The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 10 3378-3382
Copyright © 1997 by The Endocrine Society
A Case of Metastatic Medullary Thyroid Carcinoma: Early Identification Before Surgery of an RET Proto-Oncogene Somatic Mutation in Fine-Needle Aspirate Specimens1
Diego Russo,
Franco Arturi,
Eusebio Chiefari,
Domenico Meringolo,
Davide Bianchi,
Bartolomeo Bellanova and
Sebastiano Filetti
Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e
Clinica (F.A., S.F., E.C.), Cattedra di Farmacologia, Facoltà di
Farmacia (D.R.), Università di Reggio Calabria, Via T.
Campanella, 88100 Catanzaro, Italy; Unità Operativa di
Endocrinologia (D.M., D.B.), Ospedale Bentivoglio; and Servizio di
Medicina Nucleare Policlinico S. Orsola-Malpighi (B.B.), Bologna,
Italy
Address all correspondence and requests for reprints to: Sebastiano Filetti, Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica, Via T. Campanella, 88100 Catanzaro, Italy. E-mail: filetti{at}mbox.vol.it
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Abstract
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Medullary thyroid carcinoma (MTC) management requires determination of
the sporadic or familial nature of the disease. RET
proto-oncogene mutation analysis in the tumor tissue obtained at
surgery and in the peripheral blood identifies somatic
vs. germinal mutations. We now report a case of MTC in
which a RET somatic mutation at codon 918 was detected
in fine-needle aspiration specimens obtained from both the thyroid
nodule and two enlarged neck lymph nodes but not in peripheral blood.
Therefore, a diagnosis of sporadic MTC was made before surgery. Thus,
this approach, by excluding preoperatively multiple endocrine neoplasia
disease, permitted immediate thyroidectomy without search for
pheochromocytoma. PCR-based genetic analysis in fine-needle aspiration
biopsy specimens, therefore, preoperatively identifies genetic
abnormalities at an early and easily manageable stage and may well
contribute to the management strategy of MTC.
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Introduction
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MEDULLARY thyroid carcinoma (MTC) may arise
as sporadic or familial disease, eventually occurring in association
with other endocrine tumors in multiple endocrine neoplasia (MEN)
syndrome 2A and 2B. In these inherited disorders, germline
abnormalities (mostly point mutations) in the RET
proto-oncogene have been identified in the majority of patients
affected (1). Somatic RET mutations occur as well in
sporadic MTC with a frequency variable between 25 and 70% in different
studies (1, 2). Identification and assessment of the nature of the
RET mutation (somatic or germinal) in all of the MTC is,
therefore, a critical tool for discriminating sporadic cases from
unrecognized familial disease and could replace biochemical screening
based on the calcitonin (CT) serum level at basal concentrations and
after pentagastrin stimulation (3, 4).
This investigation was undertaken to assess the possibility of
preoperative detection of genetic abnormalities in fine-needle
aspiration specimens from a patient with MTC. A major problem in the
management of these patients is the necessity of excluding the presence
of a pheochromocytoma. In fact, the asymptomatic normotensive patient
with MEN2 syndrome is at risk of dangerous and fatal paroxysms during
surgery if an unsuspected pheochromocytoma is present (5). Thus, we
report a case of a patient with a MTC presenting with enlarged neck
lymph nodes. In this patient, we performed the RET
proto-oncogene mutation analysis in the complementary DNA (cDNA) from
the fine-needle aspiration biopsy (FNAB) of the thyroid nodule and the
lymph nodes, as well in the peripheral blood DNA.
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Case Report and Methods
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A 47-yr-old male was referred to us in April 1996 because
of a right neck mass. Physical examination revealed several palpable
enlarged lymph nodes in the right cervical and submandibular region and
a 2-cm diameter nodule in the right thyroid lobe. Ultrasound evaluation
confirmed the presence of a solid right thyroid mass (cold at
99Tc scintigraphy) and of multiple enlarged lymph nodes in
the right cervical region. Serum-free T3 and TSH were
normal. Subsequently, FNAB (under US guidance) on both the thyroid
nodule and the enlarged lymph nodes was performed. An aspirate aliquot
was smeared for cytological examination, and another was utilized for
messenger RNA extraction.
