Full text of DOI:10.1007/BF03343809

J. Endocrinol. Invest. 24: 51-55, 2001
CASE REPORT
RET proto-oncogene mutation in a mixed medullary-
follicular thyroid carcinoma
F. Orlandi*, E. Chiefari**, P. Caraci*, A. Mussa*, I. Gonzatto*, P. De Giuli***,
D. Giuffrida****, A. Angeli*, and S. Filetti**
*Department of Clinical and Biological Sciences, University of Torino, Torino; **Department of
Experimental and Clinical Medicine, Chair of Endocrinology, University of Catanzaro, Catanzaro;
***Department of Pathology, San Luigi Hospital, Orbassano, Torino; ****Division of Medical Oncology,
San Luigi Hospital, Catania, Italy
ABSTRACT. A case of a patient with an uncom-
mon thyroid carcinoma, showing histological and
immunohistochemical features of both follicular
and parafollicular cells is described. Somatic
point mutation (ATG to ACG heterozygotic mu-
tation at codon 918) of the RET proto-oncogene
was detected in tumor tissue, as confirmed by
immunohistochemical expression of RET onco-
protein. Our findings suggest that constitutive
RET proto-oncogene activation may be involved
in the development of mixed medullary-follicu-
lar thyroid carcinoma.
(J. Endocrinol. Invest. 24: 51-55, 2001)
©2001, Editrice Kurtis
INTRODUCTION
are matter of debate: they may arise from uncom-
mitted stem cells of the ultimo-branchial body (10) or
may represent a special type of thyroid tumor un-
dergoing divergent differentiation. Alternatively, they
may originate from neoplastic transformation of dif-
ferent cell populations under common oncogenic
stimuli (11); finally, the so called “collision hypothe-
sis” suggests that the coexistence of different tumors
may realize a confluence in a single nodule (12, 13).
Some cases of MMFTC have been reported in the
setting of MEN 2A syndrome (6). Since somatic RET
mutations have been recently described in MMFTC
(16), we found of interest to confirm this novel ob-
servation in a patient with a thyroid tumor sharing
follicular-parafollicular phenotypic appearance.
The thyroid gland displays two distinct endocrine
cell types: the thyroglobulin-producing follicular
cells and the calcitonin-producing parafollicular
cells. Neoplastic transformation may involve both
cell types. Follicular and papillary carcinomas de-
velop from follicular cells, whereas medullary car-
cinomas (MTC) develop from parafollicular cells (1).
MTC may occur sporadically or as familial syn-
drome, with or without extrathyroid involvement
(2). Somatic or germline mutations of RET proto-
oncogene have been identified in sporadic and fa-
milial MTC, respectively (3-5).
An uncommon thyroid carcinoma, sharing histologi-
cal and/or immunohistochemical features of both fol-
licular and parafollicular cells, has been identified and
classified as mixed medullary-follicular thyroid carci-
noma (MMFTC) (6-9). A co-expression of thyroglob-
ulin (TG) and neuroendocrine peptides (calcitonin,
calcitonin gene related protein, chromogranin, neu-
rotensin and others) is the hallmark of this tumor. The
histogenesis and the pathogenesis of mixed tumors
CASE REPORT
A 76-year-old woman, with no familial history of thy-
roid tumors, was admitted to our hospital because
of a nodule in the right lobe of the thyroid. Fine
needle aspiration cytology was consistent with a
follicular lesion. Routine laboratory tests, FT3 and
FT4 were in normal range, while high serum levels
of both calcitonin (CT) (6230 pg/ml, range: 0-10
pg/ml; ELSA-hCT kit, CIS Bio International, CEDEX,
France) and carcinoembryonic antigen (130 ng/ml,
range: 0-5 ng/ml) were found.
Key-words: Mixed thyroid carcinoma, thyroid neoplasm, RET oncogene.
Correspondence: Dr. Fabio Orlandi, Department of Clinical and Biological
Sciences, University of Torino, Regione Gonzole 10, 10043 Orbassano, To-
rino, Italy.
E-mail: orlandi@molinette.unito.it
The patient underwent total thyroidectomy and cen-
tral neck lymph node dissection. Histopathological e-
Accepted August 2, 2000.
