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Original Studies |
-, ß-, and 
-Catenins in Differentiated Thyroid Carcinoma
Department of Pathology and Forensic Medicine (J.B., K.K., J.K., V.-M.K.), University of Kuopio and Kuopio University Hospital, FIN-70211 Kuopio; and Departments of Medicine and Clinical Nutrition (L.N.), Otorhinolaryngology , Oral and Maxillofacial Unit (J.K.), and Surgery (M.E., E.A.), Kuopio University Hospital, FIN-70211 Kuopio; and Department of Pathology (S.H.), Satakunta Central Hospital, FIN-28500 Pori, Finland
Address correspondence and requests for reprints to: Veli-Matti Kosma, M.D., Ph.D., Department of Pathology and Forensic Medicine, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: VeliMatti.Kosma{at}uku.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, and 
) are a group of intracellular cell adhesion
molecules that unite cytoskeleton with extracellular adhesion system.
Abnormal expression of these molecules may have prognostic relevance in
various carcinomas, including differentiated thyroid carcinoma (DTC).
We have, therefore, evaluated the prognostic value of -, ß-, and
-catenins along with traditional risk factors in 206 consecutive DTC
patients by immunohistochemistry.
Papillary carcinomas showed normal staining pattern for -, ß-, and
-catenins in 124 (60%), 136 (67%), and 94 (46%) cases,
respectively. Follicular carcinomas expressed -, ß-, and
-catenins normally in 16 (48%), 18 (55%), and 8 (32%) cases,
respectively. Follicular type of tumor showed more often reduced
staining for all catenins than papillary carcinoma
(P = 0.009, P = 0.004, and
P = 0.002, respectively). Age (>60 yr) and
pTNM-stage were related to reduced - and ß-catenin expression
levels (P = 0.027 and P =
0.026, respectively) and larger size of the tumor to reduced ß- and
-catenin expressions (P = 0.039 and
P = 0.007, respectively). Nodal metastases at the
time of primary treatment related to reduced -catenin expression and
distal metastases to reduced ß- and -catenin staining signals
(P = 0.022, P = 0.014, and
P = 0.039, respectively). Reduced -catenin
associated with tumor recurrence (P = 0.002) and
reduced ß-catenin with cancer-related mortality
(P = 0.005). The multivariate analysis for
recurrence-free survival showed that -catenin and serum
thyroglobulin level 1 yr after primary treatment were prognostic of
recurrent disease (hazards ratio, 3.42, P = 0.022;
and hazards ratio, 10.03, P = 0.0001). In addition,
-catenin retained its prognostic significance in low-stage patients
(P = 0.0151). We propose that the evaluation of
-catenin expression by immunohistochemistry in DTC patients has
prognostic value in addition to that obtained by traditional prognostic
factors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and -catenins (100 kD, 94 kD, and
80 kD, respectively). -Catenin shows homology to vinculin
and links E-cadherin to the actin-cytoskeleton, either directly or via
-actinin (1). ß-catenin is a multifunctional protein
that is both an integral component of adherens junctions and homologous
to a pivotal component of a wingless (wnt) signal transduction pathway
in Drosophila (2). -Catenin (plakoglobin) is
a major component of desmosomal plaque proteins (3). ß-
and -catenins also link -catenin to E-cadherin in a mutually
exclusive fashion. In addition, catenins are involved in cell
proliferation, differentiation, and migration (4). The role of catenins in cell adhesion seems to be very important, because changes in their expression or function lead to disturbances also in E-cadherin-mediated extracellular adhesion (5). It has been suggested that immunohistochemical detection of catenins more directly reflects loss of cell-cell adhesion than that of E-cadherin alone (6). Indeed, catenins have been found to have prognostic significance in several cancers (7, 8, 9, 10).
Differentiated thyroid carcinoma (DTC) is a rather common endocrine malignancy with favorable prognosis, in general. However, because some patients do have poor outcome, new biological and clinical predictive and prognostic markers are needed to pinpoint the individuals who need more aggressive treatment (11, 12).
