[Cancer Research 61, 19-22, January 1, 2001]
© 2001 American Association for Cancer Research
More Frequent ß-Catenin Exon 3 Mutations in Gallbladder Adenomas Than in Carcinomas Indicate Different Lineages1
Nobuyuki Yanagisawa2,
Tetuo Mikami,
Makoto Saegusa and
Isao Okayasu
Department of Pathology, Kitasato University School of Medicine, Kanagawa 228-8555, Japan
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ABSTRACT
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To clarify the contribution of ß-catenin, which is related to cell
adhesion and intranuclear transcription, to gallbladder carcinogenesis,
we investigated its expression using immunohistochemistry, and
ß-catenin exon 3 mutations by DNA
direct sequencing, in 18 gallbladder adenomas and 82 adenocarcinomas.
Membranous expression was significantly lower in moderately and poorly
differentiated than in well-differentiated adenocarcinoma cases
(P < 0.001). The gallbladder adenomas
showed significantly stronger expression in the cytoplasm and the
nucleus than carcinomas (P < 0.05 and
P < 0.001, respectively), and exon 3
mutations were observed in 62.5% (10 of 16) of adenomas, but only
4.8% (1 of 21) of carcinomas. With ß-catenin as a molecular marker,
the adenoma-carcinoma sequence can be considered to be a minor pathway
in gallbladder carcinogenesis.
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Introduction
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ß-Catenin links E-cadherin and 
-catenin, playing a role in
control of E-cadherin-adhesion functions. It is also involved in the
Wingless/Wnt signaling cascade, a transcription-activating pathway,
impacting on cell proliferation, polarity, and migration
(1)
. The ß-catenin molecule itself is metabolized by a
degradation system with
APC3
protein and GSK-3ß (2
, 3)
. Therefore, if APC
or ß-catenin mutations occur, this regulation
system may break down, resulting in ß-catenin nuclear accumulation
and activation of the transcription pathway. It is, thus, important to
investigate the ß-catenin status in considering carcinogenesis
related to APC. The most important site of
ß-catenin is considered to be encoded by exon
3, regarded as the GSK-3ß phosphorylation region (4)
. In
the present study, we investigated ß-catenin protein expression using
immunohistochemistry and ß-catenin gene exon 3
mutations by DNA direct sequencing to shed light on their significance
for gallbladder tumorigenesis.
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Materials and Methods
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Eighty-two cases of gallbladder carcinomas, 18 of gallbladder
adenomas (17 pyloric gland type and 1 intestinal type), and samples of
control mucosa (44 were background normal mucosa in carcinoma cases,
and 22 were from cholecystectomy specimens without gallbladder
carcinoma), surgically resected at Kitasato University Hospital,
Kitasato University East Hospital, Tokyo Metropolitan Komagome
Hospital, and Kudanzaka Hospital from 1986–2000, were investigated.
The WHO histological typing and the American Joint Committee on Cancer
pT classification for invasion were applied to all 82 carcinoma cases
(5, 6, 7)
. Ten percent of buffered formalin-fixed,
paraffin-embedded tissue samples were examined in this study.
Immunohistochemistry.
A streptavidin-biotin immunoperoxidase complex method using a
commercial kit (LSAB2/HRP; DAKO, Carpinteria, CA) was applied.
Four-micrometer thick histological sections were deparaffinized and
heated in citrate buffer solution (0.01 M, pH 6.0) for five 3-min
cycles using a microwave oven to retrieve antigens. Endogenous
peroxidase activity was inhibited by incubation with 0.3%
H2O2 in methanol for 30 min
at room temperature. After nonspecific reactions were blocked with 10%
normal porcine serum, the sections were incubated with an
anti-ß-catenin antibody (1:1000 diluted; Transduction Laboratories,
Lexington, KY) at 4°C overnight. After washing in 0.01 M PBS,
the slides were incubated with a biotinylated second antibody for 30
min and finally incubated with streptavidin-peroxidase for 30 min.
3–3'-Diaminobendizine (0.05%) was used as the final chromogen.
Nuclear counter staining was achieved with 0.3% methyl green solution.
