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Original Studies |
Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro (M.T., P.Vit., P.A., G.C., A.Pe., R.C., B.M., A.Pi., L.C.), Dipartimento di Oncologia Sezione di Anatomia Patologica (A.G.N., P.Via.), Dipartimento di Clinica Chirurgica (P.M.), Università di Pisa, Pisa, Italy
Address correspondence and requests for reprints to: Massimo Tonacchera, Dipartimento di Endocrinologia, Università degli Studi di Pisa, Via Paradisa 2, 56124, Cisanello, Pisa.
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Abstract |
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 Top Abstract IntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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genes may confer TSh-independent growth advantage to
neoplastic thyroid cells, we searched for somatic mutations of these
genes in a series of hyperfunctioning and nonfunctioning follicular
thyroid adenomas specifically selected for their homogeneous gross
anatomy (single nodule in an otherwise normal thyroid gland). TShR gene
mutations were identified by direct sequencing of exons 9 and 10 of the
TShR gene in genomic DNA obtained from surgical specimens. Codons 201
and 227 of the Gs gene were also analyzed. At histology, all
hyperfunctioning nodules and 13 of 15 nonfunctioning nodules were
diagnosed as follicular adenomas. Two nonfunctioning thyroid nodules,
although showing a prevalent microfollicular pattern of growth, had
histological features indicating malignant transformation (a minimally
invasive follicular carcinoma and a focal papillary carcinoma).
Activating mutations of the TShR gene were found in 12 of 15
hyperfunctioning follicular thyroid adenomas. In one hyperfunctioning
adenoma, which was negative for TShR mutations, a mutation in codon 227
of the Gs gene was identified. At variance with hyperfunctioning
thyroid adenomas, no mutation of the TShR or Gs genes was detected
in nonfunctioning thyroid nodules. In conclusion, our findings clearly
define a different molecular pathogenetic mechanism in hyperfunctioning
and nonfunctioning follicular thyroid adenomas. Activation of the cAMP
cascade, which leads to proliferation but maintains differentiation of
follicular thyroid cells, typically occurs in hyperfunctioning thyroid
adenomas. Oncogenes other than the TShR and Gs genes are probably
involved in nonfunctioning follicular adenomas. |
Introduction |
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TopAbstractIntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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Pituitary thyroid-stimulating hormone (TSh) is the main stimulator of
growth and function of normal follicular thyroid cells (2). TSh after
binding to its membrane receptor (TShR) activates
guanine-nucleotide-binding proteins Gs and Gq and stimulates the
adenylate cyclase (AC)-cAMP pathway and the phospholipase
C-diacylglycerol regulatory cascade, respectively (2, 4).
Gain-of-function (activating) mutations have been detected in up to
80% of toxic adenomas in TShR gene and in up to 25% in the Gs
gene, suggesting that these genetic anomalies may play a role in the
pathogenesis of hyperfunctioning nodules (5). However, discrepancy
still exists on the frequency of TShR mutations in hyperfunctioning
thyroid nodules (5). The lower prevalence observed in Japan, the United
States, and in some Italian series (6) leaves room for other
pathophysiological mechanisms or gene targets (5). In particular,
studies in naturally occurring toxic adenomas suggested that
constitutive activation of the Gs protein-AC pathway may not be
sufficient to generate these benign tumors (7).
Activation of the cAMP pathway, although not directly linked to malignant transformation, may concur in the development of malignant neoplasms, as shown by experimental animal models (8, 9, 10). Constitutive activation of the AC-cAMP pathway in transgenic mice expressing the A2 adenosine receptor in thyroid cells (8) results in the development of goiter and hyperthyroidism. Transgenic mice expressing the E7 protein of the human papillomavirus type 16, which inactivates the retinoblastoma gene product (Rb1) in the thyroid, develop simple goiter (9). Transgenic mice coexpressing the two oncogenes develop malignant thyroid nodules giving lung metastases (10). This animal model demonstrates an additive affect of the two oncogenes on neoplastic thyroid growth.
