Clinical and Genetic Features of Adrenocortical Lesions in Multiple Endocrine Neoplasia Type 1*
BRITT SKOGSEID, CATHARINA LARSSON, PER-GUNNAR LINDGREN, EVA KVANTA, JONAS RASTAD, ELVAR THEODORSSON, LEIF WIDE, ERIK WILANDER, AND KJELL ÖBERG
Departments of Internal Medicine (B.S., K.O.), Diagnostic Radiology (P .- G.L.), Surgery (J.R.), Clinical Chemistry (L.W.), and Pathology (E.W.), University Hospital, and the Ludwig Institute for Cancer Research (K.O.), Uppsala; and the Departments of Clinical Genetics (C.L., E.K.) and Clinical Chemistry (E.T.), Karolinska Hospital, Stockholm, Sweden
ABSTRACT
In multiple endocrine neoplasia type 1 (MEN-1), benign enlarge- ment of the adrenal cortex has been found in about one third of necropsy cases. To elucidate the clinical and genetic characteristics of the MEN-1 adrenal lesion, we have investigated 33 MEN-1 patients. Twelve individuals (37%) demonstrated adrenal enlargement, which was bilateral in 7 of them. Histopathology revealed diffuse and nodular cortical hyperplasia, adenomas, and a single case of adrenocortical carcinoma. The apparently benign adrenal enlargements were not associated with presently ascertainable biochemical disturbances in the hypothalamic-pituitary-adrenocortical axis, and they were without ra- diological signs of progression during follow-up. The individual devel- oping unilateral adrenocortical carcinoma showed rapid adrenal expan-
sion, feminization, and an abnormal urinary steroid profile after 4 yr of observation for bilateral minor adrenal enlargements. Pancreatic endocrine tumors were significantly overrepresented and present in all MEN-1 individuals with adrenal involvement. In agreement with find- ings in sporadic cases, the MEN-1 adrenocortical carcinoma genome showed loss of constitutional heterozygosity for alleles at 17p, 13q, 11p, and 11q. The benign adrenal lesions retained heterozygosity for the MEN-1 locus at chromosome 11 q 13. Despite its prevalence and malignant potential, the pituitary-independent adrenocortical prolif- eration does not appear to be a primary lesion in MEN-1, but might represent a secondary phenomenon, perhaps related to the pancreatic endocrine tumor. (J Clin Endocrinol Metab 75: 76-81, 1992)
T HE GENETIC defect responsible for multiple endocrine neoplasia type 1 (MEN-1) has been mapped to chromo- some 11 q 13 (1). Tumorigenesis may result from unmasking of a recessive mutation (1, 2), and this sequence of events has been shown in the parathyroid and pancreatic endocrine lesions morphologically defined as hyperplasia as well as the pancreatic endocrine neoplasia of MEN-1 (1, 3-5). Involve- ment of the adrenal gland has been reported in a considerable proportion (36-41%) of patients with MEN-1 (6-9). This lesion is often characterized as bilateral hyperplasia or ade- nomas, and occasionally even carcinoma (7, 9), of the adrenal cortex without consistent signs of adrenocortical hormone excess. These findings raise the question of whether prolif- eration of the adrenal cortex, despite its mesodermal origin and steroid hormone production, should be regarded as one of the primary lesions in MEN-1. The present study eluci- dates genetic alterations and clinical features of the adrenal lesions in MEN-1.