The expression of catenins has been rarely studied in DTC (13, 14, 15), and their prognostic role is still unknown. However, the prognostic relevance of E-cadherin has previously been reported (16, 17). Therefore, we have evaluated the prognostic value of catenins in a large series of consecutive DTC patients collected from a defined area taking also into account the effects of traditional prognostic factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Representative samples of primary tumors were obtained from 206 consecutive patients with DTC who were treated and followed-up between 1976 and 1995 in the eastern part of Finland (Kuopio University Hospital, Pohjois-Karjala, and Savonlinna Central Hospitals, the only hospitals in the area treating DTC). The material consisted of 173 papillary and 33 follicular carcinomas. Evidence of tumor recurrence was based on either ultrasound, radioiodine (RaI), or magnetic resonance image findings; recurrence was defined as a new evidence of local disease or distant metastases occurring more than 6 months after the primary operation (18). If the serum thyroglobulin was elevated, the low-dose RaI scan was performed, and, in case of evident RaI accumulation, ablation therapy was given (17 cases). Ultrasound imaging with a fine-needle biopsy was made in approximately half of the patients. If the RaI accumulation was detected in the mediastinum or abdominal region, computed tomography or magnetic resonance imaging was carried out to determine the exact localization and size of the tumor before ablative therapy. Thus, none of the recurrences was based solely on elevated serum thyroglobulin. Tumor stage was assessed retrospectively according to the tumor, node, metastasis (pTNM) classification system (UICC, 1987) (19). The formation of the study population and the clinical prognostic factors have been reported elsewhere (11). The study plan was approved by the Ethics Committee of the University of Kuopio.
Treatment and follow-up
All the patients in this study underwent thyroidectomy. The type of operation was total or near total thyroidectomy in 180 (87%) patients, palliative resection of the thyroid gland in 14 (7%) patients, and 9 (5%) patients whose quality of operation could not be assessed from the patient files. Hemithyroidectomy was performed on three (1%) patients with occult DTC. The standard procedure was always to perform as complete thyroidectomy as possible. After surgical operation, the low-dose RaI scan (74 MBq) was performed. The ablation therapy was given if there was an accumulation of RaI (range, 1850–5550 MBq; median, 3700 MBq). The whole-body scan was performed a few days after RaI ablation, and the uptake was recorded. In our study group, 174 (85%) patients received the RaI ablation therapy. Fifty-four patients received ablation therapy twice or more. The mean follow-up time of the patients was 83.6 months (median, 60; range, 1–237) and the mean age of the patients 48.0 yr (range 8–92 yr).
Serum TSH and thyroglobulin
From 1976–1981 serum TSH was determined using the TSH RIA kit (Amersham Pharmacia Biotech, Buckinhamshire, England), from 1981–1986 using the TSH(125I) RIA kit (Farmos Diagnostica, Turku, Finland), and thereafter the Spectria TSH immunoradiometric assay 125I kit (Farmos Diagnostica). The suppression of serum TSH 1 yr after operation was defined as 1.0 mU/L or less and/or no response to TRH during the years 1976–1986 and as serum TSH 0.1 mU/L or less from 1986–1995 (11).
Serum thyroglobulin was assessed 1 yr after operation. Serum thyroglobulin values were determined in a central laboratory (Oy Medix Ab, Kauniainen, Finland) using a RIA kit (HTGK2; Biosorin, Saluggia, Italy). The cut-off level was 3 µg/L, and levels over that were considered elevated. Thyroglobulin was measured during the follow-up on Lthyroxine treatment. All thyroglobulin measurements were checked, and four patients with interfering serum antibodies were excluded from the analyses concerning serum thyroglobulin.
