MEN1 Gene Analysis in Sporadic Adrenocortical Neoplasms*
CHRISTINA HEPPNER, MARTIN REINCKE, SUNITA K. AGARWAL,
PATRICIA MORA, BRUNO ALLOLIO, A. LEE BURNS, ALLEN M. SPIEGEL, AND STEPHEN J. MARX
Metabolic Diseases Branch (C.H., S.K.A., A.L.B., A.M.S., S.J.M.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; Abteilung Innere Medizin II (M.R., P.M.), Universitätsklinik Freiburg, Freiburg, Germany; and Medizinische Klinik (B.A.), Universitätsklinik Würzburg, Würzburg, Germany
ABSTRACT
Adrenocortical tumors occur as sporadic tumors, as part of the multiple endocrine neoplasia type 1 (MEN1) syndrome or as part of other hereditary disorders. We recently cloned the MEN1 gene, a tumor-suppressor gene located on chromosome 11q13. Subsequently, we showed that sequential somatic inactivation of both alleles of the MEN1 gene contributes to the development of some sporadic endo- crine neoplasms (parathyroid, enteropancreatic neuroendocrine, bronchial carcinoid, and pituitary tumors). We now studied whether somatic inactivation of the MEN1 gene contributes to the pathogen- esis of sporadic adrenocortical neoplasms. Seven adrenocortical car- cinomas, 2 adrenocortical carcinoma cell lines, and 11 aldosterone-
secreting, 8 cortisol-secreting, and 5 nonsecreting benign adreno- cortical tumors were studied. Seven tumors (5 of 5 carcinomas, 2 of 21 nonsecreting benign adenomas; P < 0.001) exhibited loss of het- erozygosity on 11q13. All 33 tumors and cell lines were screened for mutation throughout the MEN1 open-reading frame and adjacent splice junctions. None exhibited a mutation within the MEN1-coding region. We conclude that somatic MEN1 mutation within the MEN1- coding region does not occur commonly in sporadic adrenocortical tumors, although the majority of adrenocortical carcinomas exhibit 11q13 loss of heterozygosity. (J Clin Endocrinol Metab 84: 216-219, 1999)
S PORADIC adrenocortical tumors are relatively common neoplasms, occurring in 2-9% of the population (1). The majority of adrenocortical neoplasms are benign tumors. Ma- lignant adrenal tumors occur rarely and exhibit aggressive local and metastatic tumor progression. Adrenal adenomas and carcinomas are mostly monoclonal (2), suggesting that a genetic alteration in a progenitor cell contributes to tumor- igenesis. However, the molecular pathogenesis of these tu- mors remains unclear (for a recent review, see Ref. 3).
Loss of one allele in adrenocortical tumors has been ob- served for several chromosomal loci, most frequently in- volving 17p (4), 11p (4, 5), 11q (6, 7), and 13q (4). Frequent allele loss at a specific locus suggests a tumor-suppressor gene in this region. 11q13 harbors the MEN1 gene (8), and 11q LOH (loss of heterozygosity) thus suggested that MEN1 gene mutation may contribute to the pathogenesis of sporadic adrenocortical tumors (6, 7).
Multiple endocrine neoplasia type 1 (MEN1), an autoso- mal dominant inherited tumor syndrome, typically presents with combinations of neoplasms of the parathyroid glands, enteropancreatic neuroendocrine cells, and the anterior pi-
tuitary gland (9). Frequent involvement of the adrenal glands in patients with MEN1 is a feature recognized only recently (10, 11). Diffuse adrenocortical hyperplasia, hormone-secret- ing adrenocortical neoplasms, and malignant adrenocortical neoplasms have been reported to occur in patients with MEN1 (10-13). However, 11q13 LOH has been surprisingly rare in adrenal neoplasms of cases with MEN1 (10).
We recently cloned the MEN1 gene, a tumor-suppressor gene located on chromosome 11q13 (14). Subsequently, we showed that somatic inactivation of the MEN1 gene, by se- quential loss of both alleles, contributes to the tumorigenesis of some sporadic endocrine neoplasms. We found somatic mutation of the MEN1 gene in 21% of sporadic parathyroid tumors (15), 33% of gastrinomas (16), 17% of insulinomas (16), 5% of sporadic anterior pituitary neoplasms (17), and 36% of sporadic carcinoid tumors of the lung (18).