Materials and Methods
The study comprises 33 MEN-1 patients (15 women and 18 men) from 20 different families. Their age at inclusion was 21-71 yr (mean ± SD, 50 ± 14 yr), and they were recruited due to a clinically overt MEN-1
Address all correspondence and requests for reprints to: Britt Skog- seid, M.D., Department of Internal Medicine, University Hospital, S-751 85 Uppsala, Sweden,
* This work was supported by the Swedish Cancer Foundation.
syndrome (n = 23) or screening among kindreds (10). At initiation of the present study, three individuals were hypercalcemic due to hyper- parathyroidism, and another 28 patients had undergone parathyroid surgery (Table 1). Twenty-four (73%) of the patients had pancreatic endocrine tumors diagnosed at a mean age of 46 ± 14 (±sD) yr and recognized biochemically during an average of 6.0 ± 4 yr. Sixteen (48%) of the individuals had pituitary enlargements with PRL (n = 13) or GH (n = 3) excess during a mean of 8.8 ± 8 yr before biochemical normali- zation upon surgery, irradiation, and/or medical therapy. The patients were followed for 1-6 yr (mean ±sD, 4.5 ± 1.4 yr) after inclusion in the study and were subjected to annual biochemical and radiological inves- tigations.
After thorough biochemical evaluation of the MEN-1 syndrome (10), the patients were examined with abdominal computed tomography (CT; Siemens Somatom DR 2) using a 4-mm slice thickness and by abdominal ultrasonography (Acuson 128; 3.5 mHz). The films were read independ- ently of knowledge of clinical status. The adrenals were considered enlarged when the width of the adrenal wings was more than twice the normal size or when a localized enlargement of more then 7 mm in diameter was found. In patients with adrenal enlargement, the hypo- thalamic-pituitary-adrenal axis and adrenal cortical and medullary func- tion were investigated by measurements of plasma CRF (competetive RIA in acid-ethanol-extracted plasma using antiserum RAS8561 from Peninsula Laboratories, Belmont, CA), plasma proopiomelanocortin (POMC; Neurochemical Laboratory, St. Lars Hospital, Lund, Sweden), plasma ACTH and serum dehydroepiandrosterone sulfate (Scandinavian Immune Laboratory, Malmo, Sweden), aldosterone and testosterone (solid phase RIA kits, Diagnostic Products Corp., Los Angeles, CA), and plasma neuropeptide-Y (11). The diurnal serum cortisol profile and the 24-h urinary cortisol excretion (Farmos Diagnostica, Turku, Finland) were determined as well as the 24-h urinary adrenaline and noradren- aline values corrected for urinary creatinine (Calab Medical Laboratory, Stockholm, Sweden), and a 24-h urinary steroid profile was obtained (12, 13). A 12-h dexamethasone (1 mg) suppression test was performed, in which at least 50% reduction of the serum cortisol values was
MEN-1 ADRENAL CORTEX
| Patient no./family | Age at diagnosis (yr) | Pancreatic tumor markers | Adrenal lesion | ||||
|---|---|---|---|---|---|---|---|
| HPT | PT | EPT | Adrenal | Size (cm) | Histology | ||
| 1/A | 31 | 31 | 34 | 38 | PP, insulin, proinsulin | 1/1 | Diffuse hyperplasia |
| 2/A | 54 | 42 | 60 | PP, insulin, proinsulin, gastrin | 1/1 | ||
| 3/B | 50 | 50 | 55 | PP, gastrin | 1/1 | ||
