 11-deoxycortisol, and cortisol were measured simultaneously preoperatively and post- operatively by automated Sephadex LH-20 chromatog- raphy and subsequent radioimmunoassays as re- ported.13,14 Plasma samples were collected between 8
PLASMA 11-DEOXY - CORTICOSTERONE (DOC)
ng/ml nmol/l
1.2+3.6
0.9+2.7
0.6+1.8
o
O
0.3+0.9
.
8
0.1+0.3
T
PRE OP
POST OP
PLASMA 11-DEOXYCORTISOL (S)
ng/ml nmol/l
10
30
.
10
O 0
1.
1
ES
1
S
0.1 — 0.3
PRE OP
POST OP
AM and 10 AM by rapid venipuncture or through an intravenous catheter, thereby avoiding prolonged stress on the children. The postoperative samples were col- lected 3 to 4 weeks after surgery when the child was already in good clinical condition, neither showing clin- ical evidence of adrenocortical insufficiency nor having had glucocorticoid replacement therapy. Blood was col- lected into EDTA tubes, immediately centrifuged, and the plasma was stored at -20° C until assayed. For sta- tistical comparison between the means of two samples, Student’s t-test was used.
Results
11-Deoxycorticosterone (DOC)
In all patients with adrenocortical tumors (carcinoma and adenoma) plasma 11-DOC levels were preopera- tively clearly above the highest value measured in nor- mal controls (range, 0.29 to 1.01 ng/ml vs. 0.03 to 0.28 ng/ml). Postoperatively, however, all DOC levels re- turned to normal (Fig. 1).
11-Deoxycortisol (S)
Prior to surgery, plasma 11-S levels exhibited a pat- tern very similar to that of DOC (Fig. 2). All values ranging from 2.44 to 5.50 ng/ml were clearly above the
upper limit in healthy children (range, 0.21 to 1.50 ng/ml). After surgery, all levels were normal.
Corticosterone (B) and Cortisol (F)
Preoperatively and postoperatively, B plasma levels were not different from normal controls. Plasma F levels, however, were elevated before surgery in four of eight patients and in two of eight patients after surgery (Table 2).
Corticosterone/11-Deoxycorticosterone (B/DOC) Ratios
Whereas the ratios of all the carcinoma patients rang- ing between 1.9 and 7.7 were clearly below normal (8.8 to 125), those of three adenoma patients were within the normal range before surgery (Fig. 3). After surgery, the ratios of the carcinoma patients increased, while those of three adenoma patients slightly decreased or did not change. Only the ratio of Patient 1 (adenoma) showed the same pattern as the carcinoma patients.
Cortisol/11-Deoxycortisol (F/S) Ratios
The ratios of all carcinoma and of three adenoma patients were below normal. The ratio in Patient 3 (ade- noma) was within the low normal range. However, after removal of the tumor, all ratios increased significantly (2P < 0.001) into the normal range (Fig. 4).
Discussion
Most reports on childhood adrenocortical tumors are case reports because these tumors are so rare. Therefore, overall experience with these tumors in the pediatric age group is limited. Recent reports dealing mainly with adults have suggested that there are variable defects in the enzymatic equipment of the tumor cells resulting in a wide variety of highly abnormal steroid patterns simi- lar to those found in congenital adrenal hyperplasia. 15-19
In adults 11 beta-hydroxylase defects have more fre- quently been reported.9-11.20,21 In children, however, only two case reports so far have dealt with an 11-beta- hydroxylase deficiency.22.23 Our results before surgery showed markedly elevated plasma levels of both 11- DOC and S in all eight children. This suggests a reduced activity of 11 beta-hydroxylase. Such an enzymatic de- fect could be a typical feature of the tumor itself; or, the highly elevated plasma androgen levels, usually found in adrenocortical tumors, may competitively inhibit the 11 beta-hydroxylase enzyme system as suggested by Sharma et al.24 This suggestion was again recently ques- tioned by New and Levine.25
The F/S and B/DOC ratios represent only an indirect measure of 11 beta-hydroxylase activity.26 A signifi-
| Patient | Corticosterone | Cortisol | ||
|---|---|---|---|---|
| Preop | Postop | Preop | Postop | |
| 1 | 2.60 | 1.30 | 103.6 | 43.4 |
| 2 | 4.71 | 0.41 | 61.8 | 29.2 |
| 3 | 8.60 | 0.59 | 207.6 | 78.5 |
| 4 | 7.56 | 0.62 | 169.5 | 41.3 |
| 5 | 7.79 | - | 129.5 | - |
| 6 | 3.21 | 1.51 | 177.3 | 171.0 |
| 7 | 1.88 | 1.04 | 194.0 | 158.0 |
| 8 | 0.84 | 0.46 | 38.0 | 57.6 |
| Normal range | Normal range | |||
| = 0.29-9.37 | = 32.0-136.0 | |||
cantly reduced activity of this enzyme has indeed re- cently been demonstrated29 by direct studies in vitro, including one in this series (Patient 8). The clearly sub- normal F/S ratios observed in seven of eight patients further support the concept of a deficiency in 11 beta- hydroxylation. However, the B/DOC ratios were differ- ent between the patients with carcinoma and those with adenoma. While the B/DOC ratios of all carcinoma pa-
B / DOC
500
100
10
1
PRE
POST OP
OP
F/S
1000
100
.
