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Short Communication |
From the Department of Pathology* and the
Division of Cell and Molecular Pathology,
University of Zurich, Zurich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent molecular genetics studies, especially on some rare hereditary syndromes with associated adrenocortical lesions, including the multiple endocrine neoplasia type 1 (MEN1), the Li-Fraumeni (LFS) and the Wiedemann-Beckwith syndromes (WBS), have given some insights into the genetic changes underlying tumorigenesis and development of adrenocortical tumors. The susceptibility gene for MEN1 that was mapped to 11q13 has been cloned.4 LFS harbors germ line mutations in p53 located on 17p135 and the molecular basis of WBS is associated with structural abnormalities and allelic losses of 11p15.6 These genetic aberrations have also been reported in some sporadic adrenocortical tumors.7,8 Furthermore, other genetic alterations have occasionally been observed in sporadic adrenocortical tumors, such as loss of heterozygosity of 11p, 13q, and 17p9 as well as mutations of gip210 and ACTH receptor genes.11 The clinical significance of these genetic changes, however, remains to be clarified. More recently, comparative genomic hybridization (CGH), which allows a detection of all relative DNA sequence copy number alterations of the entire genome of a tumor in one examination,12 was used to identify genomic alterations in adrenocortical tumors.13,14 Because only small series of tumors were investigated in both of the reported studies, it is difficult to recognize characteristic genomic imbalances. In the present study, we used CGH to examine 41 adrenocortical lesions including 12 carcinomas, 23 adenomas, and 6 hyperplasias. In addition, we carried out fluorescence in situ hybridization (FISH) experiments to independently confirm some of the CGH results.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical data of the patients examined are summarized in Table 1
. Adrenocortical lesions of 41 patients
(39 frozen samples and 2 paraffin-embedded tumors) were analyzed. The
samples included 12 adrenocortical carcinomas, 23 adenomas, and 6
hyperplasias. The average diameter of the adrenocortical adenomas and
carcinomas was 4 (range, 2–9) and 8.4 (range, 4.5–13) cm,
respectively. Sections of formaldehyde-fixed, paraffin-embedded samples
from each tumor were stained with hematoxylin-eosin and used for
histological assessment and classification according to previously
published criteria.3,15
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Isolation of genomic DNA from frozen tumor samples was performed using the D-5000 Puregene DNA Isolation Kit (Gentra Systems Inc., Minneapolis, MN). Approximately 2 mm3 of frozen tumor material was homogenized and DNA extraction carried out according to the manufacturer's recommendations. DNA extraction from paraffin-embedded tumors was performed as previously described.16 Direct fluorescence labeling of DNA was performed by nick translation using a commercial kit (BioNick kit, Life Technologies, Gaithersburg, MD).
CGH Analysis
CGH was carried out as previously described.16 The hybridization mixture consisted of 200 to 400 ng of Spectrum Green-labeled tumor DNA, 200 ng of Spectrum Red-labeled normal reference DNA, and 10 µg of unlabeled human Cot-1 DNA dissolved in 10 µl of hybridization buffer (50% formamide, 10% dextran sulfate, 2X SSC, pH 7.0). Hybridization took place over 3 days at 37°C to sex- matched normal metaphase spreads (Vysis, Downers Grove, IL). Digital images were collected from 6 to 7 metaphases using a Photometrics cooled CCD camera (Microimager 1400, Xillix Technologies, Vancouver, British Columbia, Canada). The VYSIS software program was used to calculate average green to red ratio profiles for each chromosome. At least four observations per autosome and two observations per sex chromosome were included in each analysis.
Thresholds used for definition of DNA sequence copy number gains and
losses were based on the results of CGH analyses of normal tissues. A
gain of DNA sequences was assumed at chromosomal regions where the
hybridization resulted in a green to red ratio 
1.20.
Over-representations were considered amplifications when the
fluorescence ratio values exceeded 1.5 in a subregion of a chromosome
arm. A loss of DNA sequences was presumed at chromosomal regions where
the tumor to normal ratio was 
0.80. Since some false positive results
were found in normal tissues at chromosomes 1p, 16p, 19, and 22, gains
at these G-C-rich regions were excluded from all analyses.