| 4/C | 50 | 50 | 50 | 50 | PP, insulin, proinsulin, glucagon | 1/- | |
| 5/D | 40 | 51 | 52 | 58 | PP, insulin, proinsulin, gastrin, hCGa | 1/2,5 | Atypical adenoma |
| 6/E | 42 | 47 | 47 | 50 | PP, insulin, proinsulin, gastrin | 10/1.5 | Unilateral carcinoma hyperplasia |
| 7/F | 34 | 53 | 53 | PP, insulin, gastrin, hCGa | 3/10 | Hyperplasia, adenoma, cyst | |
| 8/G | 57 | 57 | 57 | 57 | PP, insulin, gastrin, amylin | 4/- | Hyperplasia |
| 9/H | 55 | 54 | 60 | PP, neurotensin | -/3 | ||
| 10/I | 39 | 49 | 47 | 52 | Insulin, gastrin, glucagon | 1/1.5 | Diffuse hyperplasia |
| 11/J | 54 | 64 | 71 | Proinsulin, gastrin, somatostatin | -/1.5 | ||
| 12/K | 50 | 19 | 49 | 55 | PP, insulin, proinsulin, gastrin | -/1 | |
| 13/A | 28 | 25 | PP, proinsulin | ||||
| 14/B | 51 | 60 | PP, gastrin, VIP, hCGa | ||||
| 15/B | 48 | 52 | PP, gastrin, VIP, neurotensin | ||||
| 16/B | 28 | 37 | PP | ||||
| 17/B | 31 | None | |||||
| 18/B | 19 | 15 | 20 | PP, insulin, gastrin | |||
| 19/B | 19 | 19 | PP | ||||
| 20/C | 30 | 30 | None | ||||
| 21/C | 28 | 28 | None | ||||
| 22/D | 45 | 59 | None | ||||
| 23/D | 19 | 19 | PP, insulin, proinsulin | ||||
| 24/E | 26 | 20 | None | ||||
| 25/L | 39 | 50 | PP, gastrin | ||||
| 26/M | 55 | 54 | 60 | PP, proinsulin | |||
| 27/N | 48 | 48 | None | ||||
| 28/0 | 55 | 55 | PP | ||||
| 29/P | 38 | 35 | None | ||||
| 30/Q | 36 | None | |||||
| 31/R | 46 | Gastrin, hCGa | |||||
| 32/S | 65 | 65 | None | ||||
| 33/T | 66 | 66 | PP, gastrin | ||||
PP, Pancreatic polypeptide; VIP, vasoactive intestinal polypeptide.
considered to be a normal response. A CRF stimulation test was con- ducted with measurements of plasma ACTH and serum cortisol re- sponses for 90 min after iv injection of 100 ug CRF (Corticoliberin human, Bissendorf Peptide, Hannover, Germany). These results were compared with those of eight healthy controls (four women and four men), aged 17-61 yr (mean ± SD, 37 ± 18 yr).
Adrenocortical tissue specimens were obtained from six of the patients with adrenal enlargement. These were obtained by ultrasonographically guided cutting biopsies of 1.2 mm width (patient 5; Table 1) (14, 15) as well as during adrenalectomy with or without pancreatic resection in five patients. Microscopic investigations were performed on formalin- fixed 4-um thin paraffin sections stained with hematoxylin-eosin, van Gieson stain, the argyrophil reaction (16), and antibodies to neuron- specific enolase (Dako Corp., Carpinteria, CA) and chromogranin to exclude medullary sampling (17).
After histological investigation of slices from all tissue specimens, high mol wt DNA was isolated from the adrenocortical tissue as well as from peripheral leukocytes of the same individuals. The DNA samples were digested to completion with restriction enzymes, electrophoresed, transferred to nylon membranes, and hybridized to radiolabeled probes, as previously described (18). The restriction fragment length polymor- phisms (RFLP) markers, their location (19), and restriction enzymes used are listed in Table 2.
Statistics
Two-tailed Student’s t test for unpaired samples, the nonparametric Mann-Whitney U test, and the x2 test were used for statistical evaluation. P < 0.05 was considered significant.
