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Original Studies |
Departments of Surgery (M.K., L.-O.F., M.B.), Molecular Medicine (M.K., L.R., B.T., C.L.), Clinical Neuroscience (S.G.), Woman and Child Health (M.K., M.H.), and Pathology (A.H.) Karolinska Hospital, S-171 76 Stockholm, Sweden; and Laboratory for Cancer Genetics (O.-P.K.), Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33521 Tampere, Finland
Address all correspondence and requests for reprints to: Dr. Magnus Kjellman, Department of Surgery, Karolinska Hospital P9:03, S-171 76 Stockholm, Sweden. E-mail: kmak{at}kir.ks.se
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Studies of genetic alterations in adrenocortical tumors have demonstrated the involvement of multiple chromosomal regions, which to some extent overlap with the mapping of the familial forms of these tumors. Thus, both familial and sporadic adrenocortical tumors show loss of heterozygosity/allelic imbalance (LOH/AI) for markers in 11p, 11q, and 17p. Comparative genomic hybridization performed on 22 sporadic tumors showed an increased number of sequence copy number aberrations by tumor size in carcinomas, whereas the benign tumors were genetically more stable (9). Moreover, losses were most often found on chromosomes 2, 11, and 17, whereas gains were detected on chromosomes 4 and 5. The results suggest that putative tumor suppressor genes (TSG) and oncogenes are located on these chromosomes. Other studies have focused on genetic alterations within candidate genes such as TP53, p21, ACTH receptor, CYP-21, and IGF2 (10, 11, 12, 13, 14, 15, 16).
Here we have applied LOH analysis to a panel of adrenocortical adenomas and carcinomas to identify chromosomal regions that may be involved in the development and progression of this tumor form. With the aim to get an insight into the importance of the MEN1 gene and CC locus, the 11q13 and 2p16 regions were analyzed in more detail. Furthermore, mutation analysis of the MEN1 gene was performed.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This study includes matched pairs of constitutional and tumor
samples from 60 patients with primary sporadic adrenocortical tumors
(39 carcinomas and 21 adenomas) operated on between 1973 and 1995. The
tumors were classified as malignant or benign based on
histopathological findings (invasion, necrosis, pleomorphism, and
number of mitosis), tumor size (median, 7.5; range, 0.5–22.0 cm), and
clinical outcome. Endocrine symptoms were present in 38 of the
patients; 17 showed signs of Cushing’s disease (11 carcinomas and 6
adenomas), 4 showed virilization (carcinomas), 2 showed feminization
(carcinomas), and 15 were classified as having Conn’s disease (4
carcinomas and 11 adenomas). Fifty-six of the tumors were screened for
LOH/AI on chromosome 1 to X (the Y chromosome was not analyzed). Four
additional carcinomas from patients 2, 6, 11, and 12 were included
after the initial screening had been performed and were only analyzed
for LOH/AI on chromosomes 2, 4, 11, 14, 17, 18, and 22 (Table 2
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Further analysis of 11q13 and 2p16 was performed in 27 tumors (13
carcinomas and 14 adenomas) for which normal and tumor tissue was still
available. The MEN1 gene was subsequently screened for
mutations, using single strand conformation analysis (SSCA), in the 11
carcinomas and in the 2 adenomas that showed chromosome 11 LOH/AI.
Informed consent was obtained from all living patients, and the study
was approved by the ethical committee of the Karolinska Hospital.
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Immediately after surgical removal, adrenocortical tissue was frozen in liquid nitrogen and stored at -70 C. High mol wt DNA was extracted from 27 frozen tumor tissues by phenol chloroform extraction and ethanol precipitation according to standard methods. To ensure representativity of the tumor material, pieces were cut from all specimens for histopathological examination, and the samples that were included in the study contained 70% or more tumor cells. In 33 cases where no frozen tumor tissue was available, tumor DNA was obtained from sections of routinely processed paraffin-embedded tumor tissue as previously described (17). In short, 5-µm sections were deparaffinized with lemonene, and tumor cell areas were dissected and incubated with proteinase K in lysis buffer [500 µg/mL proteinase K, 0.05 M Tris-HCl (pH 7.9), 0.15 M NaCl, 5 mM ethylenediamine tetraacetate, and 1% SDS] at 45 C overnight, followed by phenol-chloroform extraction and ethanol precipitation. Constitutional DNA was extracted from peripheral leukocytes or paraffin-embedded normal tissues.
