Thyroid Transcription Factor-1 in Normal, Hyperplastic, and Neoplastic Follicular Thyroid Cells Examined by Immunohistochemistry and Nonradioactive In Situ Hybridization

Article (PDF Available)inModern Pathology 13(5):570-6 · June 2000with21 Reads
DOI: 10.1038/modpathol.3880098 · Source: PubMed
Abstract
Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes. We examined the expression of TTF-1 in non-neoplastic and neoplastic thyroid tissues. By immunohistochemistry, the nuclei of normal and hyperplastic follicular cells strongly reacted with the antibody against TTF-1. Immunohistochemistry also revealed a distinctive nuclear positivity of TTF-1 in all 33 differentiated follicular cell tumors, including 15 follicular adenomas, 5 follicular carcinomas, and 13 papillary carcinomas. No immunoreactions were observed in three of four undifferentiated carcinomas, whereas an isolated and weak nuclear positivity was obtained in one. In normal and hyperplastic tissues, the distribution of TTF-1 was fairly related to that of thyroid-specific proteins thyroglobulin and thyroperoxidase. However, discrepancies in the distribution were observed in tumor tissues. By in situ hybridization, the riboprobe hybridized distinctively with the cytoplasm of neoplastic cells as well as normal follicular cells. Papillary carcinoma cells expressed TTF-1 mRNA in all but two cases, and its expression was also demonstrated in one of four undifferentiated carcinomas. Reverse transcription-polymerase chain reaction confirmed the presence of TTF-1 mRNA in two human undifferentiated carcinoma cell lines, TTA-1 and TTA-2. In conclusion, the investigation of TTF-1 provides useful information on the functional activities and/or differentiation of thyroid tumors and may lead to an increase in our understanding of the biologic nature of thyroid tumors.
3 Figures
Thyroid Transcription Factor-1 in Normal, Hyperplastic,
and Neoplastic Follicular Thyroid Cells Examined by
Immunohistochemistry and Nonradioactive
In Situ
Hybridization
Ryohei Katoh, M.D., Ph.D., Akira Kawaoi, M.D., Ph.D., Eri Miyagi, Ph.D., Xin Li, M.D.,
Koichi Suzuki, Ph.D., Yasushi Nakamura, M.D., Ph.D., Kenichi Kakudo, M.D., Ph.D.
Department of Pathology, Yamanashi Medical University School of Medicine (RK, AK, EM, XL), Tamaho,
Japan; Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health (KS), Bethesda, Maryland; and Department of
Pathology, Wakayama Medical College (YN, KK), Wakayama, Japan
Thyroid transcription factor-1 (TTF-1) has been
known to regulate the transcriptional activity of
thyroid-specific genes. We examined the expression
of TTF-1 in non-neoplastic and neoplastic thyroid
tissues. By immunohistochemistry, the nuclei of
normal and hyperplastic follicular cells strongly re-
acted with the antibody against TTF-1. Immunohis-
tochemistry also revealed a distinctive nuclear pos-
itivity of TTF-1 in all 33 differentiated follicular cell
tumors, including 15 follicular adenomas, 5 follicu-
lar carcinomas, and 13 papillary carcinomas. No
immunoreactions were observed in three of four
undifferentiated carcinomas, whereas an isolated
and weak nuclear positivity was obtained in one. In
normal and hyperplastic tissues, the distribution of
TTF-1 was fairly related to that of thyroid-specific
proteins thyroglobulin and thyroperoxidase. How-
ever, discrepancies in the distribution were ob-
served in tumor tissues. By in situ hybridization, the
riboprobe hybridized distinctively with the cyto-
plasm of neoplastic cells as well as normal follicular
cells. Papillary carcinoma cells expressed TTF-1
mRNA in all but two cases, and its expression
was also demonstrated in one of four undifferen-
tiated carcinomas. Reverse transcription–polymer-
ase chain reaction confirmed the presence of TTF-1
mRNA in two human undifferentiated carcinoma
cell lines, TTA-1 and TTA-2. In conclusion, the in-
vestigation of TTF-1 provides useful information on
the functional activities and/or differentiation of
thyroid tumors and may lead to an increase in our
understanding of the biologic nature of thyroid tu-
mors.
KEY WORDS: Immunohistochemistry, In situ
hybridization, Reverse transcription–polymerase
chain reaction, Thyroid neoplasms, Thyroid tran-
scription factor-1.