| Locus | Location | Enzyme | Patient no. | |||
|---|---|---|---|---|---|---|
| 1 | 5 6 | 7 | 8 10 | |||
| D11S12 | 11p15.5 | TaqI | ☒ | |||
| HRAS | 11p15.5 | TaqI | ☒ | ☒ | ||
| INS | 11p15.5 | TaqI/PstI | ☒ | ☒ ☒ | ||
| CALCA | 11p15.4 | TaqI | ☒ ☒ | |||
| PTH | 11pter-p15.5 | TaqI | ||||
| CD20 | 11q12-q13.1 | MspI | ☒ | |||
| PYGM | 11q12-q13.2 | TaqI/MspI | ☒ | |||
| D11S146 | 11q12-q13.2 | TaqI | ☒ | ☒ | ||
| INT2 | 11q13 | TaqI | ☒ | ☒ ☒ | ||
| D11S97 | 11q13 | HaeIII | ☒ | ☒ | ||
| D11S29 | 11q23-qter | TaqI | ☒ | |||
| DIS57 | 1 | TaqI | ☒ | |||
| D2S44 | 2p | TaqI | ☒ | |||
| D10S25 | 10q | TaqI | ☒ | |||
| D13S1 | 13q12-q14 | TaqI/MspI | ||||
| D17S5 | 17p13.3 | BamHI | ||||
| D17S58 | 17p11.2-cen | TagI | ☒ | |||
| D22S10 | 22q11.1-q11.2 | TaqI | ☒ | |||
| MB | 22q11.2-qter | TaqI | ☒ | |||
Results
Twelve (37%) of the patients (7 women and 5 men) from 11 of the families displayed adrenal enlargement upon CT and ultrasonography. Comparison of these patients with those lacking signs of adrenal lesions demonstrated no sig- nificant difference with respect to mean age (55 ± 8 vs. 50 ± 16 yr) or recognized mean duration of the MEN-1 syn- drome (14 ± 8 vs. 12 ± 7 yr). Those with adrenal enlarge- ment, however, had histories of combined hyperparathyroid- ism and pituitary and endocrine pancreatic involvement at greater frequency (58% vs. 9.5%). This discrepancy was mainly due to a significant overrepresentation of pancreatic endocrine tumors (P < 0.05, by x2 test), which were present in all patients with adrenal lesions (Table 1). These pancreatic lesions were multiple and associated with peripheral excess of mainly pancreatic polypeptide, insulin, proinsulin, and gastrin in different combinations (Table 1). Only the presence of elevated insulin and proinsulin levels, either alone or in combination, was significantly (P < 0.05, by x2 test) more common than in the pancreatic involvement of the individ- uals lacking signs of adrenal enlargement. None of the patients with adrenal lesions displayed severe endocrine symptoms related to their pancreatic tumors, which were associated with metastatic disease in 6 of them. Seven of the patients with adrenocortical involvement had histories of hyperprolactinemia. The mean duration of pituitary hyperse- cretion before normalization due to therapy was statistically indistinguishable between these individuals and those lack- ing signs of adrenal enlargements (10 vs. 8 yr).
The size of adrenal lesions averaged 2.5 ± 2.8 (±SD) cm, and they were located bilaterally in 7 of the patients. During the course of follow-up, the size of the lesions remained stable in all but one individual. This patient developed feminization in association with rapid (13 months) adrenal enlargement due to a unilateral adrenocortical carcinoma after 4 yr of observation for minor bilateral adrenal enlarge- ments (Fig. 1). None of the others showed any symptoms or signs of adrenal hormone excess. The biochemical analysis revealed normal basal values in blood for aldosterone, tes- tosterone, dehydroepiandrosterone sulfate, neuropeptide-Y, POMC, CRF, and ACTH as well as in urine for cortisol, adrenaline, and noradrenaline in all MEN-1 patients with adrenal involvement. The urinary steroid profile was abnor- mal only in the patient harboring an adrenocortical carci- noma (Table 3).
The diurnal serum cortisol variation and the dexametha- sone test were normal in all subjects. The responses of plasma ACTH and serum cortisol during the CRF test were statisti- cally indistinguishable between the MEN-1 patients exhibit- ing adrenal enlargement and the control subjects.