10
1
PRE
POST
OP
OP
tients were below normal, those of three of four ade- noma patients were within the control range, suggesting a normal 11 beta-hydroxylase activity.
Regarding the F/S and B/DOC ratios of all patients, it appears that the 11 beta-hydroxylase defect is more pro- nounced in the cortisol than in the aldosterone biosyn- thetic pathway. This coincides with the suggestion that there is more than one 11 beta-hydroxylase enzyme sys- tem27 and with the two-gland concept of the adrenal cortex,25 that includes separately regulated biosynthetic pathways within the zona fasciculata and glomerulosa. However, aldosterone levels (data not shown) were within normal limits in all cases studied. This would suggest that glomerulosa function is not affected at least under basal conditions, and that the 11 beta-hydroxy- lase deficiency is mainly restricted to the fasciculata zone. It is still unclear, however, why B/DOC ratios in carcinoma patients tended to be lower than in adenoma patients. We can therefore only speculate that a de- creased B/DOC ratio may indicate malignancy, but this hypothesis will have to be evaluated in many more of patients.
After surgery, DOC and S levels returned to normal, and F/S ratios in all patients and B/DOC ratios in the carcinoma patients increased, indicating the success of the surgical removal of the tumor. Therefore, DOC and S levels as well as the F/S and B/DOC ratios seem also to be useful markers in the postoperative monitoring of adrenocortical tumors in children.
REFERENCES
1. Bulger AR, Correa RJ. Experience with adrenal cortical carci- noma. Urology 1977; 10:12-18.
2. Lefevre M, Gerard-Marchant R, Gubler JP, Chaussain JL, Le- merle J. Adrenal cortical carcinoma in children: 42 patients treated from 1958 to 1980 at Villejuif. In: Humphrey GB, Gridey GB, Dehner LP, Acton RT, Pysher TJ, eds. Adrenal and Endocrine Tumors in Children. Boston: Martinus Nijhoff Publishers, 1984; 265-276.
3. Weatherby RP, Carney JA. Pathologic features of childhood ad- renocortical tumors. In: Humphrey GB, Gridey GB, Dehner LP, Acton RT, Pysher TJ, eds. Adrenal and Endocrine Tumors in Chil- dren. Boston: Martinus Nijhoff Publishers, 1984; 217-248.
4. Young JL, Miller RW. Incidence of malignant tumors in U.S. children. J Pediatr 1975; 86:254-258.
5. Neville AM, Symington T. Bilateral adrenocortical hyperplasia in children with Cushing’s syndrome. J Pathol 1972; 107:95-106.
6. Korth-Schuetz S, Levine LS, New MI. Dehydroepiandrosterone sulfate levels: A rapid test for abnormal adrenal androgen secretion. J Clin Endocrinol Metab 1976; 42:1005-1013.
7. Slooten van H, Schaberg A, Smeenk D, Moolenaar AJ. Morpho- logic characteristics of benign and malignant adrenocortical tumors. Cancer 1985; 55:766-773.
8. Honour JW, Price DA, Taylor NF, Marsden HB, Grant DB. Steroid biochemistry of virilising adrenal tumours in childhood. Eur J Pediatr 1984; 142:165-169.
9. Lipsett MB, Hertz R, Ross GT. Clinical and pathophysiological aspects of adrenocortical carcinoma. Am J Med 1963; 35:374-383.
10. Minowada S, Kinoshita K, Hara M, Isurugi K, Uchikawa T, Nijima T. Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma. Endocrinol Japon 1985; 32:29-37.