FISH Analysis
Eight tumors (5 adenomas and 3 carcinomas) were analyzed for
interphase cytogenetics by using a combination of two centromere probes
specific for chromosomes 1 and 17, with the goal to independently
confirm the CGH results of chromosome 17 abnormalities. Furthermore,
eighteen tumors (8 adenomas and 10 carcinomas) were subjected to FISH
analysis by an 11q13 probe (MEN1 gene), in
combination with chromosome 11-specific centromeric probe, to compare
deletions of this locus and the CGH findings of chromosome 11. Touch
preparations from frozen tumor material were used. Centromere probes
specific for chromosomes 1 and 17 were labeled using spectrum
green-dUTP and spectrum red-dUTP (Vysis), respectively. The centromere
probe specific for chromosome 11 was labeled using biotin (Boehringer
Mannheim, Mannheim, Germany) and the 11q13 probe with spectrum
green-dUTP. Hybridization, posthybridization washes, and detection of
the hybridized signals were carried out as previously
described.17
At least 100 interphase nuclei with strong
hybridization signals were scored for each tumor. Normal frozen adrenal
or connective tissue in the vicinity of tumors served as control and
exhibited two centromere and 11q13 signals in 95% of nuclei. An
aneusomy was assumed if more than 30% of nuclei exhibited an abnormal
number of centromere signals. It was considered a deletion when more
than 30% of nuclei demonstrated only one 11q13 signal.
Statistics
Contingency table analysis and Student's t-test were used to compare the number of aberrations and the frequency of individual changes between tumors of different types. Regression analysis was applied to compare the number of genetic changes and the tumor size.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA copy number changes were observed in all carcinomas and 15 of
23 adenomas (Table 1)
. The average number of alterations per carcinoma
and adenoma was 14 ± 10.6 and 2 ± 3.2
(P = 0.0001), respectively. Of the 6
adrenocortical hyperplasias, one demonstrated a gain of chromosome 17
as only alteration and another a gain at 17q. The number of chromosomal
alterations was strongly associated with malignancy. Comparison of the
number of genetic changes and the tumor size by regression analysis
showed only a weak correlation (r2 =
0.4) which, however, was statistically significant
(P = 0.0001, Figure 1
).
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The chromosomal regions with DNA copy number alterations (losses,
gains, and amplifications) identified in all 41 tumors by CGH are
illustrated in Figure 2 (A and B)
. The
most frequent DNA copy number changes in adrenocortical carcinomas
included losses of 1p21–31 (67%), 9p (58%), 3p (50%), 2q, 3q, 6q,
and 11q14-qter (42% each), 1q23–41, 2p21-pter, and 18q (33% each),
as well as gains of 20q (50%), 5q12–13, 5q22-ter, 9q32-qter,
12q13–14, and 12q24 (42% each), and Xq13–21 (33%). Several
amplifications were found at the regions of 4p16, 5p15, 5q13
(n = 2), 5q32-qter, 8q24, 9q32-qter, and
12q13–14 (n = 2) in 4 carcinomas. The genomic
aberrations prevalently occurring in adrenocortical adenomas were gains
at regions of 17q11.2–21 and 17q24–25 (35% each), 17p (26%), and
9q32-qter (22%). Gains occurring in 2 of 6 adrenocortical hyperplasias
involved chromosome 17 or 17q only.
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. The majority of the detected genomic
aberrations occurred in carcinomas as compared to adenomas or
hyperplasias. This difference reached statistical significance for
losses of 1p, 1q, 2p, 2q, 3p, 3q, 6p, 9p 11q, and 18q and for gains at
5q, 12q, 20q, and Xq. There was no significant difference for 9q+
between carcinomas and adenomas as well as for 17p+ and 17q+ among
carcinomas, adenomas and hyperplasias. Additionally, an association
between chromosomal losses at the distal part of 9p and gains of
9q32-qter in carcinomas was revealed.