In the present study, we searched for mutations of the TShR and Gs
genes in a group of hyperfunctioning and nonfunctioning follicular
thyroid adenomas specifically selected to be homogeneous in their gross
anatomy (single nodule in an otherwise normal thyroid gland). The
histological features of most of these nodules were undistinguishable,
but some presented as scintigraphically "hot" hyperfunctioning
nodules, whereas others were scintigraphically "cold"
nonfunctioning nodules.
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Patients, Materials, and Methods |
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TopAbstractIntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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Included in this study were 30 patients submitted to surgery for
a solitary thyroid nodule originating in an otherwise normal gland.
Before surgery, 15 patients were diagnosed as having a hyperfunctioning
(toxic) thyroid adenoma (a hot nodule at 131I
scintiscan with suppression of the extranodular thyroid parenchyma),
and 15 patients were diagnosed as having a nonfunctioning
(scintigraphically cold) nodule. Cytological smears by fine-needle
aspiration showed a pattern of follicular adenoma (11) in all nodules.
Patients with hyperfunctioning thyroid adenomas were thyrotoxic with
high serum FT4 and FT3 concentrations and undetectable serum TSh by a
sensitive method. All patients with nonfunctioning thyroid nodules were
euthyroid. Thyroid autoimmunity was excluded for the absence of
circulating thyroglobulin, thyroperoxidase, and TShR antibodies.
Patients with hyperfunctioning thyroid adenomas were prepared to
surgery with methimazole and iodide. Most patients with a
nonfunctioning thyroid nodule had been treated with L-thyroxine at
TSh-suppressive doses before surgery. Thyroid volume was calculated by
ultrasonography using to the formula of the ellipsoid model (12).
Normal reference values for thyroid volume were obtained by measuring
thyroid size in 130 healthy adults residing in areas with sufficient
iodine intake. Mean ± SD thyroid volume was 11.3
± 3.4 mL in 65 males and 8.6 ± 2.2 mL in 65 females. Age and sex
of patients and findings of thyroid ultrasonography are shown in Tables 1
and 2.
Thyroid lobectomy was performed in all patients.
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FT4 and FT3 were measured by a RIA after chromatographic separation of the free hormone (FT4 RIA, FT3 RIA, Lysophase; Technogenetics S.r.l., Milan, Italy). TSH was assessed by a sensitive assay (AutoDELFIA hTsh Kit; Pharmacia s.p.a., Milan, Italy). Thyroperoxidase and thyroglobulin antibodies were measured by passive agglutination (SERODIA-AMC and SERODIA-ATG; Fujirebio, Tokyo, Japan). TShR antibodies were searched for using a commercial radioreceptor assay (TRAK assay; B.R.A.H.M.S., Berlin, Germany).
Sequence determination
Genomic DNA was extracted from hyperfunctioning and
nonfunctioning thyroid nodules and from the normal extranodular tissue
when available, as described previously (13). Direct sequencing of
exons 9 and 10 of the TShR and of codon 201 and 227 of the Gs gene
were performed using previously described methods (13). At least two
different PCR amplifications from genomic DNA were sequenced on both
strands with sense and antisense primers.
No other coding region of the two genes were sequenced. All the mutations identified were subcloned in a plasmid, and sequences were repeated on individual clones. Contamination problems were ruled out by including PCR control samples with no DNA as template. Extraction of DNA and pre-PCR reactions were performed in different rooms with respect to post-PCR reactions.