Mod Pathol 2000;13(5):570 –576
The thyroid-differentiated phenotype is character-
ized by the expression of a variety of proteins that
are specifically synthesized by the thyroid follicular
cells. Four genes encoding thyroid-specific proteins
have been cloned: thyroglobulin (Tg), thyroperoxi-
dase (TPO), thyroid-stimulating hormone receptor
(1), and, more recently, thyroid iodine transporter
(2). This tissue-restricted protein production is reg-
ulated, at least in part, by the thyroid-specific nu-
clear factors, thyroid transcription factors 1 and 2
(TTF-1 and TTF-2) (3, 4) and Pax-8 (5). All of them
bind to Tg and TPO promoters (5, 6). TTF-1 and
Pax-8, however, preferentially bind to the Tg and
TPO promoters, respectively (5). In addition, TTF-1
binds to the thyroid-stimulating hormone receptor
gene promoter (7, 8). An important feature of TTF-1
is that it is a homeoprotein (4). This kind of protein
plays very important roles in development, cell
growth, and differentiation processes (9). Other
than thyrocytes, TTF-1 is expressed in some regions
of the central nervous system and in the lung,
where it regulates the activity of tissue-specific
genes not related to the thyroid (10–12).
The role of TTF-1 as a critical transcription factor
regulating the gene expression of thyroid-specific
proteins has been well documented in cell culture
systems, particularly the FRTL-5 cell line (1–4, 6,
13–15). However, little is known about the localiza-
Copyright © 2000 by The United States and Canadian Academy of
Pathology, Inc.
VOL. 13, NO. 5, P. 570, 2000 Printed in the U.S.A.
Date of acceptance: November 12, 1999.
Address reprint requests to: Ryohei Katoh, M.D., Ph.D., Department of
Pathology, Yamanashi Medical University School of Medicine, 1110 Shi-
mokato, Tamaho-cho, Nakakoma-gun, Yamanashi 409–38, Japan; e-mail:
rkatoh@res.yamanashi-med.ac.jp; fax: 81-552-73-9534.
570
tion or distribution of TTF-1 protein and mRNA in
human thyroid tumor tissues (16). Especially, there
has been no investigation on the in situ expression
of TTF-1 mRNA and its correlation with the pres-
ence of thyroid-specific proteins in human thyroid
tissue specimens. Therefore, we investigated the
expression and localization of TTF-1 in normal,
hyperplastic, and neoplastic human thyroid tissues
using immunohistochemistry (IHC) and noniso-
topic in situ hybridization (ISH). Furthermore, we
sought to determine whether TTF-1 expression pro-
files are of any value for estimating the biologic
nature of thyroid neoplasms.
MATERIALS AND METHODS
Tissue Preparation
We reviewed the surgically resected specimens of
various histologic types of thyroid tumors from the
files of the Department of Pathology at Yamanashi
Medical University and Kofu City Hospital and se-
lected 47 cases for immunohistochemical and ISH
studies. The materials consisted of tissues from 5
cases of normal thyroid, 5 multinodular goiters, 15
follicular adenomas, 5 follicular carcinomas, 13
papillary carcinomas, and 4 undifferentiated carci-
nomas. The criteria for selection included represen-
tative morphologic characteristics and adequate
tissue mass. In tumor cases, the lesions were clas-
sified according to the diagnostic criteria of the
World Health Organization classification proposed
in 1988. All specimens were routinely processed,
and paraffin blocks were available in all cases.
Immunohistochemistry
For immunohistochemical analysis, the following
antibodies were used: mouse anti–TTF-1 monoclo-
nal antibody (Neomarkers, Fremont, CA), rabbit an-
tihuman thyroglobulin polyclonal antibody (origi-
nally produced) at a dilution of 1:500, and mouse
antithyroid peroxidase monoclonal antibody (Ca-
nadian Bioclinical, Scarborough, Canada) at a dilu-
tion of 1:100. Antirabbit-IgG or antimouse IgG con-
jugates (DAKO, Glostrup, Denmark) were applied to
the sections as a second antibody.
For TTF-1 staining, heat pretreatment of the par-
affin sections was used for antigen retrieval. Sec-
tions were cut and mounted on poly-L-lysine–
coated slides. Deparaffinized sections were placed
first in plastic Coplin jars filled with citrate buffer
(pH 6.0) and incubated for 10 min at 120° C in an
autoclave. After autoclave pretreatment, the sec-
tions were allowed to cool to room temperature and
were then exposed to a 0.3% solution of hydrogen
peroxide in absolute methanol to inactivate endog-
enous peroxidase.