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Eight tumors that showed alterations of chromosome 17 as
identified by CGH, which included 4 gains and 1 loss of the whole
chromosome 17, and 2 gains of 17q and 1 gain of 17p, were additionally
analyzed by FISH using a combination of specific centromere probes for
chromosomes 17 and 1. FISH analysis revealed trisomy and tetrasomy of
chromosome 17 in 3 of the 4 tumors with a gain of this chromosome
(Table 1)
. The tumor showing loss of chromosome 17 exhibited a monosomy
for this chromosome. The other 3 tumors, harboring 17q+ or 17p+,
exhibited a diploidy pattern, which was as expected, since alterations
involving only short or long arms of chromosomes cannot be detected by
FISH using centromere probes. Two tumors, which showed a chromosome 17
gain (1 adenoma and 1 hyperplasia), could not be analyzed by FISH
because of lack of tissues.
Among the 18 tumors (8 adenomas and 10 carcinomas) examined with an
11q13 probe (MEN1 gene), FISH revealed deletions of
this locus in 4 carcinomas, in 3 of which CGH also detected losses of
chromosome 11 or 11q (Table 1)
. Both FISH and CGH did not show
detectable changes in the 8 adenomas analyzed.
Collectively, these findings indicate that both CGH and FISH provided
comparable results in 7 of the 8 tumors analyzed for chromosome 17, and
in 16 of 18 tumors examined for the 11q13 locus. Representative
examples of CGH images and corresponding profiles and interphase
cytogenetics are shown in Figure 2 (C and D)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The prevalent genomic aberrations found in adrenocortical adenomas were chromosomal gains at 17q, 17p, and 9q. Interestingly, of the 6 patients with adrenocortical hyperplasia, 2 showed a DNA copy number gain involving 17q or the whole chromosome 17, which was at the same time the sole CGH finding in these lesions. This suggests that genes on chromosome 17 or 9q may be important during early tumorigenic events occurring in the adrenal cortex. It is known that the chromosomal area 17q11.2–21 harbors numerous putative candidate oncogenes such as erbB2, GAS, BRCA1, TOP2A, and NGFR.18 Among them, erbB2 is the most promising candidate gene, because it appears to be overexpressed in a variety of human tumors.19 However, we cannot exclude that other oncogenes possibly located on chromosomes 17q, 17p, or 9q may also participate in the early tumorigenesis of adrenocortical lesions. Recently, Figueiredo et al reported recurrent gains of 9q34 that were found in 8 of 9 adrenocortical tumors examined in their pilot CGH study.14 Since the 9q34 locates at the telomeric region of chromosome 9, at which a false positive gain may appear, color ratio changes at this region should be cautiously interpreted.
The present study revealed extensive genomic alterations in
adrenocortical carcinomas, including several high-level DNA copy number
gains, which have not been reported previously. Most common sites with
genomic deletions were 1p21–31, 1q23–41, 2p, 2q, 3p, 3q, 6q, 9p,
11q14-qter, and 18q, whereas the main regions with DNA copy number
gains and/or amplifications were 5q, 9q32-qter, 12q, 20q, and Xq.
Comparison of carcinomas and adenomas showed statistically significant
differences in genomic changes at these regions, except for gains of
9q, 17p, and 17q (Table 2)
. It can be speculated that such genomic
imbalances may be important for tumor progression and malignant
transformation leading to adrenocortical carcinomas. Studies are under
way to gain a more detailed insight into these chromosomal changes.