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Results |
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TopAbstractIntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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Each thyroid nodule was examined by two pathologists (P. Via and AGM), who were blinded for the clinical diagnosis. The histological diagnosis was always concordant. All thyroid nodules were circumscribed by a complete fibrous capsule. The capsule was thin in all hyperfunctioning adenomas, whereas 5 of 15 nonfunctioning nodules showed a thick fibrous capsule. All nodules showed a prevalent microfollicular architecture, but macrofollicular areas of variable sizes were also observed. All hyperfunctioning nodules and 13 of 15 nonfunctioning nodules were diagnosed as follicular adenomas. Two nonfunctioning thyroid nodules showed features of malignancy. One nodule was a minimally invasive follicular carcinoma, and the other nodule was a micromacrofollicular nodule with a focal area (15 mm) of papillary carcinoma.
Genetic analysis
A TShR gene mutation was found in 12 of 15 hyperfunctioning
thyroid adenomas (Table 3). One
hyperfunctioning adenoma, which was negative for TShR mutations, showed
a mutation in codon 227 of the Gs gene (Table 3). The mutations of
the TShR and Gs genes identified in the present study were
heterozygotic and somatic. All TShR and Gs mutations had been
described already in our (13) and other laboratories (5) and had been
found to activate constitutively the AC-cAMP cascade after transient
expression in COS-7 cells. The I486M mutation of the TShR gene was
previously shown to also activate the phospholipase C-diacylglycerol
cascade (14). At variance with hyperfunctioning thyroid adenomas, no
mutation of the TShR or Gs genes was detected in nonfunctioning
thyroid nodules.
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Discussion |
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TopAbstractIntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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genes in thyroid follicular adenomas lacking the ability to trap
iodine as shown by 131-I scintiscan. None of these nonfunctioning
thyroid nodules, including the two with malignant transformation,
showed a TShR or Gs mutation. At variance, 12 of 15 hyperfunctioning
thyroid adenomas harbored an activating TShR mutation, and in another
an activating Gs mutation was found. These results are in agreement
with previous studies indicating that gain-of-function mutations of the
TShR or Gs genes are found in most hyperfunctioning follicular
thyroid adenomas (5, 13). Other studies reported a much lower frequency
of TShR gene mutations, suggesting that intricate aberrations of the
many complex growth-controlling mechanisms are implicated in the
pathogenesis of these hyperfunctioning nodules (5, 6). In this regard,
data of ADP ribosylation of stimulatory and inhibitory G protein
subunit, together with AC activity in a group of toxic adenomas with
and without mutations in the TShR or Gs genes, suggested that a
constitutive activation of the AC pathway may not be sufficient to
generate these adenomas (7).
Our results also show that TShR or Gs gene mutations are not present
in nonfunctioning follicular adenomas and, conceivably, they are not
involved in the generation of these tumors both as an initial and a
secondary molecular event.
This study was performed because experimental models in transgenic mice
clearly demonstrated the additive effect of two oncogenes, one of which
produced a constitutive activation of the AC-cAMP cascade, on the
development and growth of malignant thyroid nodules (8, 9, 10). Mutations
of the TShR or Gs genes producing thyroid cell growth might also be
permissive for the subsequent development of additional mutations (2, 8, 10), which may abolish some thyroid-differentiated functions or even
lead to malignant transformation (2, 8, 10). Activation of the oncogene
gsp was described in up to 27% of nonfunctioning follicular adenomas,
in up to 12% of follicular thyroid carcinomas, and in up to 13% of
papillary carcinomas (15, 16). The presence of activating TShR
mutations was also reported in three of six differentiated thyroid
carcinomas with high basal AC activity and a poor response to the TSh
(17). Other studies failed to detect mutations of TShR or Gs genes
in benign and malignant nonfunctioning thyroid tumors (15, 16, 18, 19, 20).
However, in most of these studies, screening methods such as
single-stranded conformational polymorphism or probes were used to
search for gene mutations. Single-stranded conformational polymorphism
has a low sensitivity (21), and probes can only identify mutations that
have already been described (21).