Indirect immunoperoxidase staining was used ac-
cording to standard protocols (17). The sections were
rinsed with phosphate-buffered saline (PBS), pH 7.2,
and incubated overnight at C with primary anti-
body. Tissue sections were then washed, reacted with
secondary antibody for1hatroom temperature,
washed, reacted with 3,3-diaminobenzidine sub-
strate, washed, and stained with methyl green or he-
matoxylin.
For serum controls, normal rabbit serum or PBS
was used instead of the primary antibody. The tis-
sues from liver and kidney were used as a negative
control.
In Situ
Hybridization
For riboprobe preparation, a 757 bp SacII fragment
(149–905 bp) of TTF-1 cDNA was subcloned into
Bluescript SK
(Stratagene; La Jolla, CA). TTF-1 cDNA
was a kind gift from Dr. R. Di Lauro (Stazione Zoo-
logica A. Dohrn, Villa Comunale, Naples, Italy). Anti-
sense and sense riboprobes were transcribed in the
presence of digoxigenin-11-uridine-5-triphosphate
(Boehringer Mannheim GmbH Biochemica, Mann-
heim, Germany) using T7 or T3 RNA polymerases,
respectively.
The ISH protocol was a modification of a method
described by Nomura et al. (18). In brief, sections
were deparaffinized with xylene, rehydrated with a
graded series of ethanol solutions, rinsed in PBS at
pH 7.4, postfixed with 4% paraformaldehyde for 5
min, rinsed in PBS, and digested with 20
g/mL
proteinase K (Sigma, St Louis, MO) at 37° C for 20
min. Slides were fixed again with 4% paraformalde-
hyde for 5 min, rinsed with PBS, treated with an
0.02% glycine solution for 15 min, rinsed with PBS,
dehydrated in graded solutions of ethanol, and air
dried.
Probes were diluted with hybridization solution
(10 mM Tris-Cl pH 7.6, containing 50% formamide,
200
/mL transfer RNA, 1 Denhardt’s solution,
10% dextran sulfate, 600 mM NaCl, 0.25% sodium
dodecyl sulfate, and 1 mM EDTA) to a final concen-
tration of 10 ng/mL, and sections were hybridized
with heat-denatured riboprobe for 18 h at 55° C.
After hybridization, samples were washed sequen-
tially in 5 standard saline citrate (SSC) for 5 min
at 55° C, then 2 SSC containing 50% formamide
for 30 min at 55° C; 1 SSC is composed of 0.15 M
NaCl and 0.015 M sodium citrate. To degrade un-
bound riboprobe, sections were treated with RNase
A(1
g/mLin10mM Tris-Cl pH 7.6, 500 mM NaCl,
1mM EDTA) at 37° C for 30 min, then sequentially
washed once in 2 SSC for 20 min at 55° C and
twice in 0.2 SSC for 20 min at 55° C. Hybridized
probe was detected using a Nucleic Acid Detection
Kit (Boehringer) according to the manufacturer’s
instructions. Briefly, slides were incubated with
TTF-1 in Follicular Cell Thyroid Tumors (R. Katoh
et al
.) 571
1.5% blocking reagent for 30 min at room temper-
ature, then incubated with a 1:500 dilution of alka-
line phosphatase-labeled sheep antidigoxigenin
Fab fragment for1hatroom temperature. After
washing, slides were incubated with substrate solu-
tion containing nitroblue tetrazolium salt and
X-Phosphate (5-bromo-4-chloro-3-indoyl phos-
phate, toluidium salt) for 2 to 12 h, counterstained
with methyl green and mounted with a glycerin-
gelatin solution.
Reverse Transcription–Polymerase
Chain Reaction
Total RNAs were prepared by the acid
guanidinium-phenol-chloroform system (ISOGEN,
Nippon Gene Co., Ltd., Toyama, Japan) as de-
scribed in the manufacturer’s instructions. Isolated
total RNA (20
g) was fractionated by electrophore-
sis on 1.5% agarose in 18% formaldehyde gels. The
quality of the extracted RNAs was checked by
ethidium bromide staining; only RNAs showing two
clear bands of 28S and 18S, corresponding to ribo-
somal RNAs, were selected for further studies. Five
micrograms of total RNA was reverse-transcribed
using murine leukemia virus reverse transcriptase.