51

F. Orlandi, E. Chiefari, P. Caraci, et al.
METHODS
DNA extraction and PCR amplification
xamination revealed a medullary carcinoma, consi-
sting of solid areas with polygonal cells, organized in
compact lobules and trabeculae; the stroma was de-
void of amyloid deposits. In the same nodule an
histopathological pattern of follicular carcinoma, with
vascular invasion and concomitant immunohistoche-
mical expression of thyroglobulin and calcitonin was
observed (Fig. 1, Fig. 2 panel A). The surrounding thy-
roid tissue did not show any sign of C-cell hyperplasia.
The final diagnosis was mixed medullary-follicular thy-
roid carcinoma. After thyroidectomy, radioiodine ab-
lation therapy was given (131I, 30 mCi; 1,11 GBq) and
the following total body scintiscan was negative. Ultra-
sound examination and computed tomography scan
excluded residual disease and/or metastatic lesions. L-
thyroxine suppressive treatment was begun. During
the 5-year follow-up, specific serum markers for diffe-
rentiated thyroid cancer (TG<1.5 ng/ml with negative
anti-thyroglobulin antibody) and for MTC (CT<10
pg/ml and CEA<5 ng/ml) were undetectable, exclu-
ding disease progression.
Genomic DNA was extracted from peripheral
leukocytes and from paraffin embedded tissue us-
ing DNA extraction kit, under conditions suggested
by the supplier (Amersham, Milano, Italy).
Genomic amplicons encompassing exons 10, 11,
13, 14, 15 and 16 of the RET proto-oncogene were
obtained by PCR using primers designed on the
basis of the intronic sequence (14). The primers
were the following:
10S: 5’-GAATTCGCTGAGTGGGCTACGTCT-3’
10A: 5’-CTGCAGACCCACTCACCCTGGATG-3’
11S: 5’-GTGCCAAGCCTCACACCA-3’
11A: 5’-GGAGTAGCTGACCGGGAA-3’
13S: 5’-CTCTCTGTCTGAACTTGGGC-3’
13A: 5’-TCACCCTGCAGCAGGCCTTA-3’
14S: 5’-TGGCTCCTGGAAGACCCA-3’
14A: 5’-AGAGCCATATGCACGCAC-3’
15S: 5-CTCGTTCATCGGGACTTGGCA-3’
15A: 5-CTGGCTCCTCTTCACGTAGGA-3’
A
Fig. 1 - Panel A: Mixed medullary-follic-
ular carcinoma with wide vascular inva-
sion ( ). Vessel lumen (*); Vessel wall (▼)
(H&E 100x). Panel B, C: Higher magnifi-
cation (400x) of the framed area marked
in Panel A. Panel B: Immunohistoche-
mical expression of calcitonin immuno-
reactivity (3,3’-diaminobenzidine tetrahy-
drochloride). Panel C: Immunohisto-
chemical expression of thyroglobulin im-
munoreactivity (3-amino-9-ethylcarbazo-
le). The arrows ( ) in Panels A, B, C indi-
cate the same follicle in paraffin embed-
ded sections consecutively obtained.
B
C
52

Mixed thyroid carcinoma
Single strand conformation polymorphism (SSCP)
analysis
A
Amplicons of exons 10, 11, 13, 14 and 15 were
screened for mutations by SSCP analysis. An aliquot of
each sample was incubated with specific buffer (10 ml
formamide, 0.5M etilendiamminetetraacetate (EDTA),
0.3% bromophenol blue, 0.3% xylene cyanol) for 8
min at 100 C and run on 15% Tris-Borate-EDTA gel
using a Minigel apparatus (Bio-Rad Laboratories,
Segrate, Milano, Italy), at 100 V at 4°C. After electro-
phoresis, the gel was examined by DNA Silver Stain-
ing Kit (Pharmacia Biotech, Cologno Monzese, Milano,
Italy) according to the manufacturer’s instructions.
Restriction analysis
B
Amplicon of exon 16 was directly screened for mu-
tations by restriction enzyme analysis with Fok 1.
The samples were incubated for 3 h at 37°C and
run on 3% agarose-Tris-Borate-EDTA ethidium bro-
mide gel containing.