At cytology, diagnosis of suspected medullary carcinoma and lymph node
metastases was made. High serum CT, 2800 ng/L (normal = 0–10
ng/L) and carcinoembryonic antigen, 70 ng/mL (normal = 0–5 ng/mL)
levels confirmed the diagnosis of MTC. An accurate family history,
extended to second-degree relatives, was negative for endocrine or
neoplastic diseases. The presence of other neoplasia associated with
the MTC in the MEN syndromes was excluded. In fact, there was no
evidence of pheochromocytoma (by assessment of plasma and urinary
catecholamines, by computerized tomographic scan of the abdomen, and
scintigraphy by 131I-labeled metaiodobenzylguanidine) or
hyperparathyroidism (serum calcium and PTH levels in the normal range).
The patient underwent total thyroidectomy and central neck lymph node
dissection. Histological examination (kindly provided by Dr. C. Gallo)
confirmed the diagnosis of MTC and MTC metastatic right cervical and
submandibular lymph nodes; neither multifocal diseases or C cell
hyperplasia were detected; immunohistochemistry showed neoplastic cell
positivity for CT and negativity for thyroglobulin (Fig. 1
). Presence of amyloid was evident after
staining with Congo red (Fig. 2).
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Figure 1. Immunohistochemical staining of tumoral
tissue. A, Immunostaining with anti-CT antibodies (immunoperoxidase)
shows high positivity of tumoral cells. B, Immunostaining with
antithyroglobulin antibodies (immunoperoxidase) shows high positivity
of follicular peritumoral cells whereas tumoral cells are negative.
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Genetic analysis
RT-PCR amplification were performed as previously
described (6). PCR conditions were the following: cycle 1 at 42 C for
15 min and 95 C for 15 sec; 40 cycles at 95 C (1 min), 61 C (1 min) and
72 C (1 min); the last cycle at 72 C for 7 min (42 total cycles).
The following 3' and 5' oligonucleotides (from Genosys,
Cambridge, UK) were used according to the published sequence of
RET cDNA (7): A) 10F (5'-ATTGTTGGGGGACACGAG-3') sense
primer; 11R (5'-GCAGTGGATGCAGAAGGC-3') antisense primer. B) 12F
(5'-GCCGTGAAGATGCTGAAAGAG-3') sense primer; 14R
(5'-AAATGAGATGAGGTCGCCCAT-3') antisense primer. C) 15F
(5'-CTCGTTCATCGGGACTTGGCA-3') sense primer; 17R
(5'-CCATACATCACTTTGCGTGGT-3') antisense primer.
The amplification products were purified and examined by
restriction enzyme analysis and/or direct sequencing. Genomic DNA was
extracted by 2 mL peripheral blood of the same patient using a blood
DNA extraction kit (Qiagen, M Medical Genenco, Firenze, Italy). A
search for RET gene abnormalities in exons 10, 11, 13, 14,
and 16 was performed as described (8).
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Results
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The purified 198-bp DNA fragment, obtained by PCR amplification of
a region containing codon 918 of exon 16 of the RET gene,
which is the more frequently altered site in sporadic MTC (1, 2), was
digested with Fok1. Figure 3
shows the presence of a stronger undigested band together with weak
bands (of 156 and 42 bp), a pattern consistent with the loss of the
Fok1 site in one allele of the gene. The same pattern was
detected in the amplified cDNA from FNAB of both the tumor and the
lymph nodes (Fig. 3B). The nucleotide sequence of the same fragment
confirmed the presence of an ATG to ACG heterozygotic mutation at codon
918 (Fig. 4). Analysis of genomic DNA
extracted from peripheral blood of the same patient showed maintenance
of the Fok1 site, revealing the somatic nature of the
mutation (Fig. 3C).