PCRs were carried out according to the Gene Amp
DNA amplification reagent kit instructions (Perkin-
Elmer Cetus, Norwalk, CT), with modifications as
described. Thirty cycles were performed. During
each cycle, the samples were heated to 94° C for 30
TABLE 1. Summary of Immunohistochemistry (IHC) for TTF-1, Tg, and TPO and In Situ Hybridization (ISH) for
TTF-1 mRNA
Histologic type
IHC
ISH for TTF-1 mRNA
TTF-1 Tg TPO
1. normal thyroid ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
2. normal thyroid ⫹⫹⫹ ⫹⫹ ⫹⫹⫹
3. normal thyroid ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
4. normal thyroid ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
5. normal thyroid ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
6. nodular goiter ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
7. nodular goiter ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
8. nodular goiter ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
9. nodular goiter ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
10. nodular goiter ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
11. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹
12. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹
13. follicular adenoma ⫹⫹ ⫹⫹ ⫹⫹⫹
14. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹
15. follicular adenoma ⫹⫹ ⫹⫹
16. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
17. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
18. follicular adenoma ⫹⫹ ⫹⫹ ⫹⫹
19. follicular adenoma ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
20. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
21. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹
22. follicular adenoma ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
23. follicular adenoma ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
24. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹
25. follicular adenoma ⫹⫹⫹ ⫹⫹ ⫹⫹
26. follicular carcinoma ⫹⫹ ⫹⫹ ⫹⫹
27. follicular carcinoma ⫹⫹ ⫹⫹ ⫹⫹
28. follicular carcinoma ⫹⫹
29. follicular carcinoma ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
30. follicular carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
31. papillary carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹⫹
32. papillary carcinoma ⫹⫹
33. papillary carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹
34. papillary carcinoma ⫹⫺
35. papillary carcinoma ⫹⫹ ⫹⫹⫹
36. papillary carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹
37. papillary carcinoma ⫹⫹ ⫹⫹
38. papillary carcinoma ⫹⫹ ⫹⫹
39. papillary carcinoma ⫹⫹ ⫹⫹
40. papillary carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹
41. papillary carcinoma ⫹⫹⫹ ⫹⫹⫹
42. papillary carcinoma ⫹⫹ ⫹⫹
43. papillary carcinoma ⫹⫹⫹ ⫹⫹ ⫹⫹⫹
44. undifferentiated carcinoma ⫹⫺
45. undifferentiated carcinoma ⫺⫺
46. undifferentiated carcinoma ⫺⫺
47. undifferentiated carcinoma ⫺⫺
TTF-1, thyroid transcription factor-1; Tg, thyroglobulin; TPO, thyroperoxidase; , negative; , 10% tumor cells positive; ⫹⫹, 1040% tumor cells
positive; ⫹⫹⫹, 50% tumor cells positive.
572 Modern Pathology
seconds, cooled to 56° C for 60 seconds, and heated
to 72° C for 60 seconds. The reverse transcription
PCR (RT-PCR) reagent blank yielded no detectable
products. A 16-
l sample of each 50
l PCR solution
was fractionated by electrophoresis in a 2% agarose
gel. Gels were photographed using Polaroid 655
film. The following oligonucleotide primers located
at exon 1 and exon 2 of TTF-1 gene were used:
TTF-1 5 oligonucleotide, CCAGGACACCATGAG-
GAACA; TTF-1 3 oligonucleotide, TGCACTCGT-
TCTTGTACCGG. RNAs extracted from stomach, co-
lon, and liver were used as a negative tissue control.
Cell Culture
Two cell lines, TTA-1 and TTA-2, were used as
models of human undifferentiated thyroid carci-
noma (19). These two cell lines maintained the
properties of the parent tumor. The medium was
changed every other day, and cells were passaged
every 6 to 8 days.
RESULTS
Immunohistochemistry
A summary of the IHC results is shown in Table 1.
Immunoperoxidase staining with anti–TTF-1 anti-
body revealed that normal and hyperplastic thyroid
tissues exhibited positive reaction products in the
nuclei of most follicular cells as a fine granular
pattern (Figs. 1A, B). The cell nuclei in kidney, co-
lon, and stomach were nonreactive (data not
shown). A strong nuclear positivity of TTF-1 protein
FIGURE 1.
Immunohistochemical analysis of TTF-1 protein. The nuclei of almost all normal (A; magnification, 200) and hyperplastic (B;
magnification, 200) follicular cells strongly reacting with the antibody against TTF-1. C, follicular adenoma tissues showing positive reaction
products in the nuclei of cells in a fine granular pattern (magnification, 200). D, positive reaction products are found in the nuclei of papillary
carcinoma cells (magnification, 200). E, isolated cells of undifferentiated carcinoma are positive for TTF-1 (original magnification, 400).