We now have evaluated whether somatic inactivation of the MEN1 gene contributes to the pathogenesis of common- variety adrenocortical neoplasms.
Subjects and Methods
Subjects
Tumor tissue was obtained at adrenalectomy. Tumors were frozen in liquid nitrogen immediately after surgical removal. Diagnosis was es- tablished by clinical and histological criteria, as reported (19). Only sporadic adrenocortical neoplasms were included, and none of the pa- tients had a history of hereditary disease. Seven adrenocortical carci- nomas (nos. 1-7) and 11 aldosterone-secreting (nos. 8-18), 8 cortisol- secreting (nos. 19-26), 5 nonsecreting adenomas (nos. 28-32), and 2 adrenocortical carcinoma cell lines (no. 33, described in Ref. 20; and no. 34, SW-13 cell line, ATCC, Manassas, VA) were included in the series. Except for 1 carcinoma (no. 7) and for the 2 cell lines, the corresponding
Address all correspondence and requests for reprints to: Dr. Christina Heppner, National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Building 10/Room 9C101, 10 Center Drive, Bethesda, Maryland 20892-1802. E-mail: christinah@bdg10. niddk.nih.gov.
* This work was supported by a grant from the Fritz Thyssen Stiftung, FRG (to C.H.) and by funding from the Dr. Mildred Scheel Stiftung (to M.R.).
germline DNA was available. Informed consent was obtained from every patient, and the study was approved by the ethical review board of the University of Würzburg. The US Office of Human Study Research exempted this study from need for review by an NIH institutional review board.
Methods
Tumor and germline DNA was extracted using the Qiagen blood and tissue kits (Qiagen, Hildesheim, Germany). A representative number of 13 tumors (2 nonsecreting, 2 aldosterone-secreting, 4 cortisol-secreting, and 6 carcinomas) were assessed for admixture of normal tissue by microscopic analysis. Admixture of normal tissue among tumor tissue ranged from 5-20%.
LOH analysis
Samples were screened for LOH using the polymorphic markers PYGM(CAGA), D11S4946, and D11S449, spanning the MEN1 locus. For tumors showing LOH in this region, the analysis was extended to in- clude the 11q13 markers D11S480, D11S4908, and INT2. D11S480 is located centromeric to PYGM, whereas D11S4908 and INT2 are located telomeric to D11S449 (see also Fig. 1). Furthermore, 11p15 markers (D11S2344, D11S1349) and 11q22/23 markers (D11S1817 and D11S1353) were used for tumors with 11q13 LOH. Primer sequences were as pub- lished in the Genome Data Base (http://www.gdb.org). PCR conditions and LOH scoring have been described previously (21).
Mutation analysis
Exons 2-10 and the adjoining splice junctions of the MEN1 gene were screened for mutation by dideoxy fingerprinting (ddF). Germline DNA of 3 healthy subjects was included as a control. In addition, ddF patterns were compared with the ddF reactions of 142 normal chromosomes obtained previously (14). Amplified DNA fragments exhibiting an ab- errant ddF pattern were sequenced directly and compared with the sequence of the same subject’s germline DNA when available.
Primer sequences used for PCR-amplification, ddF, or direct sequenc- ing can be found as supplementary material at http://www.niddk. nih.gov/new/releases/primers.htm. PCR, ddF, and direct sequencing were performed as previously described (15).
Independent confirmation of the sequence change in tumor 7 was obtained by restriction analysis. The mutant sequence abolishes an RsrII restriction site. To assess whether this sequence change is a frequent
polymorphism, PCR-amplified fragments, derived from 98 normal chro- mosomes, were screened by restriction analysis.
Results
LOH analysis
Paired tumor and leukocyte DNA of 30 tumors was screened for 11q13 LOH. Four tumors (nos. 4, 10, 15, and 18) showed microsatellite instability for all used markers and were thus excluded from the LOH analysis. All remaining tumors except one (no. 6) were informative for at least 1 of the 3 markers used. 11q13 LOH was found in 6 tumors in the initial screening. Of these, 2 were nonsecreting benign ade- nomas (nos. 28 and 31) and 4 (nos. 1, 2, 3, and 5) were adrenocortical carcinomas.