Most of the chromosomal losses and gains observed in this study are also common to other human tumor types. Some changes, however, may be specific for adrenocortical tumors, including 1p21–31 deletions, co-occurrence of losses at the distal part of 9p and gains of 9q32-qter, and gains and amplifications (high-level gains) of chromosomes 5 and 12q. The prevalent losses of 1p21–31 found in 62% of adrenocortical carcinomas coincide with this region reported to undergo frequent loss of heterozygosity in other human tumors, such as breast cancer20 and germ cell tumors.21 This may imply that alterations of one or more tumor suppressor genes in this region may play a role in the development of adrenocortical tumors. The observed association between losses of the distal part of 9p and gains of 9q32-qter in adrenocortical carcinomas is noteworthy. These areas may harbor genes associated with each other in some specific pathways (eg, via rearrangements). The p15/p16 tumor suppressor genes are candidates on 9p21, whereas the ABL oncogene on 9q34 may be the overexpressed target at the region of 9q32-qter, although the oncogenic significance of the latter gene has only been established in hematopoietic cells thus far. The region of 12q13–14, where high-level gains were seen in 2 carcinomas, harbors several oncogenes such as MDM2, SAS, GLI, and CDK4, frequently amplified in different sarcoma types.22,23 The biological significance of these genes in the development and progression of adrenocortical tumors, however, remains to be evaluated. Remarkably, the present study identified amplifications of three different loci on chromosome 5 (5p14, 5q13 and 5q32-qter), of which, to our knowledge, the two 5q areas have rarely been reported to be amplified in adrenocortical carcinomas as well as in other tumors. These amplification sites may harbor novel genes with a possible role in the progression of adrenocortical tumors.
Our FISH results showed aneusomies of chromosome 17 alone or together
with aneusomies of chromosome 1 in 4 (2 adenomas and 2 carcinomas) out
of 8 tumors examined, where DNA copy number gains of chromosome 17 were
also detected by CGH. This implicates that aneusomies affecting only
some chromosomes are involved in the development and progression of
adrenocortical tumors, since CGH cannot detect aneusomies that
simultaneously involve all chromosomes. The FISH analysis also revealed
losses of 11q13 in 4 of 10 carcinomas examined, but not in adenomas,
consistent with previous reports.17
These data suggest
that the MEN1 gene may not be a causative gene in
adrenocortical tumorigenesis and that losses of this gene locus may
represent only a late event in the development and progression of
adrenocortical tumors. The good correlation between the results of FISH
and CGH further supports the potential application of the CGH technique
in screening for genomic alterations of tumors. In one adrenocortical
carcinoma (tumor 38), CGH did not detect a deletion at the 11q13
region, whereas FISH revealed a loss of heterozygosity at this locus
(Table 1)
. This discrepancy can be explained by a limited sensitivity
of CGH for alterations smaller than 5 to 10 megabases. This
limitation might also explain why our CGH study revealed only few
deletions involving 11p15 (2 tumors) and 17p (one tumor), in contrast
to previous allelotyping studies.9,24
The contradictory
data between CGH and FISH observed in tumors 33 and 39 (Table 1)
may be
due to intratumoral heterogeneity.
CGH studies have demonstrated that an increased number of chromosomal alterations is generally associated with poor prognosis in different tumors types, such as renal cell carcinomas.25 In agreement with this, we found a strong relationship between the average number of genomic alterations and tumor behavior. The size of adrenocortical tumors is used as a predictor of malignant potential. Our data exhibit that the number of genomic alterations is correlated, albeit not very strikingly, with tumor size, thus supporting the predictive value of tumor size. Surgical resection of an adrenocortical tumor is recommended if it exceeds 3 cm. However, we found genetic changes in 3 tumors smaller than 3 cm. This implicates that the biological behavior of adrenocortical tumors could be predicted earlier, based on the number of identified genetic changes.
In conclusion, frequent gains of 17q and 17p were found in sporadic adrenocortical adenomas and even in hyperplastic lesions of the adrenal cortex, indicating that genes important for early adrenocortical tumorigenesis may exist on this chromosome. The extensive genomic imbalances encountered in adrenocortical carcinomas indicate that the molecular pathogenesis of sporadic adrenocortical tumors is highly complex and that tumor progression and malignant transformation could be attributed to the accumulation of multiple genetic changes. Our data narrow down possible chromosomal loci frequently involved in sporadic adrenocortical lesions and thereby provide a basis for the search for novel genes playing a role in the initiation and progression of sporadic adrenocortical tumors.
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Acknowledgements |
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Footnotes |
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Supported by Swiss National Science Foundation Grant 31–53625.98 and the Hartmann Müller Foundation Grant 717.
Accepted for publication June 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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