TTF-1 in Follicular Cell Thyroid Tumors (R. Katoh
et al
.) 573
was also observed in all 33 differentiated follicular
cell tumors, including 15 follicular adenomas, 5
follicular carcinomas, and 13 papillary carcinomas,
whereas a weak and isolated positivity was demon-
strated in one of four undifferentiated carcinomas
(Figs. 1C–E). Immunostainings for thyroid-specific
proteins Tg and TPO were performed on the serial
sections for comparison with TTF-1 results (Table
1). Discrepancies in positive-cell distribution be-
tween TTF-1 and thyroid-specific proteins were ob-
served in cancerous tissues (Table 1). All four un-
differentiated carcinomas examined showed a lack
of Tg and TPO proteins.
In Situ
Hybridization
Specificity was established by comparing the ISH
using the antisense and sense probes (data not
shown). An antisense TTF-1 riboprobe but not a
counterpart sense probe exhibited a positive signal
for TTF-1 mRNA in the perinuclear region of the
thyroid follicular epithelium. Expression levels of
TTF-1 mRNA among follicles were not uniform in
normal thyroid tissue (Fig. 2A). Slight expression of
TTF-1 mRNA was detected in the flat thyroid epi-
thelium lining the lumen of large follicles. In con-
trast, the cuboidal or columnar cells of small folli-
FIGURE 2.
Findings of in situ hybridization of TTF-1 mRNA. A, in normal thyroid, little expression of TTF-1 mRNA is demonstrated in the flat
thyroid epithelium lining the lumen of large follicles, whereas the cuboidal cells of small follicles show strong expression (magnification, 200).
Multinodular goiter (B; magnification, 40), follicular adenoma (C; magnification, 200), and papillary carcinoma (D; magnification, 200) showing
diffuse expression of TTF-1 mRNA. E, distinctive expression of TTF-1 mRNA is observed in isolated tumor cells of undifferentiated carcinoma
(magnification, 400).
574 Modern Pathology
cles showed strong expression of TTF-1 mRNA (Fig.
2A). Hyperplastic thyroid tissues such as multinod-
ular goiter (Fig. 2B) showed diffuse expression of
TTF-1 mRNA in follicular cells. In follicular adeno-
mas and follicular carcinomas, the expression of
TTF-1 mRNA varied among tumors. The tumors
that were composed mainly of small follicles exhib-
ited stronger expression of TTF-1 mRNA than the
tumors that were composed of large follicles (Fig.
2C). In all but two cases of papillary carcinoma, the
expression of TTF-1 mRNA in tumor cells was iden-
tified by ISH (Fig. 2D). In one case of undifferenti-
ated carcinomas, isolated tumor cells exhibited ex-
pression of TTF-1 mRNA (Fig. 2E).
Reverse Transcription–Polymerase
Chain Reaction
We examined two cell lines of human undiffer-
entiated thyroid carcinoma, TTA-1 and TTA-2. RT-
PCR analysis revealed a band of 149 bp correspond-
ing to exon 1 and exon 2 of the TTF-1 gene after 30
cycles in the TTA-1 and TTA-2 specimens (Fig. 3).
DISCUSSION
The present study was undertaken to examine
the expression and localization of TTF-1 in normal,
hyperplastic, and neoplastic thyroid tissues using
IHC and nonisotopic ISH. In normal thyroids, the
expression intensity of TTF-1 mRNA in follicular
cells varied among thyroid follicles, whereas TTF-1
protein distributed in almost all nuclei of follicular
cells. This heterogeneous expression of TTF-1
mRNA among normal thyroid follicles may indicate
the presence of an autoregulation mechanism for
thyroid-specific gene transcription. Recently, we
hypothesized that TG-initiated, transcriptional reg-
ulation of thyroid-restricted genes is a normal,
feedback, compensatory mechanism that limits fol-
licular function and contributes to follicular heter-
ogeneity (20).
In the present study, IHC and ISH revealed that
most differentiated neoplastic tissues, including
follicular adenoma, follicular carcinoma, and pap-
illary carcinoma, express TTF-1 protein and mRNA.
Staining patterns of TTF-1 were fairly consistent
with those of Tg and TPO in normal and hyperplas-
tic thyroid tissues. However, discrepancies in the
localization or distribution of TTF-1 and thyroid-
specific proteins occasionally were observed, espe-
cially in cancerous tissues. This suggests that the
activity of TTF-1 might need to be regulated by a
variety of mechanisms, such as phosphorylation
(21), redox state (22), and interaction with other
factors and/or co-activators (23) that could be sub-
jected to regulation themselves and that may be
lacking in cancer cells. Therefore, we assume that
the presence of TTF-1 alone is not sufficient to
produce the differentiated phenotype of thyroid
neoplastic cells, although TTF-1 is necessary for full
expression of the thyroid-differentiated phenotype.