RET immunohistochemical analysis
Tissue sections (5 μm) were first dewaxed in xylene,
rehydrated in decreasing ethanol concentrations and
microwave processed for three times in citrate buffer
(pH 6.0) for 5 min. Endogenous peroxidase activity
was inhibited by immersing the sections in 0.5% hy-
drogen peroxide for 30 min. Samples were briefly
rinsed in phosphatase buffer saline (PBS), incubated
with blocking serum and subsequently with primary
antibody anti-RET oncoprotein (Clone 3F8, Novo-
castra Laboratories Ltd., Newcastle, UK) diluted 1:100
in PBS at 4°C overnight. After a thorough washing in
PBS the sections were incubated with biotinylated sec-
ondary antibody (DAKO, Copenhagen, Denmark) for
30 min and processed for peroxidase-conjugated
streptavidin (LSAB2, DAKO, Copenhagen, Denmark).
The sections were washed three times and incubated
in AEC solution, washed in tap water for 10 min, coun-
terstained with hematoxylin and mounted in glycerin.
C
Fig. 2 - Panel A: Thyroglobulin (black, ) and calcitonin (red, )
double staining immunoreactivity of the neoplastic cells show-
ing their dual expression in the same follicle. Thyroglobulin is re-
vealed by 5-Bromo-4-Chloro-3-Indolyl-Phosphate/Nitro Blue
Tetrazolium in alkaline phosphatase procedure, whereas calci-
tonin by 3-amino-9-ethylcarbazole in peroxidase procedure.
Panels B-C: Immunohistochemical immunoexpression of RET
oncoprotein. Some neoplastic cells (red, ▼) are stained both in
follicular (panel B) and in solid areas (panel C).
RESULTS AND DISCUSSION
Thyroid cancer with simultaneous histological and im-
munohistochemical features of medullary and follic-
ular thyroid carcinomas is a rare condition (16). In the
management of MTC, the identification of somatic
or germinal RET mutations is required to discriminate
sporadic cases from unrecognized familial disease
(17). In sporadic MTC, somatic point mutations of the
RET proto-oncogene have been detected (3,18).
Thus, the presence of somatic RET mutations was ex-
amined in the thyroid neoplastic tissue of our patient,
carrying an histological diagnosis of MMFTC.
16S: 5’-AGGGATAGGGCCTGGGCTTC-3’
16A: 5’-TAACCTCCACCCCAAGAGAG-3’
PCR amplification was performed as previously de-
scribed (15).
Genomic DNA was extracted from paraffin-embed-
53

F. Orlandi, E. Chiefari, P. Caraci, et al.
ded cancer tissue and peripheral blood. A purified
198 bp DNA fragment was obtained by PCR amplifi-
cation of a region containing codon 918 of exon 16 of
the RET gene. Since point mutations at this codon
determine the loss of a Fok1 restriction site, the frag-
ment was digested with Fok1. Figure 3 shows the
presence of a stronger undigested band and weak
bands, a pattern in accordance with the loss of the
Fok1 site in one allele of the gene. The nucleotide
sequence of the same fragment confirmed the pres-
ence of ATG to ACG heterozygotic mutation at
codon 918. Analysis of genomic DNA extracted from
peripheral blood showed persistence of the Fok1 site,
demonstrating, therefore, the somatic nature of the
mutation. No evidence of mutations in other domains
of the RET gene was found (data not shown).
A variable degree of cytoplasmic RET protein ex-
pression was observed by using a specific monoclonal
antibody. The immunostaining was detected in about
25% of the neoplastic cells both in follicular (Fig. 2
panel B) and in solid areas (Fig. 2 panel C).
A lot of evidence supports the existence of MMFTC
as a distinct entity. First, the co-expression of TG and
CT in the same neoplastic cells (19) may indicate
mixed cellular differentiation. Second, the presence of
mRNAs for both TG and CT in the tumoral cells as
detected by in situ hybridization or Northern Blotting
(20) may support the concomitant expression of CT
and TG by the same neoplastic cell. Third, the con-
current expression of these different proteins can be
demonstrated in the metastatic tissues (19, 21). As in
our case, the observation of concomitant CT and TG
immunostaining in the same follicle on the site of vas-
cular invasion may be considered as an adjunctive cri-
terion for the diagnosis of MMFTC. The simultane-
ous immunoexpression staining in the area of vascu-
lar invasion does not fit with the hypothesis of nor-
mal thyroid tissue entrapped in medullary carcinoma
and supports the diagnosis of true MMFTC.