For the tumors exhibiting 11q13 LOH, the analysis was extended using additional 11q13 markers. In addition, tumor no. 6, which was noninformative for the 11q13 markers used for the initial screening, was included in the extended LOH analysis. Tumor no. 6, an adrenocortical carcinoma, also ex- hibited 11q13 LOH when using the marker D11S4908. To assess whether loss of one allele is restricted to 11q13 or extends to distant regions of chromosome 11, 11p15 and 11q22/23 markers were used. All tumors showed LOH pat- terns suggesting the loss of large regions of chromosome 11 (Fig. 1). Three tumors (nos. 3, 28, and 31) showed LOH spanning from 11p15 to 11q22/23, suggesting that 1 copy of the entire chromosome was lost. Two tumors (nos. 5 and 6) showed loss of 11p15 and 11q13 but retention of 11q22. Two tumors (nos. 1 and 2) exhibited loss of 11q13 and 11q22/23 in the presence of retention centromeric of PYGM in one case (no. 1). Six tumors showed LOH on both sides of the MEN1 gene, and the seventh (no. 1) showed LOH including D11S4946, which is within 1200 bp of the 5’ end of the MEN1 open reading frame.
FIG. 1. Chromosome 11 allelotypes in five adrenocortical carcinomas (tumors no. 1, 2, 3, 5, and 6) and two nonsecret- ing benign adrenocortical adenomas (nos. 28 and 31) with 11q13 LOH. Open circles, Loss of 1 allele; closed circles, retention of both alleles; dashes, nonin- formative loci; blanks, not evaluable or not done. The MEN1 gene is located between the markers PYGM and D11S4946.
| Tumor # | 1 | 2 | 3 | 5 | 6 | 28 | 31 | |||
|---|---|---|---|---|---|---|---|---|---|---|
| 11p15 | D11S2344 | - | ☐ | |||||||
| D11S1349 | ☒ | - | ☐ | - | ☐ | ☐ | ☐ | |||
| D11S480 | ☒ | ☐ | - | ☐ | - | - | ||||
| PYGM | ☒ | ☐ | ☐ | - | ☐ | ☐ | ||||
| D11S4946 | ☐ | ☐ | ☐ | - | ☐ | ☐ | ||||
| D11S449 | ☐ | ☐ | ☐ | ☐ | - | ☐ | ☐ | |||
| 11q13 | D11S4908 | - | ☐ | ☐ | ☐ | - | - | |||
| INT 2 | ☐ | ☐ | ☐ | - | ☐ | ☐ | ||||
| D11S1817 | - | - | ☐ | ☒ | ☒ | - | - | |||
| 11q22/23 | D11S1353 | ☐ | ☐ | ☐ | ☐ |
Mutation analysis
None of the 33 tumors or cell lines exhibited a mutation within the coding region of the MEN1 gene. Polymorphisms of the MEN1-coding region (22) were readily detected by ddF. Frequency of the polymorphisms within the tumors was as follows: S145S, 1 case (heterozygous); R171Q, 1 case (het- erozygous); and D418D, 15 cases (all heterozygous).
Tumor 7 exhibited a ddF pattern change in the 3’ untrans- lated region of exon 10 that was not seen in 142 normal chromosomes. Sequencing revealed a G to A substitution at nucleotide position 1959, located 16 nucleotides downstream of the stop codon. Corresponding germline DNA was not available. The sequence change was confirmed by restriction analysis. We did not observe the restriction pattern seen in the tumor among an additional 98 normal chromosomes. It is thus unlikely that the nucleotide substitution represents a frequent polymorphism. Whether this sequence change has functional relevance, e.g. by affecting RNA stability or other mechanism, remains to be established.
Discussion
Seven of 26 informative adrenocortical tumors exhibited 11q13 LOH. Two of the tumors were nonsecreting benign adenomas; 5 were adrenocortical carcinomas. Thus, 11q13 LOH was more frequent in carcinomas (5 of 5 = 100%) than in benign adenomas (2 of 21 = 10%) (P < 0.001 by x-square test).