It has been reported that TTF-1 mRNA is always
absent in the undifferentiated (anaplastic) thyroid
carcinoma tissues (16) and their cell lines (24).
However, in the present study, IHC and ISH re-
vealed the presence of TTF-1 protein and mRNA in
one of four specimens of undifferentiated carci-
noma. In addition, we examined two cell lines of
human undifferentiated thyroid carcinoma, TTA-1
and TTA-2 (19), and demonstrated the presence of
TTF-1 mRNA expression after 30 cycles of RT-PCR.
These findings suggest that undifferentiated carci-
noma cells express TTF-1 mRNA but at very low
levels that may not be detectable by Northern blot-
ting. Recently, it was reported that transfection of
TTF-1 expression vectors in undifferentiated Fisher
rat thyroid cells results in activation of thyroglobu-
lin gene expression (25). Thus, it is conceivable that
the loss of the thyroid-specific expression of Tg and
TPO in undifferentiated carcinoma tissue may be
related to the reduced expression of TTF-1. How-
ever, further investigations are required to clarify
this matter.
In conclusion, most thyroid tumors express
TTF-1 in connection with their functional ability.
Therefore, the investigation of TTF-1 provides use-
ful information on the biologic nature of thyroid
tumors.
Acknowledgments: We thank Dr. Akira Yoshida
(Department of Surgery, Kanagawa Cancer Center)
for donating the cell lines TTA-1 and TTA-2, and
Mrs. Miyuki Ito for skillful technical assistance.
FIGURE 3.
Reverse transcription–polymerase chain reaction analysis
of TTF-1 mRNA. Undifferentiated carcinoma cell lines TTA-1 and TTA-2
expressing a band of 149 bp corresponding to exon 1 and exon 2 of the
TTF-1 gene. NT (lanes 3 and 4), normal thyroid; M, marker.
TTF-1 in Follicular Cell Thyroid Tumors (R. Katoh
et al
.) 575
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576 Modern Pathology
    • Several immunohistochemical stains have been investigated for their possible role as diagnostic markers for PTC. They are cytokeratin19 (CK19), HBME1, galectin-3 and RET and thyroid transcription factor 1.[4–9] Although galectin-3 was initially shown to have utility in the differential diagnosis between benign and malignant thyroid lesions,[1011] recent studies suggest that it is not reliable.[12–14] Several studies have shown conflicting results regarding the usefulness of CK19 as a diagnostic marker in PTC.[13–20]
    [Show abstract] [Hide abstract] ABSTRACT: The diagnosis of papillary thyroid carcinoma (PTC) is based on nuclear features. These features may be present in focal areas in benign thyroid diseases and follicular adenoma (FA), leading to diagnostic difficulty. To evaluate the expression and pattern of the distribution of cytokeratin 19 (CK19) in PTC and compare its reactivity with other neoplastic and non-neoplastic conditions to assess its potential as a useful marker for PTC. Twenty two cases of papillary carcinoma (usual type, follicular and diffuse sclerosing variant), eight follicular adenomas, eight multinodular goiters (MNG) were collected for a period of two years and six months. Sections were taken from thyroidectomy specimens fixed in 10% buffered neutral formalin. Hematoxylin and eosin staining and immunohistochemical staining for CK19 were done using standard protocol. Results were semiquantitatively scored as follows: 1+ (<5% positively stained cells), 2+ (5-25%), 3+ (25-75%) and 4+ (>75%), and then analyzed. STATISTICAL ANALYSIS AND RESULTS: All 22 (100%) papillary carcinomas showed diffuse and strong (3+ and 4+) CK19 expression. Six out of eight (75%) FAs and four out of eight (50%) MNG were positive for CK19, but it was of weaker intensity (1+ and 2+) and focal in distribution. Focal CK19 staining may be found in benign disease, but diffuse and strong positivity is characteristic of PTC, which can be used in the diagnosis of PTC in lesions of equivocal morphological appearances.
    Full-text · Article · Apr 2012
    • In dogs, we were not able to find studies considering the regulation of Tg and TPO expression in healthy thyroid glands. In contrast, various studies aimed to find a proper molecular marker for thyroid carcinoma differentiation and reported that TTF-1 is a good marker of thyroid differentiation that can be used in conjunction with Tg (Katoh et al., 2000; Ramos-Vara et al., 2002; Aupperle et al., 2003). In this study we investigated the expression of thyroid specific genes in carcinoma and healthy thyroid glands.
