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From the Department of Molecular Medicine,*
Endocrine
Genetics Unit, and the Departments of
Surgery
and
Pathology,§
Karolinska Hospital, Stockholm,
Sweden; the Department of Endocrine Surgery,
Tokyo Women’s Medical University, Tokyo, Japan; and The Laboratory of
Cancer Genetics,¶
Institute of Medical Technology,
University of Tampere and Tampere University Hospital,
Tampere, Finland
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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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Most parathyroid carcinomas occur sporadically, but familial forms of the disease are also recognized. The hyperparathyroidism-jaw-tumor syndrome and a subset of familial isolated hyperparathyroidism are both linked to chromosomal region 1q21-q32, and are associated with an increased risk of parathyroid carcinoma.3-8 Loss of heterozygosity involving the wild-type allele for markers in 1q21-q32 has been detected in tumors from 1q-linked families suggesting the inactivation of a tumor suppressor gene in this region.5,7,8
Recently, loss of heterozygosity and comparative genomic hybridization (CGH) studies have identified chromosomal regions putatively involved in tumorigenesis of sporadic parathyroid adenoma. Loss of the MEN1 region at 11q13 is the most common abnormality found9-12 and in half of these cases a somatic MEN1 mutation can be demonstrated.13-15 Losses of chromosomes 1p, 6q, 9p, 11p, 13q, and 15q as well as gains of chromosomes 7, 16p, and 19p have frequently been demonstrated.11,12,16 Because parathyroid carcinoma is so infrequently encountered (<1% of all hyperparathyroidism) and often the diagnosis is difficult to establish only a few genetic studies have been reported. Therefore the genetic mechanism underlying the development of malignant parathyroid tumors remains primarily elusive and no specific genetic alterations are known. Somatic loss of the retinoblastoma gene, Rb1, as well as loss of pRb immunostaining was reported in some carcinomas, but was also demonstrated in parathyroid adenomas.17-19 Furthermore, alterations of the TP53 gene have been found in a few carcinomas.20
CGH studies have proven to be powerful in identifying regions harboring oncogenes and tumor suppressor genes of importance for tumor development. Agarwal et al21 have previously reported differences in numerical chromosomal imbalances detected in a set of 10 parathyroid adenomas and 10 carcinomas. With the hope of gaining a better understanding of the molecular tumorigenesis of parathyroid carcinomas we have furthered the CGH studies by analyzing a total of 29 parathyroid carcinomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-nine parathyroid carcinomas from 29 patients (28 sporadic
and one familial) were fully characterized by CGH (Table 1)
. The tumors were divided into two
groups, unequivocal carcinoma and equivocal cases, because of their
histopathological features and clinical course. Cases 1 to 14 and 20 to
29 were all evaluated at the Karolinska Hospital by one of the authors
(LG) and classified according to the criteria previously
reported.22
However, because these cases were collected at
different locations from multiple centers worldwide and then returned
to the respective clinics, no histopathological re-evaluation was
performed in connection to the present study and the detailed
information for each case is not presently available. Cases 15 to 19
came from the same endocrine surgical unit at Tokyo Women’s Medical
University and all had a clinical course with distant and/or lymphatic
metastasis making the carcinoma diagnosis certain.23
In
addition to microscopically infiltrative growth pattern and/or evidence
of recurrence, the 19 cases of unequivocal carcinomas (cases 1 to 19;
Table 1
) also often showed other pathological features occurring in
parathyroid carcinoma such as marked fibrosis often with hyaline bands
splitting the parenchyma and focally spread necrosis as well as
cytological features such as marked cellular atypia, macronucleoli, and
large nuclei. Ten patients had tumors that showed histopathological
features of carcinoma, as described above, but lacked microscopically
infiltrative growth pattern as well as evidence of recurrence, ie,
equivocal cases (cases 20 to 29; Table 1
). These cases were therefore
classified as equivocal in line with the previously published
classification criteria.22
By histopathological
investigation all tumor samples were shown to contain a minimum of 70%
tumor cells. The study was approved by the local ethics committee.