Histological classification
Two experienced histopathologists (V.-M.K. and S.H.) reexamined hematoxylin and eosin-stained sections simultaneously, being unaware of the clinical data. Histopathological diagnosis was performed by reviewing one to four original sections of the primary tumor. Papillary carcinomas were subdivided into common type, follicular variant, or occult carcinomas. Invasion type of follicular carcinoma was evaluated as minimal or widely invasive. Minimally invasive carcinomas were encapsulated and showed unequivocal vascular or full-thickness capsular invasion. Widely invasive carcinomas lacked complete encapsulation and showed widespread infiltration of adjacent thyroid tissue and/or cancer extension into surrounding tissue. Three follicular adenomas were also reevaluated in addition to metastases (n = 12) of the primary DTCs included in the current material.
Immunohistochemical staining for -, ß-, and -catenins
Sections were deparaffinized, rehydrated, and washed twice for 5
min with PBS (pH = 7.2). They were then heated in a microwave oven
for 3 x 5 cycles in Tris-Hcl buffer (pH = 9.2) (-catenin)
or in citrate buffer (pH = 6.0) (ß- and -catenins).
Endogenous peroxidase was blocked by 5% hydrogen peroxide for 5 min,
followed by washing for 5 min with PBS. The sections were preincubated
in 1.5% normal horse serum (Vectastain; Vector Laboratories, Inc., Burlingame, CA) for 30 min at room
temperature and then incubated overnight at 4 C with the primary
antibody for - or -catenin (Transduction Laboratories, Lexington,
UK; 1:100 dilution) and for ß-catenin (Transduction Laboratories;
1:1000 dilution) in PBS with 1% BSA, washed with PBS, and incubated
for 35 min with a biotinylated secondary antibody
(Vectastain; Vector Laboratories, Inc.).
Next, the sections were washed in PBS, incubated for 45 min in
preformed avidin-biotin peroxidase complex, washed twice with PBS, and
developed for 5 min with 0.05% 3,3-diaminobenzidine
tetrahydrochloride (Sigma, St. Louis, MO) and
H2O2. Finally, the sections
were dehydrated, cleared, and mounted with DePex (BDH, Poole, UK). In
each staining batch normal follicular epithelium was used as a positive
control. In the negative controls the primary antibody was omitted.
Evaluation of -, ß-, and -catenin stainings
Immunostainings were evaluated by three independent observers (J.B., K.K., and V-M.K.) without knowledge of any clinicopathological data. Disagreement in the assessment of staining was found in 9% of the slides examined, and consensus was reached on further review. The fraction of cancer cells with membranous staining was primarily analyzed on a continuous scale. For statistical analyses, tumors having 90% or greater membranous staining pattern were considered normal, whereas the others with reduced staining (<90%) were considered abnormal (20). In tumors having an abnormal catenin expression, discontinuous or absent membranous staining was seen, with or without cytoplasmic staining. Nuclear staining of ß-catenin was also taken into account.
Statistical analysis
The SPSS, Inc. program package was used for
statistical analyses. The associations between catenins and different
clinicopathological factors were tested by Mantel-Haenzel or Pearson
2 tests. Correlations between catenin
stainings were tested by Pearson correlation coefficient. Kaplan-Meier
analysis and log-rank test were used to clarify the significant
univariate predictors. The Cox regression model was used for
multivariate analyses, and P value less than 0.05 was
considered statistically significant. The recurrence-free survival
(RFS) was defined as the time from surgical resection to the time of
DTC recurrence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and -catenins and association with
clinicopathological factors
In normal thyroid follicular epithelium in close vicinity of the
neoplastic tissue, strong immunoreactivity for -, ß-, and
-catenins was seen on cell membranes. All three follicular adenomas
analyzed showed also high immunoreactivity for -, ß-, and
-catenins.
In carcinomas the staining was concentrated on cell membranes of the
neoplastic follicular epithelium (Fig. 1
). No nuclear staining of
ß-catenin was noted. Papillary carcinomas showed normal staining
pattern for -, ß-, and -catenins in 124 (60%), 137 (67%), and
94 (46%) cases, respectively. Follicular carcinomas expressed -,
ß-, and -catenins normally in 16 (48%), 18 (55%), and 8 (32%)
cases, respectively (Table 1). Staining
percentages of -, ß-, and -catenin expressions correlated with
each other (r = 0.277–0.472, P = 0.0001 for
all).