    [Show abstract] [Hide abstract] ABSTRACT: Thyrotropin receptor (TSH-R), thyroglobulin (Tg), thyroperoxidase (TPO), thyroid specific transcription factor-1 (TTF-1), paired box 8 transcription factor (PAX-8), insulin like growth factor-1 (IGF-1) and estrogen receptor alpha (ERα) transcripts were determined by real-time PCR in follicular carcinoma and contralateral (CL) lobes, and healthy thyroid canine glands. Concentrations of TSH-R, PAX-8, and ERα mRNA were not different among groups; the carcinoma group had lower Tg and TPO mRNA than healthy and CL groups, while no differences were found between the two latter groups, suggesting that the carcinoma tissue presents an altered capacity to synthesize thyroid hormones. The transcription factor that promotes thyrocytes proliferation, TTF-1 as well as IGF-1, presented a greater mRNA expression in the CL group, suggesting that the CL lobe may function in a compensatory state.
    Full-text · Article · Jul 2011
    • The thyroid transcription factor 1 (TTF-1) is a protein belonging to the homeobox-containing gene family. This kind of protein plays very important roles in development, cell growth and differentiation processes[1]. TTF-1 is required for expression of the thyroid stimulating hormone receptor gene in thyroid cells and essential for the morphogenesis of the thyroid, lung and ventral forebrain[2]. One of the most important factors evaluating the biological aggressiveness of carcinoma is the cell proliferating activity.
    [Show abstract] [Hide abstract] ABSTRACT: We report the immunohistochemical diagnosis, including TTF-1 (thyroid transcription factor 1) and Ki-67, of a rare mixed thyroid neoplasm composed of minimally invasive well differentiated follicular areas and highly aggressive undifferentiated anaplastic areas. A 75 old female presented to our clinic with a rapidly growing neck mass. Considering the dynamics of the disease and the multiple challenges presented by the patient: advanced age, tumor size, history of a longstanding goiter we decided to transfer her to the department of surgery. The intraoperative findings were an enlarged right lobe with tracheal and surrounding tissues infiltration. Total thyroidectomy, radical neck lymph nodes dissection and tracheostomy were performed. The histopathological and immunohistochemical examination revealed a coexistent anaplastic and follicular thyroid carcinoma. The proliferation index Ki-67, a cell proliferation marker, was found to be significantly higher in the anaplastic areas (30 +/- 5%) in the comparison with the follicular areas (2 +/- 1%). The evaluation of the thyroid transcription factor 1 (TTF-1) expression revealed a correlation with the tumor cells aggressiveness accordingly to the cancer areas. After a radical surgery an external adjuvant radiation was applied. The patient is alive and more than five years after diagnosis she presented an increase of the serum thyroglobulin level suggesting, probably, a recurrence of the follicular form of the cancer. According to our survey we suggest that in thyroid cancers TTF-1 and Ki-67 could provides useful information on the differentiation activities of thyroid tumor cells and may be helpful to distinguish well differentiated and undifferentiated areas in a mixed thyroid cancer.
    Full-text · Article · Jan 2009
    • The thyroid transcription factor 1 (TTF-1) is a protein belonging to the homeobox-containing gene family. This kind of protein plays very important roles in development, cell growth and differentiation processes[1]. TTF-1 is required for expression of the thyroid stimulating hormone receptor gene in thyroid cells and essential for the morphogenesis of the thyroid, lung and ventral forebrain[2]. One of the most important factors evaluating the biological aggressiveness of carcinoma is the cell proliferating activity.
    [Show abstract] [Hide abstract] ABSTRACT: We report the immunohistochemical diagnosis, including TTF-1 (thyroid transcription factor 1) and Ki-67, of a rare mixed thyroid neoplasm composed of minimally invasive well differentiated follicular areas and highly aggressive undifferentiated anaplastic areas. A 75 old female presented to our clinic with a rapidly growing neck mass. Considering the dynamics of the disease and the multiple challenges presented by the patient: advanced age, tumor size, history of a longstanding goiter we decided to transfer her to the department of surgery. The intraoperative findings were an enlarged right lobe with tracheal and surrounding tissues infiltration. Total thyroidectomy, radical neck lymph nodes dissection and tracheostomy were performed. The histopathological and immunohistochemical examination revealed a coexistent anaplastic and follicular thyroid carcinoma. The proliferation index Ki-67, a cell proliferation marker, was found to be significantly higher in the anaplastic areas (30 +/- 5%) in the comparison with the follicular areas (2 +/- 1%). The evaluation of the thyroid transcription factor 1 (TTF-1) expression revealed a correlation with the tumor cells aggressiveness accordingly to the cancer areas. After a radical surgery an external adjuvant radiation was applied. The patient is alive and more than five years after diagnosis she presented an increase of the serum thyroglobulin level suggesting, probably, a recurrence of the follicular form of the cancer. According to our survey we suggest that in thyroid cancers TTF-1 and Ki-67 could provides useful information on the differentiation activities of thyroid tumor cells and may be helpful to distinguish well differentiated and undifferentiated areas in a mixed thyroid cancer.