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DNA was extracted from fresh-frozen tumor tissue in five cases and from formalin-fixed paraffin-embedded tumors in the remaining 24 cases. DNA extraction from 20 to 30 paraffin sections (thickness, 3 to 4 µm) was performed using the QIAamp Tissue Kit (Qiagen, GmbH, Germany). The yield of DNA was maximized with a prolonged proteinase-K digestion according to a previously published protocol.24
CGH was performed as previously described.25 Briefly, tumor DNA samples were labeled with fluorescein isothiocyanate-dUTP (DuPont, Boston MA) by nick translation, and normal reference DNA was labeled with Texas Red (Vysis Inc., Downers Grove, IL). In each case the tumor and reference DNA samples were always sex-matched. Tumor and reference DNA were mixed with unlabeled Cot-1 DNA (Gibco BRL), denatured, and applied onto slides with denatured metaphases of normal lymphocytes (Vysis Inc.). After hybridization at 37°C for 48 hours, the slides were washed in 0.4x standard saline citrate (SSC)/0.3% Nonidet P-40 at 74°C for 2 minutes and in 2x SSC/0.1% NP-40 at room temperature for 1 minute. After air drying, the slides were counterstained with 4,6-diamino-2-phenylindole (Vysis Inc.). Two control hybridizations were also performed including normal female DNA against normal male DNA and DNA from a previously characterized breast cancer cell line (MPE 600; Vysis Inc.) against normal female.
Digital Image Analysis
Six to 10 three-color digital images (4,6-diamino-2-phenylindole, fluorescein isothiocyanate, and Texas Red fluorescence) were collected from each hybridization using a Zeiss Axioplan 2 (Carl Zeiss Jena GmbH, Jena, Germany) epifluorescence microscope and Sensys (Photometrics) charge-coupled-device camera interfaced to a IPLab Spectrum 10 workstation (Signal Analytics Corp., Vienna, VA). Relative DNA sequence copy number changes were detected by analyzing the fluorescence intensities of tumor and normal DNAs along the length of all chromosomes in each metaphase spread. The absolute fluorescence intensities were normalized so that the average green-to-red ratio of all chromosomes in each metaphase was 1.0. The final results were plotted as a series of green-to-red ratio profiles and corresponding standard deviations (SDs) for each human chromosome from p-telomere to q-telomere. At least 12 ratio profiles were averaged for each chromosome to reduce noise. Green-to-red ratios >1.20 were considered as gains of genetic material, and ratios <0.80 as losses. Heterochromatic regions, the short arm of the acrocentric chromosomes and chromosome Y were not included in the evaluation.
Comparison of CGH Alterations in Carcinomas versus Adenomas
Individual chromosome copy number changes of parathyroid adenomas12 and parathyroid carcinomas were compared using the Fisher’s exact test in the StatView 4.02 software. Probabilities of <0.05 were accepted as significant. The two groups of tumors were previously classified histopathologically as adenomas and carcinomas by one of the authors (LG), and analyzed by CGH side by side by two of the authors (SK and FF) using identical laboratory procedures and cut-off levels for identification of gains and losses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA samples from all five fresh frozen tumors and from 24
of the 30 paraffin embedded tumors were successfully analyzed by CGH
(success rate 100% and 80%, respectively). The chromosomal regions
with increased and decreased DNA sequence copy numbers are illustrated
in Figure 1
and detailed for each tumor
in Table 2
. The 29
cases of parathyroid carcinomas were subdivided into cases with
unequivocal and equivocal diagnosis of carcinoma, however there were no
differences in the numbers of aberrations detected or the
subchromosomal regions involved in the two groups of tumors. The number
of detected alterations fell within a range of 0 to 15 with a mean
value of 4.9 aberrations per sample. Chromosomal imbalances were
identified in 25 of the 29 tumors analyzed (86%), with gains and
losses detected in comparable frequencies (67 out of 141 and 74 out
of 141, respectively).