However, the morphological criteria together with
a molecular analysis of the microdissected cancer
tissue, by examining the gene expression of both
follicular and neuroendocrine cells, may allow a
more certain and unambiguous diagnosis of mixed
tumors. In this regard, Volante et al. (16), by using
laser-based microdissection technique was recent-
ly able to isolate the two-cell type components of
12 mixed tumors. By analyzing them for mutations
of the RET proto-oncogene, allelic losses and for
clonal expression, an hyperplastic rather than a neo-
plastic histological feature and a policlonal pattern
of the follicular structures were frequently detect-
ed. This suggests that a true MMFTC tumor is a
very rare thyroid cancer. Furthermore, since me-
dullary and follicular components exhibited a diffe-
rent pattern of mutations and allelic losses, the
“stem cell theory” (derivation from a single proge-
nitor cell) as responsible of MMFTC appears unlike.
Our data show, in a mixed tumor, a somatic muta-
tion of RET proto-oncogene at codon 918, which
represents a common abnormality in sporadic MTC
(17). Therefore, we confirmed the recent observa-
tion of Volante et al. (16), which reported the same
RET mutation in two mixed sporadic tumors and a
different mutation (at codon 883) in a third patient.
These findings confirm the central role of malignant
transformation of parafollicular cells in the oncoge-
nesis of mixed tumors. However, MMFTC is a rare
but polymorphic entity with different pathogenetic
mechanisms that require further studies on larger
number of tissues to better clarify the controversial
pathogenesis.
→
200 bp →
50 bp →
ACKNOWLEDGMENTS
This work was supported by a grant from Associazione Italiana
per la Ricerca sul Cancro (AIRC) to S. Filetti and from Ministero
per l’Università e la Ricerca Scientifica e Tecnologica (MURST)
to F. Orlandi. We thank Dr. Susanna Cappia for immunohisto-
chemical evaluation of the RET oncoprotein.
1
2
3
4
5
Fig. 3 - Restriction analysis of PCR products of RET exon 16 af-
ter Fok 1 digestion (3 h at 37°C). Samples were run on 3%
agarose-tris-borate-etilendiamminetetraacetate gel containing
ethidium bromide. Lane 1: DNA size marker. Lane 2: undigest-
ed amplicon from unaffected subject. Lane 3: digested ampli-
con from unaffected subject. Lane 4: digested amplicon from tu-
moral tissue DNA of the patient (presence of heterozigotic point
mutation at codon 918 determines loss of Fok 1 restriction site
leading to pathological pattern). Lane 5: digested amplicon
from blood DNA of the patient (presence of a normal restric-
tion pattern indicates absence of germinal mutation).
REFERENCES
1. Rosai J., Carcangiu M.L., De Lellis R.A.
Tumors of the thyroid gland.
In: Atlas of tumor pathology.
Armed Force Institute of Pathology, Washington,
1992.
54

Mixed thyroid carcinoma
2. Giuffrida D., Gharib H.
12. Lax S.F., Beham A., Kronberger-Schonecker D.,
Langsteger W., Denk H.
Current diagnosis and management of medullary
thyroid carcinoma.
Coexistence of papillary and medullary carcinoma of
the thyroid gland-mixed or collision tumour? Clinico-
pathological analysis of three cases.
Ann. Oncol. 1998, 9: 695-701.
3. Wohllk N., Cote G.J., Bugalho M.M.J., Ordonez N.,
Evans D.B., Goepfert H., Khorana S., Shultz P.,
Richards S., Gagel R.F.
Virchows Arch. 1994, 42: 441-447.
13. Pastolero G.C., Coire C.I., Asa S.L.
Concurrent medullary and papillary carcinomas of thy-
roid with lymph node metastases. A collision pheno-
menon.
Am. J. Surg. Pathol. 1996, 20: 245-250.
14. Takahashi M., Buma Y., Iwamoto T., Inaguma Y.,
Ikeda H., Hiai H.
Relevance of RET proto-oncogene mutations in spo-
radic medullary thyroid carcinoma.
J. Clin. Endocrinol. Metab. 1996, 81: 3740-3745.
4. Santoro M., Carlomagno F., Melillo R.M., Billaud M.,
Vecchio G., Fusco A.
Molecular mechanisms of RET activation in human
neoplasia.
J. Endocrinol. Invest. 1999, 22: 811-819.
Cloning and expression of the RET proto-oncogene
encoding a tyrosine kinase with two potential trans-
membrane domains.