    Full-text · Article · Feb 2008
    • All of the well-differentiated thyroid cancers such as the follicular and papillary carcinomas showed strong diffuse positivity, but in the poorly-differentiated carcinomas, the positivity reactivity was diminished and the most part of undifferentiated carcinomas showed a complete loss of immunoreactivity. These findings share similar characteristics to the studies with thyroglobulin, and this molecule can be interpreted as a marker of differentiation for thyroid carcinomas (23).
    [Show abstract] [Hide abstract] ABSTRACT: To examine the immunohistochemical alterations associated with the histological dedifferentiation of thyroid carcinomas, we performed staining for HBME-1, high molecular weight cytokeratin (HCK), CK 19, thyroid transcription factor-1 (TTF-1) and E-cadherin (E-CD) on 125 various types of thyroid carcinomas. The HBME-1 staining was strong and diffuse in follicular carcinoma (FC), papillary carcinoma (PC), and poorly differentiated carcinoma (PDC), while it was rare in undifferentiated carcinoma (UC) as well as in benign lesions. Strong, diffuse staining for CK19 and HCK was predominantly found in PC, and these markers were not much found in other carcinomas. TTF-1 uniformly stained the tumor cells of all cases of PC, FC and Hurthle cell carcinoma (HC) and 42% of the PDC, while there was only focal staining in one case of the UC. Compared to the strong, diffuse reactivity in the benign lesions, E-CD staining was noted in 67% of PC, 80% of FC, 83% of HC, 58% of PDC and none of the UC. These results suggest that HBME-1 may be a marker for well-differentiated carcinomas while CK19 and HCK are phenotypic markers for papillary carcinoma. The loss or reduced expression of TTF-1 and E-CD may be markers for dedifferentiation.
    Full-text · Article · Nov 2005
    • Studies demonstrated the positivities of galectin-3 in 100% of papillary carcinomas, 90 – 100% of follicular carcinomas, 50 – 80% of medullary carcinomas, 0 – 33% of follicular adenomas and 0 – 38% of nodular goitres (Beesley et al, 2002). Thyroid transcription factor-1 also expressed 100% of differentiated follicular tumours including follicular adenoma, follicular carcinoma and papillary carcinoma, but only 25% of undifferentiated carcinoma of the thyroid (Katoh et al, 2000). These markers were often expressed in benign lesions as well as malignant lesions, being due to the different mechanisms from LAR P-subunit.
    [Show abstract] [Hide abstract] ABSTRACT: Protein tyrosine phosphatase (PTPase) dephosphorylation and protein tyrosine kinase (PTKs) phosphorylation of key signal transduction proteins may be regulated by extracellular signals, making PTPases important in the regulation of cell proliferation. Leucocyte common antigen (LAR), a receptor-like PTPase, consists of E-subunit, containing the cell adhesion molecule-like receptor region, and P-subunit specific for a short segment of the extracellular region, the transmembrane peptide, and two cytoplasmic PTPase domains. We produced a monoclonal antibody against the LAR P-subunit for immunohistochemical screening of LAR expression in normal and tumourous tissues. Gliomas and gastric, colorectal, lung, breast and prostate cancers showed weak and relatively infrequent expression. Intense and diffuse expression, however, was detected in 95% (227 out of 239) of thyroid carcinomas, but only 12% (22 out of 128) of adenomas and no cases of benign thyroid disease were immunopositive. In contrast to broad staining in carcinomas, LAR expression in thyroid adenomas was often found in small focal or locally invasive areas. Western blot analysis similarly detected LAR P-subunit protein in thyroid carcinomas, but not in normal tissues. We believe this to be the first demonstration of LAR overexpression in thyroid carcinoma and may help to elucidate the role of PTPases in the development of malignancy.
    Article · May 2003
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