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. The pattern of
CGH alterations in the individual tumors varied depending on the total
number of detected alterations (Table 2)
. Gains of Xq and 1q were both
detected as single aberrations in two cases. However, the four most
frequent aberrations, ie, loss of 13q, gain of 19p, loss of 9p, and
loss of 1p were seen in tumors with at least two or more aberrations.
Furthermore loss of 6q and 4q, as well as gain of 9q and 16p were only
detected in tumors having a total of four or five alterations.
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. Gain of Xc-q13
was detected in eight of the 13 tumors from male patients, but in none
of the 15 female cases (P = 0.0004; Table 3
). On
the other hand, gain on 1q31-q32 was only seen in tumors from female
patients (P = 0.04 Table 3
).
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The distribution of losses and gains detected in the 29 carcinomas
were compared with our previously published results for 26 sporadic
parathyroid adenomas12
(Figure 2)
. This comparison
revealed highly significant differences between the two types of
tumors. Loss of 1p, 4q, and 13q as well as gains of 1q, 9q, 16p, 19p,
and Xq were significantly more common in the carcinomas than in the
adenomas (Figure 2)
. In contrast loss of 11 was much more common in the
adenomas as compared to the carcinomas (Figure 2)
. The different
genetic profiles of adenomas and carcinomas were even more evident when
the minimal regions involved were considered. For example, losses
involving 1p are characteristic of carcinomas but are also frequent in
adenomas. However the minimal regions on 1p involved are clearly
different, with involvement of 1p21-p22 in the carcinomas (Figure 2)
and of 1p34-pter in the adenomas.12
Furthermore, the
MEN1 gene region in 11q13 is a major target for losses of
chromosome 11 in adenomas, whereas the MEN1 gene locus was
not involved in any of the two 11q losses detected in carcinomas
(Figure 1)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This finding
suggests that the two entities may have diverse pathogenesis. CGH
analyses of adenomas versus carcinomas has been previously
reported for other tumor types (eg, colon, ovary, and adrenocortical),
and in all these cases the adenomas presented with few CGH imbalances
whereas the carcinomas, in addition to the alterations present in the
adenomas, also demonstrated multiple numerical
alterations.26-28
The distribution of genetic alterations on the most frequently involved
chromosome arms support the idea of a progression of genetic events in
the development of parathyroid carcinoma (Table 1)
. Because gains of Xq
and 1q were both detected as single alterations it is likely that
alterations of genes in these regions are relatively early events in
tumorigenesis. Similarly 13q loss, 9p loss, and 19p gain can be
regarded as intermediate events, whereas 6q loss, 4q loss, 9q gain, and
16p gain would represent events occurring late in the tumor
development.
The most frequently detected abnormality in this study was gain of 19p
(45%). Gain of the same chromosome arm has also been described in
benign parathyroid tumors, although at significantly lower frequencies
(Figure 2)
. The 19p region harbors a locus for familial hypocalciuric
hypercalcemia29
making this a possible candidate gene for
development of a range of parathyroid tumors including sporadic
adenomas and carcinomas, irradiation-associated adenomas, and adenomas
from familial cases.
One of the most frequently detected losses involved the 13q14-q31
region. This region harbors the RB1 gene at 13q14.3, which
is known to be altered in several different human malignancies. Whether
RB1 is involved in parathyroid malignant transformation is
still unknown. On the one hand, losses including the RB1
locus were significantly more common in the carcinomas than in the
adenomas (Figure 2)
. However, on the other hand the minimal region of
loss includes large parts of chromosome 13, leaving several other genes
as possible candidates eg, the BRCA2 tumor suppressor gene.