Morphological examination of the adrenal tissue from six patients showed different stages of cortical cell proliferation (Fig. 2). A diffuse cortical hyperplasia was found in two patients with enlargements measuring 1-1.5 cm. Bilateral nodular hyperplasia with a unilateral degenerative cyst, 10 cm in diameter, was found in one individual. Two patients had adrenocortical adenomas (2.5 and 4 cm in diameter), of which one demonstrated conspicuous atypia and irregular
A
B
| Metabolite | Values | (umol/24 h) |
|---|---|---|
| Androsterone | 5.7 | (<15.7) |
| Etiocholanolone | 4.5 | (<14.2) |
| 118-Hydroxyandrosterone and | 64.4 | (<7.4) |
| 17a-hydroxypregnanolone | ||
| Pregnanetriol | 38.4 | (<6) |
| 11-Oxopregnanetriol | 12.5 | (0) |
| Androst-5-en-38,16a,173-triol | 10.6 | (<1.0) |
| Tetrahydro-11-deoxycortisol | Traces | (0) |
| Cortisol metabolites | 30 | (<35) |
Reference values are given in parentheses.
cell arrangement. Another specimen, 10 cm in diameter, disclosed adrenocortical carcinoma with necroses, capsular invasion, cellular polymorphism, nuclear atypia, and preva- lent mitoses. This adrenalectomy specimen also exhibited diffuse adrenocortical hyperplasia distal to the carcinoma.
An appropriate amount of adrenocortical tissue for RFLP
A
O
analysis was available in six cases (Fig. 3 and Table 2). The adrenocortical hyperplasia of patient 1 (indexed Lu II: 1 in Refs. 1 and 5) as well as patients 7, 8, and 10 and the atypical adenoma of patient 5 showed retained heterozygosity at all informative loci along chromosome 11 as well as for those on 1.2p, 10q, 13q, 17p, and 22q. In contrast, the adrenocor- tical carcinoma of patient 6 demonstrated allele losses on chromosomes 11p, 11q, 13q, and 17p. For the 11p and 11q markers, there was a complete disappearance of one of the constitutional alleles. Further analysis established that the retained 11th chromosome carried the mutated MEN-1 gene, i.e. chromosome 11 being passed to her MEN-1-affected daughter (patient 24). Losses on chromosomes 13q and 17p were partial, with a 50% reduction of the hybridization signal, and were interpreted as being present in subclones of the tumor cells.
Discussion
Adrenal gland enlargement has been reported in 1.5-2.9% of necropsy cases, and especially nodular cortical hyperplasia has been considered the disease of the ageing adrenal (20, 21). This prevalence contrasts to the present findings of adrenal involvement in more than one third of the individ- uals with MEN-1, which, however, coincided with autopsy investigations of MEN-1 (6, 7). The biochemical evaluation failed to demonstrate adrenocortical hormone excess and disturbances in the hypothalamic-pituitary-adrenal axis, ex- cept in the patient developing feminization due to adreno- cortical cancer. Since altogether 42% of the individuals with adrenal enlargement lacked biochemical and radiological signs of pituitary involvement, it is likely that neither POMC, the N-terminal part of which can stimulate adrenocortical cell growth (22), nor any heretofore characterized pituitary hyperfunction causes the adrenocortical lesion in MEN-1.
Pancreatic endocrine tumors were present in 73% of the patients included in this study, which corresponds to the prevalence upon prospective screening (10) and autopsy investigations of MEN-1 (6). The tumors were overrepre- sented and were, in fact, present in all MEN-1 patients with adrenal enlargement. Insulin and proinsulin were signifi- cantly overrepresented and demonstrable in high concentra- tions within the circulation of 10 of our 12 patients with adrenal enlargement, although none of them exhibited a history of hypoglycemia. Expression of mRNA for insulin- like growth factor-II (IGF-II) has been demonstrated in adre- nocortical carcinomas, and IGF-II has been suggested to mediate autocrine growth stimulation (23). Both insulin and proinsulin can bind to the IGF-I receptor and may also stimulate the IGF-II receptor (24). Growth factors or peptides produced by the pancreatic endocrine tumors could, thus, be causally linked to the adrenocortical proliferation in MEN-1.