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Figure 3. A, Restriction analysis of cDNA derived PCR
products of RET exon 16 after FokI
digestion (3 h at 37 C). Samples were run on 2%
agarose-Tris-borate-etilendiamminetetraacetate gel containing ethidium
bromide. Lane 1, Undigested amplicon from medullary carcinoma TT cell
line; lane 2, digested amplicon from TT cells; lane 3, digested
amplicon from patient’s tumor; point mutation at codon 918 determines
loss of FokI site leading to a pathological pattern. B,
Restriction analysis of cDNA- derived PCR products of
RET exon 16 after FokI digestion. Lane 1,
DNA size marker; lane 2, undigested amplicon from medullary carcinoma
TT cell line; lane 3, digested amplicon from TT cells; lanes 4 and 5,
Digested amplicons from cervical lymph node metastases of patient;
point mutation at codon 918 determines loss of FokI site
leading to a pathological pattern. C, Restriction analysis of blood
DNA-derived PCR products of RET exon 16 after
FokI digestion. Lane 1, DNA size marker; lane 2,
undigested amplicon from unaffected individual; lane 3, digested
amplicon from unaffected individual; lane 4, digested amplicon from
patient; presence of a normal restriction pattern indicates absence of
germinal mutations.
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Figure 4. Presence of a point mutation in codon 918
of RET gene in DNA of tumoral tissue (coexistence
of C and T as indicated by
arrow).
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The other areas of the RET gene known to host abnormalities
in familial medullary thyroid carcinoma (FMTC) or MEN2 patients were
also examined in the FNAB specimens and blood DNA of the patient,
showing absence of alterations (data not shown).
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Discussion
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MTC has a malignant clinical behavior. Regional lymph node
metastases are often present at the diagnosis and are considered the
most important prognostic factors (9). In approximately 20% of the
cases, MTC is the primary manifestation of a MEN2 disease. In patients
affected by MTC, therefore, evaluation of medullary adrenal gland and
parathyroid function, as well as the assessment of a heritable disease,
is required. Even without a suspicious family history, in fact, an
inherited disease has been demonstrated in some cases by biochemical
(10) or genetic screening (11).
Recently, an alteration of RET proto-oncogene has been
identified as the genetic marker of MEN 2A and 2B and hypothesized to
be the causative event in such diseases. Point mutations of the
RET gene occur also in sporadic MTC. Thus, new protocols for
management of this illness using genetic analysis have been proposed
(12, 13). In all of these protocols, the search for mutations in
RET oncogene is first performed either in specimen obtained
at surgery or, after the histological diagnosis, by DNA extraction from
paraffin-embedded tissue blocks. Subsequently, if genetic analysis is
positive, the search is extended to peripheral blood samples from the
patients and eventually to their close relatives. In consideration of
the rare possibility of coexistence of somatic and germinal alterations
in different sites (14), the search for RET alterations must
include all of the sites known to host genetic anomalies. However, at
present, even after such a complete investigation, a 100% exclusion
diagnosis for familial disease may be not reached (4).
Our study shows the possibility and the feasibility to search for
RET mutations at a very early stage, even before performing
surgery, by analyzing the aspirates obtained from the thyroid nodule
and enlarged neck lymph nodes, when present. In our patient the genetic
analysis established a diagnosis of sporadic MTC, making it unnecessary
for either familial screening with the traditional, not well-tolerated
pentagastrin test or for further investigation for other endocrine
neoplasia as part of a MEN2. In this regard, it appears as an important
feature of our approach to exclude preoperatively the presence of MEN
diseases in our patient with MTC. In fact, pheochromocytoma, even if
asymptomatic, should be removed first, because its presence greatly
increases the surgical risk during thyroidectomy (5). On the other
hand, it is certainly difficult to exclude the presence of an
asymptomatic pheochromocytoma by both hormonal assay and different
imaging modalities, whereas the detection of a germline RET
mutation would demonstrate the presence of MEN2 disease. In addition,
detection of a RET mutation suggests a more careful follow
up of a patient with sporadic MTC, even if the role of RET
alterations as poor prognostic marker is still controversial (8, 15, 16).
Considering both the feasibility and the accuracy of the genetic
analysis, this approach may be excellent for staging patients and for
monitoring the disease before surgery, as well to achieve or confirm a
definitive diagnosis of MTC in the presence of a neck mass of unclear
histology/cytology. Thus, PCR-based genetic analysis in of FNAB
aspirates may represent a feasible and reliable tool in the management
strategy of medullary thyroid carcinoma.
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Footnotes
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1 This work was supported by a Ministero dell’Università e
delle Ricerca Scientifica e Tecnologica grant (40%) and by a grant of
Associazione Italiana per la Ricerca sul Cancro (AIRC) (to S.F.). 
Received March 21, 1997.
Revised June 3, 1997.
Accepted June 19, 1997.
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Abstract
Introduction