Loss of 1p was also seen in almost half of the carcinomas (41%). The common minimal region of loss was assigned to 1p21-p22, which is clearly different from the 1p34-pter region preferentially involved in parathyroid adenomas.11,12,30 The tumor suppressor gene or genes in this region contributing to the development of these tumors have not been identified yet. The distal portion of 1p is frequently deleted in neuroblastoma and other tumors and overlaps with the location of the p73 tumor suppressor gene, which consequently can be excluded as involved in parathyroid carcinomas.12,31 However, the 1p21-p22 minimal region of loss defined in this study overlaps with the 1cen-p31 region of loss that we have previously seen in pheochromocytomas and abdominal paragangliomas.32 These findings would support the existence of a tumor suppressor gene within 1p21-p22 that is involved in tumorigenesis of endocrine tumors.
Gains of 1q31-q32 and of Xcen-q13 were significantly more common in carcinomas than in adenomas. Sex-dependent occurrences were also apparent with exclusive involvement of 1q in female cases and of Xq in male cases. Interestingly, the 1q31-q32 region of gain overlaps with the HRPT2 locus for the hyperparathyroidism-jaw-tumor syndrome and some forms of familial isolated hyperparathyroidism, which are characterized by a predisposition to parathyroid adenoma/carcinoma in combination with ossifying jaw fibromas and renal hamartomas. Furthermore, a reduced penetrance for the parathyroid component is characteristic of the disease in female members of families linked to the HRPT2 locus.5,7 This circumstance has been suggested to indicate the involvement of an additional locus on the X chromosome in the tumor development. Tumors from HRPT2-linked families frequently demonstrate loss of heterozygosity of the wild-type alleles for polymorphic markers in the region that could indicate that the disease gene is a tumor suppressor gene. However, the finding of gain in the 1q31-q32 region by CGH in both familial and sporadic cases, suggests a more complex mechanism of tumor development. Considering the above circumstances it is tempting to speculate about a model where the tumor development is promoted in a dose-dependent manner both by the loss of the wild-type gene as well as by the amplification of the mutated allele.
Whether sporadic parathyroid carcinomas develop from benign adenomas,
or if they occur as a separate disease has not been confirmed. Although
adenomas are common, carcinomas are extremely unusual, which would in
itself speak against a progression in the majority of cases. The
diverse genetic profiles of the two entities are not supportive of a
progression pathway. In agreement with previous CGH studies of
parathyroid tumors,11,21
loss of the 11q13 region is the
most common abnormality in adenomas but was not demonstrated in a
single carcinoma (Figure 1)
. This strongly indicates that adenomas
developing along the MEN1 pathway do not have high potential
for malignant transformation. This observation is also in agreement
with the notion that families with hyperparathyroidism related to a
constitutional MEN1 mutation hardly ever develop carcinomas.
However, the genetic basis of sporadic adenomas without involvement of
the MEN1 gene locus is still unknown. Therefore the
available genetic information cannot be used to establish or exclude a
genetic relationship between those adenomas and the group of sporadic
parathyroid carcinomas. On the other end of the spectra, affected
members of 1q-linked families have a high risk of parathyroid
carcinoma. These cases are usually diagnosed as benign tumors in the
initial phase, whereas parathyroid malignancy is only recognized during
follow-up, which could possibly be related to a malignant progression.
Whether some sporadic parathyroid tumors also develop along the
HRPT2 gene pathway and are therefore characterized by a
malignant potential remains a central question which will finally be
answered after identification of the gene involved.
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Acknowledgements |
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m, M.D.; C. Organ, M.D.; C. Proye, M.D.; J. Salomon,
M.D.; E. Sarfati, M.D.; Ö. Selking, M.D.; D. M. Shapiro,
M.D.; and J. Visset, M.D. |
Footnotes |
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Supported by the Swedish Cancer Society, the Torsten and Ragnar Söderberg Foundations, the Gustav V. Jubilee Fund, the Swedish Society for Medical Research, the Swedish Medical Research Council, the Cancer Society of Stockholm, and the Fredrik and Ingrid Thuring Foundation. S. K. is supported by Karolinska Institutet and F. F. by the Wenner-Gren Foundation.
Accepted for publication April 27, 2000.
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