In aldosterone-secreting adenomas, 11q13 LOH has been reported to occur in 21% of informative cases (23). In contrast, we did not detect LOH of the MEN1 region in any of the aldosterone-secreting adenomas. Our finding is in agree- ment with a recent study using comparative genome hy- bridization (CGH). Kjellman et al. did not observe losses of chromosome 11 in aldosterone secreting-tumors (6), al- though it has to be considered that CGH detects only dele- tions larger than 10 Mb and also fails to detect allelic loss if the remaining allele is duplicated (6). Nevertheless, in aldo- sterone-secreting tumors, allelic loss of 11q13 seems to be a rather rare finding in the Caucasian population. In addition to the lack of 11q13 LOH, we did not detect MEN1 gene mutation in any of the aldosterone-secreting tumors, making it unlikely that MEN1 gene inactivation contributes to the tumorigenesis of sporadic aldosterone-secreting adrenal neoplasms, though this has been suggested previously (7, 23).
In adrenocortical carcinomas, loss of one allele on 11q13 has been reported in a CGH study in three of eight sporadic carcinomas (6). In the present study, 11q13 LOH was iden- tified in all informative adrenocortical carcinomas and in two benign nonsecreting adenomas. Extending the LOH analysis to regions distant to the MEN1 gene for all tumors with 11q13 LOH, we found that LOH is not restricted to 11q13 but seems to involve loss of one allele of the entire chromosome 11 in three cases or loss of large parts of chromosome 11 in four cases.
None of the adrenocortical tumors exhibited a mutation within the coding region of the MEN1 gene. We cannot exclude the possibility that a MEN1 mutation has been missed. However, using ddF as the screening method, we
have been able to identify MEN1 gene mutation in 47 of 50 kindreds with familial MEN1 (22), and we thus consider the sensitivity of ddF to be at least as high as 94% in detecting heterozygous MEN1 mutation. For tumors with 11q13 LOH, the sensitivity of ddF is probably even higher, because tu- mors with 11q13 LOH are hemi- or homozygous for the mutated allele.
One tumor showed a single base change in the 3’ non- coding region of the MEN1 gene. We do not know whether this change is of somatic or germline origin. Furthermore, because of the nonavailability of the patient’s germline DNA, we could not assess the 11q13 LOH status in this tumor. We cannot exclude the possibility that the single base change has functional relevance, e.g. by affecting RNA stability. How- ever, given the finding that none of the other tumors showed a MEN1 gene mutation, a functional relevance of this se- quence change seems unlikely.
Deletion of one entire copy of the MEN1 gene has been suggested in 1 MEN1 kindred (24). The approach used by us is not suitable to exclude the occurrence of a large deletion, including all or part of the MEN1 gene, in the presence of 11q13 LOH. Studying tumor DNA, the admixture of normal DNA could be sufficient to serve as a PCR template and thus obscure the detection of a large deletion of the retained copy of the MEN1 gene in the tumor sample.
For tumors exhibiting 11q13 LOH, alternative mechanisms of MEN1 gene inactivation [such as hypermethylation of the promoter region (25) or mutations within the noncoding region leading to RNA instability] might be relevant. Thus, exclusion of MEN1 gene inactivation in sporadic adrenocor- tical tumors has to await the careful assessment of MEN1- RNA and protein expression by in situ hybridization and immunohistochemistry. Furthermore, other tumor-suppres- sor genes located on or near 11q13 may contribute to the tumorgenesis of nonsecreting adrenocortical adenomas and adrenal carcinomas. However, given that adrenocortical car- cinomas exhibited loss of large regions of chromosome 11 in the present study, and of multiple chromosomes in a pre- vious study (6), LOH on 11q13 might correlate with genetic instability (26), rather than indicate the presence of a tumor- suppressor gene on this locus.
We conclude that somatic MEN1 mutation within the MEN1-coding region does not occur commonly in sporadic adrenocortical tumors although 11q13 LOH is frequent in adrenocortical carcinomas.
Acknowledgments
We thank Mike Emmert-Buck, Larissa Debelenko, and John Gillespie for technical advice.
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