LOH/AI studies
The 36 microsatellite markers used for the initial screening and
their chromsomal localizations are detailed in Table 1. Standard PCR reactions were carried
out on microtiter plates in a final volume of 50 µL containing 40 ng
constitutional or tumor DNA, 10 mM Tris base (pH 9), 50
mM KCl, 1.5 mM MgCl2, 0.1%
(vol/vol) Triton X-100, 0.01% (wt/vol) gelatin, 50 pmol of each
primer, 1.25 mM of each deoxy (d)-NTP, and 1 U
Taq polymerase. Samples were amplified using a hot start,
with Taq polymerase added after a 5-min denaturation step at
96 C, followed by 35 two-step cycles with annealing at 55 C for 30
s and denaturation at 94 C for 40 s, and a final elongation step at 72
C for 2 min. To minimize the number of sequencing gels and gel
loadings, a procedure developed by Hazan et al. (18) was
used. In short, PCR amplifications of different markers from the same
genomic DNA sample were pooled and loaded into a single well of a
sequencing gel. After migration, the products were transferred onto a
nylon filter (Hybond N+) and hybridized with one of the
primers (end labeled with 33P) used in the PCR reaction.
The filters were then subjected to autoradiography for 7–10 days.
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The location and order of markers are according to GDB (http://www.gdb.org). LOH/AI was defined as either a total absence or a reduction by 50% or more of the signal intensity of one of the constitutional alleles in the tumor compared to the constitutional DNA that could be detected visually.
SSCA
The nine coding exons of the MEN1 gene were amplified
using 15 different fragments of 200–300 bp each, as previously
described (7). Fifty nanograms of genomic DNA were amplified by PCR in
50 mM KCl; 10 mM Tris-HCl (pH 9.0); 1.5
mM MgCl2 (Promega Corp., Madison,
WI); 0.2 mM dTTP, dCTP, and dGTP; 0.05 mM dATP;
and [
-32P]dATP (Amersham, Arlington
Heights, IL) at 1 mCi/reaction and 2 U Taq DNA polymerase
(Promega Corp.) in a final volume of 15 µL.
Thermocycling conditions were 30 cycles of 1 min at 94 C, 1 min at 62
C, followed by one cycle of 5-min extension at 72 C. The PCR products
were then electrophoresed in 25% MDE (FMC, Rockland, ME)
gels at room temperature for 12 h at 6–8 watts. Gels were dried
before autoradiography.
Direct DNA sequencing
SSCA-shifted bands were excised from the MDE gel and placed in 50 µL ddH2O at 37 C for 1 h. A 5-µL aliquot of this solution was then amplified in a 50-µL reaction with the following components: 50 mM KCl; 10 mM Tris-HCl (pH 9.0); 1.5 mM MgCl2 (Promega Corp.); 0.2 mM dTTP, dCTP, dGTP, and dATP; and 15 U Taq DNA polymerase (Promega Corp.). The purified PCR products were sequenced using cycle sequencing reactions from the ABI PRISM 377 BigDye Primer cycle sequencing kit (Perkin-Elmer, Norwalk, CT) and run on the automated sequencer ABI 377 (PE Applied Biosystems, Foster City, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present study adrenocortical carcinomas and adenomas from
60 patients were screened for LOH/AI using microsatellite markers
(Table 1
). For the acrocentric chromosomes, one locus on the q-arm was
analyzed, and for most of the nonacrocentric chromosomes, markers on
both the p- and q-arms were tested. As several of the DNA samples were
isolated from paraffin-embedded tissues, all samples did not amplify
for all the markers. Overall LOH/AI was detected on most chromosomes,
and in frequencies that varied between 0–29%. Detailed study of the
LOH/AI with regard to the malignant (39 cases) and benign (21 cases)
classification revealed that the vast majority of losses occurred in
the carcinomas. In the carcinomas the LOH/AI frequency per marker
varied from 0–60%, and in the adenomas it varied from 0–14%.
Similarly, the individual variation in the number of detectable
chromosomal alterations fell within a range of 0–18 for the carcinomas
and 0–2 for the adenomas.
Allele losses were detected in 79 of 813 informative instances (Table 1). Seventy-four of these occurred in the carcinomas, and most often
involved chromosome arms 2q (50%), 4p (44%), 11p (60%), 11q (47%),
and 18p (57%; Table 1 and Fig. 1). In
addition, 1p, 1q, 3p, 6q, 8p, 14q, 17p, 17q, 18q, 22q, and Xp showed
LOH/AI in 20–36% of the informative cases. The five losses identified
in the adenomas involved five different chromosome arms,
i.e. 1q, 3q, 4q, 14q, and 18q (Table 1). Among the
carcinomas, the larger tumors showed an increased number of LOH/AI
compared to the smaller tumors. However, the pattern of chromosomes
involved did not differ between tumors of different hormonal
status.
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shows the clinical, morphological,
hormonal, and genetic data for each patient in this subgroup. LOH/AI in chromosome 11q13 and mutation analysis of the MEN1 gene
The PYGM marker located very close to the
MEN1 gene in 11q13 showed LOH/AI in all 8 informative
carcinomas (100%) and in 2 of the 13 informative adenomas (13%).
However, losses were less frequent in the distal parts of chromosome
11. One of the carcinomas and the 2 adenomas with loss of
PYGM (cases 4, 21, and 27) showed retained heterozygosity
with D11S909 in 11pter (Fig. 2).