It is important to emphasize that most thyroid nodules included in the
present study had similar pathological features, both macroscopically
and microscopically. Hyperfunctioning thyroid adenomas (toxic adenomas)
exceptionally progress toward cancer (22). In agreement with this
notion, the activation of the AC-cAMP cascade, although leading to
proliferation and hyperfunction of follicular thyroid cells, does
maintain the differentiation of these cells (2). Malignant
transformation is observed in nearly 5% of nonfunctioning
microfollicular adenomas and in 25% of nonfunctioning embrional
adenomas (1, 23). In keeping with these data, 13% of nonfunctioning
follicular thyroid adenomas in our series harbored a cancer. These
findings support the concept that nonfunctioning thyroid adenomas do
not grow because of TShR or Gs mutations that activate the AC-cAMP
pathway, but due to a different cascade that also leads to the loss of
the ability to trap iodine. Recently RET-protoncogene-activating
rearrangements have been found in 45% of benign thyroid follicular
adenomas appearing after therapeutic or accidental exposure of the
gland to ionizing radiation (24). The possible involvement of RET
rearrangements in nonfunctioning follicular adenomas originating in
nonirradiated thyroid glands is a matter of future studies. An
alternative explanation for the pathogenesis of nonfunctioning thyroid
adenomas would be the outgrowth of clonal expansion of thyrocytes that
never had iodine uptake due to the lack of an NaI symporter protein
(25). In such a scenario, even a gain-of-function mutation of the TShR
would presumably generate a nonfunctioning adenoma. This sequence of
events is not supported by our findings because no activating mutations
of TShR or Gs were found in our series of nonfunctioning thyroid
adenomas.
In conclusion, our findings suggest a different molecular pathogenetic
mechanism in hyperfunctioning and nonfunctioning follicular thyroid
adenomas. Activation of the AC-cAMP cascade, which leads to
proliferation but maintains differentiation of follicular thyroid
cells, occurs in hyperfunctioning thyroid adenomas. Oncogenes other
than the TShR and Gs are probably involved in nonfunctioning
follicular adenomas.
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Footnotes |
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Received February 4, 1999.
Revised July 13, 1999.
Accepted August 24, 1999.
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References |
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TopAbstractIntroductionPatients, Materials, and MethodsResultsDiscussionReferences |
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protein-adenylate cyclase pathway
may not be sufficient to generate toxic thyroid adenomas. J Clin
Endocrinol Metab. 81:1898–1904.[Abstract]
-subunit of Gs in human
thyroid neoplasms. J Clin Endocrinol Metab. 76:1446–1451.[Abstract]
in human thyroid cancers: low prevalence of mutations
predicts infrequent involvement in malignant transformation. J
Clin Endocrinol Metab. 81:3898–3901.This article has been cited by other articles:
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  S G Watson, A D Radford, A Kipar, P Ibarrola, and L Blackwood Somatic mutations of the thyroid-stimulating hormone receptor gene in feline hyperthyroidism: parallels with human hyperthyroidism J. Endocrinol., September 1, 2005; 186(3): 523 - 537. [Abstract] [Full Text] [PDF]
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 M. Tonacchera, P. Viacava, P. Agretti, G. de Marco, A. Perri, C. di Cosmo, M. de Servi, P. Miccoli, F. Lippi, A. G. Naccarato, et al. Benign Nonfunctioning Thyroid Adenomas Are Characterized by a Defective Targeting to Cell Membrane or a Reduced Expression of the Sodium Iodide Symporter Protein J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 352 - 357. [Abstract] [Full Text] [PDF]
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K. Krohn and R. Paschke Progress in Understanding the Etiology of Thyroid Autonomy J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3336 - 3345. [Full Text] [PDF]
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M. Tonacchera, P. Agretti, L. Chiovato, V. Rosellini, G. Ceccarini, A. Perri, P. Viacava, A. G. Naccarato, P. Miccoli, A. Pinchera, et al. Activating Thyrotropin Receptor Mutations Are Present in Nonadenomatous Hyperfunctioning Nodules of Toxic or Autonomous Multinodular Goiter J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2270 - 2274. [Abstract] [Full Text]
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