As originally proposed by Knudson (2), initiation of tu- morigenesis sometimes involves chromosomal deletions un- masking recessive mutations. Such deletions are detected as losses of constitutional alleles in tumors from both sporadic and inherited forms of the same type of tumors. However, it has been suggested that polyclonal hyperplasia, due possibly to a mitogenic factor (25), might precede the second muta-
13
1
N NP PH EPTHPT N EPT AH
1
D11S97
2
Hae III
8
7
N EPT AH AH
N EPT AA AH
1
CD 20
1
D11S97
2
Msp I
2
Taq |
10
5
6
N AH
N AA
N AC
1
1
2
1
2
2
D11S146
PYGM Taq I
PYGM Msp I
Taq I
tional event in the parathyroids of MEN-1 patients (4). This hypothesis contrasts to other analyses of DNA alterations in MEN-1-associated parathyroid lesions (3, 5). Furthermore, allele losses constitute an early event in MEN-1 pancreatic tumorigenesis, as they have been demonstrated in microad- enomas as well as pancreatic endocrine neoplasms (1). In the present study diffuse and nodular hyperplasia as well as typical and atypical adenomas of the adrenal cortex retained constitutional heterozygosity for markers flanking the MEN- 1 locus. Histological examinations excluded that this was the result of inadequate tissue sampling. Although restricted somatic deletions, undetectable by the applied methods, cannot be excluded, as the MEN-1 gene remains to be cloned, the present findings suggest that adrenocortical hyperplasia and adenoma constitute no primary lesion in MEN-1. This interpretation is strengthened by brothers numbered 1 and 13 as well as patients 7 and 8. These individuals were informative for several markers in close vicinity of the MEN- 1 locus and demonstrated loss of the wild-type allele in a parathyroid adenoma and pancreatic endocrine microaden- omas as well as in neoplasms of the endocrine pancreas, but not in the adrenocortical hyperplasias and adenomas. The possbility that the adrenal lesion may be the result of inter- action between a heretofore undefined MEN-1 allele in con-
junction with another locus seems unlikely considering that the adrenal involvement never has occurred as the presenting lesion in MEN-1 individuals. In fact, all of these patients reported in the present and previous studies (6-9) have displayed concomitant endocrine pancreatic tumors. The lack of allele losses in benign MEN-1 adrenal lesions is also in agreement with RFLP analysis of sporadic adrenocortical adenomas retaining constitutional heterozygosity for markers on chromosome 11 (26).
Acquired homozygosity for 17p, 13q, and 11p markers has been demonstrated in sporadic adrenocortical carcinomas (26) as well as in those of the Wiedemann-Beckwith syn- drome caused by a germ cell mutation on 11 p 15(27). Our one case of MEN-1 adrenocortical carcinoma displayed par- tial allelic losses at 17p and 13q, indicating the presence of subclones in the tumor tissue and a total loss of heterozy- gosity for 11p and q markers. Thus, adrenocortical tumor genesis in this MEN-1 patient might correspond to that of sporadic as well as inherited adrenocortical carcinomas of the Wiedemann-Beckwith syndrome, i.e. inactivation of both 11 p 15 alleles. On the other hand, the chromosomal re- arrangements in the MEN-1 adrenocortical carcinoma could be interpreted as if the malignancy developed due to loss of the wild-type allele at the MEN-1 locus. This seems unlikely
considering that this rearrangement was lacking in earlier stages of cortical cell proliferation and that adrenocortical carcinoma, in contrast to benign adrenal lesions, is exceed- ingly uncommon in MEN-1. Since all reported MEN-1 cases with adrenal affection have disclosed concomitant pancreatic endocrine tumors, it could be speculated that polyclonal adrenocortical cell proliferation develops secondary to the pancreatic tumor.
Acknowledgment
We are grateful to Thore Curstedt, M.D., Ph.D., who analyzed all urinary steroid profiles included in this paper.
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