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-catenin expression
(P = 0.027), whereas gender of the patient did not have
any relation to catenin expressions. pTNM-stage associated inversely
with ß-catenin expression (P = 0.026) (Table 1
). The
size of the tumor was related to ß- and -catenins
(P = 0.039, P = 0.007). The presence of
nodal metastases at the time of primary treatment was related
significantly to reduced -catenin staining signal (P
= 0.022), whereas the presence of distant metastases associated with
reduced ß- and -catenin expression levels (P =
0.014, P = 0.039). Recurrence of DTC occurred in 30
patients. -Catenin associated significantly with tumor recurrence
(P = 0.002). ß-catenin was related to cancer-related
mortality (P = 0.005) (Table 1).
Of the 12 metastases included in this material, 9 were located in
regional lymph nodes and 3 elsewhere. Eight of the metastases (67%)
showed normal -catenin staining pattern and nine (75%) and five
(42%) expressed ß- and -catenins normally, respectively.
Concordance of -, ß-, and -catenin stainings between primary
tumor and metastasis was reached in eight (67%), eight (67%), and
nine (75%) cases, respectively.
Survival analyses
-Catenin was the only catenin that associated
significantly with recurrent DTC in univariate log-rank test
(P = 0.001). The multivariate analysis for RFS included
traditional prognostic factors that previously associated significantly
with tumor recurrence (11) [i.e. age >60 yr,
type of tumor (papillary vs. follicular), pTNM stage, TSH
suppression, and thyroglobulin level]. After taking into account these
markers, the reduced -catenin expression showed a 3.42-fold risk and
high serum thyroglobulin level a 10.03-fold risk for recurrent DTC,
respectively (P = 0.022 and P = 0.001,
respectively) (Table 2). We further
assessed the effect of -catenin expression to RFS solely in stage
I–II tumors by using Kaplan-Meier analysis and log-rank test.
Interestingly, -catenin retained its prognostic significance also in
low-stage patients (P = 0.0151).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin yields prognostic
information independent of traditional clinical prognostic factors in
DTC.
Strong E-cadherin-mediated extracellular adhesion requires connection
with cellular cytoskeleton, and, therefore, normal functions of -,
ß-, and -catenins are essential for cell-cell adhesions. Abnormal
expression of these molecules leads eventually to divergent functions
of the cells. -Catenin and/or E-cadherin expression and functional
status may be altered by methylation-associated silencing of E-cadherin
(29), deletions in -catenin gene, altered tyrosine
phosphorylation, or hormonal factors (30). -Catenin is
possibly a more sensitive indicator of impaired E-cadherin-mediated
extracellular adhesion than E-cadherin expression itself
(31). E-cadherin seems to be under the control of the
TSH-cAMP-dependent pathway (32), at least in
vitro. E-cadherin and -catenin are frequently coexpressed
(33), whereas it is unknown whether TSH has any impact on
catenin expression. Interestingly, after introducing -catenin into
the multivariate analysis, the previously shown independent role of TSH
suppression (11) was reduced to a nonsignificant level in
the current study. One can speculate that the decreased -catenin
expression impairs the response to
L-T4-mediated suppression of TSH.
The expression patterns of -catenin have been studied rather rarely
in DTC and its metastases. In the study by Huang et al.
(14), most of the follicular carcinomas expressed
-catenin normally at cell-cell junctions, which contradicts our
results. This discrepancy is possibly explained by differences in the
study material and methods used. In our material, 67% (8 of 12) of the
metastatic tissues showed normal membranous staining for -catenin
that was almost the identical staining percentage as that of the
primary tumors. Due to the lack of other reports in DTC, no comparisons
can be made. In the current material, numerous lymph node metastases
were seen in tumors with reduced -catenin expression, a finding that
has also been reported in other cancers (20, 31, 34). It
is tempting to speculate that disturbed -catenin expression impairs
E-cadherin-mediated cell adhesion, subsequently leading to progressive
disease with regional lymph node and distant recurrences of DTC.