Similarly, no losses were detected with D11S968 at 11qter in 3
carcinomas and 1 adenoma displaying LOH for PYGM in 11q13
(Fig. 2). SSCA analysis of the 9 coding exons of the MEN1
gene revealed no mutations in the 11 tumors analyzed. In 6 cases, a
common polymorphism (GAC to GAT) involving codon 418 in exon 9 was
detected. This polymorphism (Asp418Asp) was detected in
both constitutional and tumor DNA and has been reported to occur in up
to 44% of the population (6).
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LOH/AI on chromosome 2 was only detected in the carcinomas, with
increasing frequency for markers within 2p16 (median, 72%; range,
54–88%) compared to D2S319 in 2pter (8%) and D2S125 in 2qter (44%).
Twelve of the 13 carcinomas showed LOH/AI for at least 1 of the 7
microsatellite markers from the 2p16 region typed (Fig. 3). In 7 of
these, all informative markers showed loss (tumors 1, 2, 3, 4, 6, 10,
and 11). However, in the remaining 5 tumors, LOH/AI only occurred at
some loci, whereas other loci showed retained heterozygosity (tumors 7,
8, 9, 12, and 13). Based on the results from tumors 9, 12, and 13, a
single minimal region of overlapping deletions could be mapped between
D2S391 and D2S288 (Fig. 3). The interval
between D2S391 and D2S288 defines a 1-centimorgan (cM) region
that is separate from the location of the 6-cM CC region, as well as
from the human MSH2, human MSH6, and
POMC genes (Fig. 3).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Adrenocortical tumors in MEN1 patients are always seen in conjunction with tumors in the endocrine pancreas (3, 4, 19). LOH at 11q13 in adrenocortical lesions from MEN1 patients has to date only been reported in a rare case of an aldosterone-producing adenoma (20) and in one adrenocortical carcinoma (3). In our study of sporadic adrenocortical tumors, two benign tumors, both aldosteronomas, showed LOH at 11q13. This has previously been described in patients with familial hyperaldosteronism type II, in one sporadic aldosteronoma as well as in the MEN1 case mentioned above (20, 21). The involvement of the MEN1 gene in the pathogenesis of sporadic adrenocortical tumors cannot be completely ruled out even though no mutations were found in this study. We employed the commonly used method of SSCA to screen for mutations. For the MEN1 gene this method has a detection rate of approximately 60–80% in family studies and somewhat higher in studies of tumors with loss of one 11q13 allele. Clearly, one allele of the MEN1 gene is frequently lost, and other mechanisms than mutations within the coding part of the gene, such as splice mutations or hypermethylation of the promoter region, may be of importance. Furthermore, another gene in this chromosomal region may be involved.
It has not yet been established whether there is a progression of adrenocortical tumors from the benign to the malignant phenotype, or if malignant and benign tumors have different origins. It could be speculated, however, that adrenocortical cell proliferation may be initiated by a mitogen stimulus, which, in turn, facilitates for key genetic alterations to take place. Mutational events in genes that are important to maintain the stability of the genome may then lead to the multiple numerical and structural chromosomal aberrations seen in tumors larger than 4–5 cm, as demonstrated in this study and previously (9). Until mutations in genes have been identified that initiate the development and are involved in the malignant transformation of this tumor form, numerical changes in chromosomes and structural genetic changes at specific chromosome regions that occur in the carcinomas may work as a preoperative tumor marker for malignancy. Fluorescence in situ hybridization and/or PCR methods can be used on fine needle aspiration material obtained preoperatively.
As LOH/AI in 2p16 was found frequently in the carcinomas but not in the benign tumors, it indicates that the region is important in the malignant progression of adrenocortical tumors. Furthermore, the defined 1-cM interval between D2S391 and D2S288 separates from the location of the 6-cM CC region as well as from the human MSH2, huamn MSH6, and POMC genes. This would exclude the CC gene as the target for the 2p16 deletions found in this study. However, the CC locus can still be involved in adrenocortical tumors because of earlier published data on Carney tumors indicating that the CC gene may have a dominant, rather than a recessive, tumorigenic function (22). The DNA repair genes located on 2p16 are other candidates, but are not likely to be involved because they are located outside the 1-cM interval, and no frequent finding of microsatellite instability was detected in this study.
In conclusion, gross genetic alterations seen in adrenocortical tumors are complex and an increased genetic instability is a feature of the malignant tumors. Further studies on the mechanisms that cause chromosomal instability in this tumor form may lead us to genes involved in the malignant transition. Isolation and characterization of the putative TSG in the 2p16 region may provide insights into such malignification of adrenocortical tumors. Furthermore, no mutations in the MEN1 gene was found in this study.
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Acknowledgments |
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Footnotes |
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Received August 14, 1998.
Revised November 9, 1998.
Accepted November 12, 1998.
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References |
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