11. West CD, Kumagai LF, Simons EL, Dominguez OV, Berliner DL. Adrenocortical carcinoma with feminization and hypertension associated with a defect in 11 beta-hydroxylation. J Clin Endocrin Metab 1964; 24:567-579.
12. Frohn-Mulder IME, Drop SLS, Meradji M, ten Kate FWJ, Doerr HG, Sippell WG. Precocious pubertal development as present- ing symptom of an adrenal tumour. J Pediatr Endocrinol (in press).
13. Sippell WG, Lehmann P, Hollmann G. Automation of multiple Sephadex LH-20 column chromatography for the simultaneous sepa- ration of plasma corticosteroids. J Chromatogr 1975; 108:305-312.
14. Sippell WG, Bidlingmaier F, Becker H et al. Simultaneous ra- dioimmunoassay of plasma aldosterone, corticosterone, 11-deoxycor- ticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol and cortisone. J Steroid Biochem 1978; 9:63-74.
15. Atwill WH, Ensor RD, Glenn GF. Massive pregnanetriol ex- cretion due to virilizing adrenal tumour. J Endocrin 1970; 46:547-548.
16. Boyar RM, Nogeire C, Fukushima D, Hellman L, Fishman J. Studies of the diurnal pattern of plasma corticosteroids and gonadotro- pins in two cases of feminizing adrenal carcinoma: Measurements of estrogen and corticosteroid production. J Clin Endocrinol Metab 1977; 44:39-45.
17. Huhtaniemi I, Kahri AI, Pelkonen R, Salmenpera M, Sivula A, Vihko R. Ultrastructural and steroidogenic characteristics of an an- drogen-producing adrenocortical tumour. Clin Endocrinol 1978; 8:305-314.
18. Cameron EHD, Hammerstein J, Jones D, Morris S, Griffiths K. Steroid synthesis in a human virilizing adrenal carcinoma: Some un- usual features. Acta Endocrinol 1970; 65:133-147.
19. Weinstein RL, Kliman B, Neeman J, Cohen RB. Deficient 17- hydroxylation in a corticosterone producing adrenal tumor from an infant with hemihypertrophy and visceromegaly. J Clin Endocrin 1970: 30:457-468.
20. Dennis PM, Cope CL, Pearson J. The production of 11-deoxy- cortisol (Reichstein’s Substance S) in patients with adrenocortical hy- perfunction. J Endocrin 1967; 39:207-212.
21. Solomon SS, Swersie SP, Paulsen CA, Biglieri EG. Feminizing adrenocortical carcinoma with hypertension. J Clin Endocrin 1968; 28:608-612.
22. Costin G, Goebelsmann U, Kogut MD. Sexual precocity due to a testosterone-producing tumor. J Clin Endocrinol Metab 1977; 45:912-919.
23. Tan S-Y, Genel M, Forman BH, Mulrow PJ. Steroid profile in a case of adrenal carcinoma with severe hypertension. Am J Pathol 1977; 67:591-593.
24. Sharma DC, Forchielli E, Dorfman RI. Inhibition of enzymatic steroid 11 beta-hydroxylation by androgens. J Biol Chem 1963; 238:572-575.
25. New MI, Levine LS. Congenital Adrenal Hyperplasia. Mono- graphs on Endocrinology. New York: Springer-Verlag, 1984.
26. Ganguly A, Meikle AW, Tyler FH, West CD. Assessment of 11 beta-hydroxylase activity with plasma corticosterone, deoxycorticoste- rone, cortisol and deoxycortisol: Role of ACTH and angiotensin. J Clin Endocrinol Metab 1977; 44:560-568.
27. Levine LS, Rauh W, Gottesdiener K et al. New studies of the 11 beta-hydroxylase and 18-hydroxylase enzymes in the hypertensive form of congenital adrenal hyperplasia. J Clin Endocrinol Metab 1980; 50:258-263.
28. Cagle PT, Hough AJ, Pysher J et al. Comparison of adrenal cortical tumors in children and adults. Cancer 1986; 57:2235-2237.
29. Degenhart HJ, Drop SLS, Hoogerbrugge J et al. In vitro studies of enzymatic activities in human adrenal tumors. Hormone Research 1982; 16:96-99.
30. Gabrilove JL, Seman AT, Sabet R et al. Virilizing adrenal ade- noma with studies on the steroid content of the adrenal venous effluent and a review of literature. Endocrine Reviews 1981; 2:462-470.