Earlier, reduced -catenin expression has been found to have an
independent prognostic value in colon, prostate, renal, and ovarian
carcinomas (7, 8, 10, 35), supporting our results. To the
best of our knowledge, the prognostic value of -catenin has not been
analyzed previously in DTC.
Abnormal ß-catenin expression on the plasmamembrane of the neoplastic
tissue associated with occurrence of distant metastases and
cancer-related mortality in the current series. This may be because
abnormal ß-catenin expression associated particularly with more
progressed follicular type of tumors that in some cases developed
distant metastases with fatal consequences. In fact, similar
ß-catenin expression patterns have been reported previously in
follicular carcinomas (14). In addition, ß-catenin has
been frequently found to be mutated and translocated in the nucleus of
the anaplastic thyroid carcinomas (36). However, in DTCs
we did not find any tumor specimen with nuclear localization of this
protein. These findings support the hypothesis that in the early stages
of a malignancy down-regulation of -catenin may be significant for
disruption of E-cadherin-mediated cell-cell adhesion and that
down-regulation of ß-catenin occurs later in the transformation
process.
-Catenin modulates the activity of desmosomal cadherins
(30). The reduction of this protein inevitably affects the
integrity of desmosomal adhesion. However, functional capabilities of
-catenin are still largely unknown. It possibly has different roles
and regulation pathways than ß-catenin (37). In the
current material, -catenin showed more often abnormal expression
pattern compared with that of - or ß-catenins, confirming previous
findings (13, 14). The rather similar clinicopathological
associations of ß- and -catenins seemed associated with factors
that are related to poor prognosis although ß- and -catenins did
not have any statistical prognostic significance. Previously, bladder
and lung carcinomas with unfavorable outcome showed less -catenin on
their cell membranes (37, 38). In addition, -catenin
expression was reduced in anaplastic thyroid carcinoma cell lines as
compared with its expression in nonneoplastic thyrocytes
(39). At present, the role of -catenin in thyroid
carcinoma, however, remains unclear.
Taken together, as a novel finding, we have shown that even a slight
reduction of -catenin expression has prognostic significance in DTC.
Importantly, abnormal -catenin expression can predict DTC recurrence
independent of traditional prognostic factors. Therefore,
immunohistochemical detection of -catenin can be a useful tool in
the assessment of DTC prognosis, and it can help us to recognize those
DTC patients who require more aggressive treatment.
Received May 11, 2000.
Revised August 1, 2000.
Accepted August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin with the cadherin/catenin cell-cell
adhesion complex via -catenin. J Cell Biol. 130:67–77.-Catenin expression has prognostic value in local and
locally advanced prostate cancer. Br J Cancer. 80:477–482.[CrossRef][Medline]
-catenin, collagen IV, and Ki-67 expression in
epithelial ovarian cancer. J Clin Oncol. 16:2591–2600.[Abstract]
-catenin is associated
with aggressive features in laryngeal squamous-cell carcinoma. Int J
Cancer. 79:546–550.[CrossRef][Medline]
catenin is associated with poor
prognosis in colorectal carcinoma. J Clin Pathol. 52:10–16.[Abstract]
-catenin, ß-catenin and plakoglobin in
esophageal carcinomas and its prognostic significance:
immunohistochemical analysis of 96 lesions. Oncology. 54:158–165.[Medline]
-catenin expression in human esophageal cancer. Cancer Res. 54:291–296.-catenin expression
in prostate cancer: correlation with patient survival. Cancer Res. 57:3189–3193.-catenin expression in colorectal
cancer correlates with invasiveness, metastatic potential, and
survival. J Surg Oncol. 68:92–99.[CrossRef][Medline]
-catenin is associated with poor prognosis
of patients with localized renal cell carcinoma. Int J Cancer. 74:523–528.[CrossRef][Medline]
-catenin expression correlates with poor
survival in patients with bladder cancer. J Urol. 160:1889–1893.[CrossRef][Medline]
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