5. Eng C., Clayton D., Schuffenecker I., Lenoir G.,
Gilbert D.V.M., Cote G., Gagel R., Ploos van Amstel
H.K., Lips C.J., Nishisho I., Takai S.I., Marsh D.J.,
Robinson B.G., Frank-Raue K., Raue F., Xue F., Noll
W.W., Romei C., Pacini F., Fink M., Niederle B.,
Zedenius J., Nordenskjold M., Komminoth P., Hendy
G.N., Gharib H., Thibodeau S., Lacroix A., Frilling A.,
Ponder B.A.J., Mulligan L.M.
Oncogene 1988, 3: 571-578.
15. Chiefari E., Russo D., Giuffrida D., Zampa G.A., Meringolo
D., Arturi F., Chiodini I., Bianchi D., Attard M., Trischitta
V., Bruno R., Giannasio P., Pontecorvi A., Filetti S.
Analysis of RET proto-oncogene abnormalities in pa-
tients with MEN 2A, MEN 2B, familial or sporadic
medullary thyroid carcinoma.
J. Endocrinol. Invest. 1998, 21: 358-364.
The relationship between specific RET Proto-onco-
gene mutations and disease phenotype in multiple
endocrine neoplasia type 2: international RET muta-
tion consortium analysis.
16. Volante M., Papotti M., Roth J., Saremaslani P., Speel
E.J.M., Lloyd R.V., Carney J.A., Heits P.U., Bussolati
G., Komminoth P.
Mixed medullary-follicular thyroid carcinoma. Mole-
cular evidence for a dual origin of tumor components.
Am. J. Pathol. 1999, 155: 1499-1509.
JAMA 1996, 276: 1575-1579.
6. Papotti M., Negro F., Carney J.A., Bussolati G.,
Lloyd R.V.
17. Komminoth P., Kunz E.K., Matias-Guiu X., Hiort O.,
Christiansen G., Colomer A., Roth J., Heitz P.U.
Analysis of RET proto-oncogene point mutations di-
stinguishes heritable from nonheritable medullary
carcinomas.
Cancer 1995, 76: 479-489.
18. Romei C., Elisei R., Pinchera A., Ceccherini I., Molinaro
E., Mancusi F., Martino E., Romeo G., Pacini F.
Mixed medullary-follicular carcinoma of the thyroid.
A morphological, immunohistochemical and in situ
hybridization analysis of 11 cases.
Virchows Arch. 1997, 430: 397-405.
7. Hales M., Rosenau W., Okerlund M.D, Galante M.
Carcinoma of the thyroid with a medullary and fol-
licular pattern: morphologic, immunohistochemical,
and clinical laboratory studies.
Somatic mutations of the RET proto-oncogene in spo-
radic medullary thyroid carcinoma are not restricted to
exon 16 and are associated with tumor recurrence.
J. Clin. Endocrinol. Metab. 1996, 81: 1619-1622.
Cancer 1982, 50: 1352-1359.
8. Sobrinho-Simoes M.
Mixed medullary and follicular carcinoma of the
thyroid.
19. Holm R., Sobrinho-Simoes M., Nesland J.M.,
Johannessen J.V.
Histopathology 1993, 23: 287-289.
9. Hedinger C., Williams E.D., Sobin L.H.
Histological typing of thyroid tumours.
In: World Health Organization (Ed.), International
histological classification of tumours.
Concurrent production of calcitonin and thyroglob-
ulin by the same neoplastic cells.
Ultrastruct. Pathol. 1986, 10: 241-248.
20. Noel M., Delehaye M.C., Segond N, Lasmoles F.,
Caillon B, Gardet P, Fragn P., Moukhtar M.S.
Study of calcitonin and thyroglobulin gene expression
in human mixed follicular and medullary thyroid carci-
noma.
Springer-Verlag, Berlin, 1988, p. 1.
10. Williams E.D., Toyu C.E., Harach H.R.
The ultimobranchial gland and congenital thyroid ab-
normalities in man.
J. Pathol. 1989, 159: 135-141.
Thyroid 1991, 1: 249-256.
11. Triggs S.M., Williams E.D.
21. Uribe M., Fenoglio-Preiser C., Grimes M., Feind C.
Medullary carcinoma of the thyroid gland. Clinical,
pathological, and immunohistochemical features with
review of the literature.
Experimental carcinogenesis in the thyroid follicular
and C cells. A comparison of the effect of variation in
dietary calcium and of radiation.
Acta Endocrinol. (Copenh.) 1977, 85: 84-92.
Am. J. Surg. Pathol. 1985, 9: 577-594.
55