The scoring method of Sinicrope et al. (8)
was
applied to evaluate both the immunohistochemical staining intensity and
the proportion of stained epithelial cells. Membranous, cytoplasmic,
and nuclear staining were independently considered. The staining
intensity was subclassified as: 1, weak; 2, moderate; and 3, strong.
The amount of positive cells was expressed as the percentage of the
total number of epithelial cells and assigned to one of five
categories: 0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4,
>75%. ISs for each case were calculated by multiplication of the
values for the two parameters. Moreover, in advanced carcinomas, both
intramucosal spreading and stromal invasive regions were examined to
evaluate the association between progression and ß-catenin protein
expression. Each lesion was examined and scored separately by two
pathologists (N. Y. and T. M.), and cases with discrepant scores were
discussed until unity was achieved.
Microdissection and DNA Direct Sequencing.
After deparaffinization, tumor cells were microdissected from six
serial 6-µm thick sections under a stereomicroscope, and cellular DNA
was extracted through proteinase K/phenol-chloroform treatment. A
148-bp fragment of exon 3 of the ß-catenin gene
was amplified in hemi-nested PCR reactions. With the outer PCR, 1 µl
of template DNA solution was amplified by Taq DNA polymerase (Takara,
Shiga, Japan) in a volume of 10 µl with the P1
(5'-ATTTGATGGAGTTG-GACATGG-3') and P2 (5'-TGTTCTTGAGTGAAGGACTGA-3')
primers. Then, 1 µl of this reaction solution was used for the second
PCR (in 20 µl total volume), with P1 and P3
(5'-TCTTCCTCAGGATGCCTT-3') primers. Both PCR procedures were performed
with 30 cycles of denaturation at 94°C for 0.5 min, annealing at
55°C for 0.5 min, and extension at 72°C for 0.5 min, with a
predenaturing time of 2 min and a final extension time of 5 min. As a
negative control, distilled water was used instead of template DNA for
each examination. After electrophoresis, the PCR products were purified
from agarose gels using a QIAquick gel extraction kit (Qiagen K.K.,
Tokyo, Japan), and sequenced using a dRhodamine dye terminator
cycle sequencing kit (Perkin-Elmer Applied Biosystems, Foster City,
CA), and analyzed with ABI PRISM 310 genetic analyzer (Perkin-Elmer),
according to the manufacturers’ protocols.
Statistical Analysis.
Data are mean ± SD values. Statistical analyses
were performed using the Fisher’s Protected Least Significant
Difference test as a post hoc test. Correlations among
immunoreactive scores were examined with the Pearson’s correlation
coefficient test. The frequency of exon 3 mutations was tested with the
2 test method. P < 0.05 and r >0.5 were regarded as statistically significant.
All statistical analyses were performed on a personal computer using
Statview software version 4.01 (Abacus Concepts, Berkeley, CA).
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Results
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The eighty-two gallbladder carcinomas [age, 42–85 years; average
age, 66.4 years; 56 female cases and 26 male cases] comprised 23
(28%) early (17 pTis and 6 pT1), and 59 (72%) advanced (37 pT2, 17
pT3, and 5 pT4) cases. Sixty-four (78%) were classified as
well-differentiated adenocarcinomas, and 18 (22%) as moderately and
poorly differentiated adenocarcinomas, according to the WHO
histological typing.
Immunohistochemistry.
The normal gallbladder mucosa showed strong membranous staining for
ß-catenin, whereas cytoplasmic staining was slight and nuclear
staining was completely absent (Fig. 1)
. Strong cytoplasmic and nuclear expression was shown by 14 of the
gallbladder adenomas (14 of 18, 78%; Fig. 1
). The carcinomas generally
showed weak membranous staining and slightly positive cytoplasm.
Diffuse nuclear staining was demonstrated in only two carcinoma cases
(2 of 82, 2.4%; Fig. 1
). In advanced carcinoma cases, the membranous
ISs for the stromal invasive regions were significantly lower than
those for intramucosal spreading regions (4.7 ± 2.4 and
6.1 ± 2.5, respectively; P < 0.001). The membranous ISs were significantly lower in moderately and
poorly differentiated than in well-differentiated adenocarcinoma cases
(Fig. 2)
. The cytoplasmic and the nuclear ISs in adenomas were higher than in
normal mucosa and carcinomas with significance (Fig. 2)
. There was a
positive correlation between cytoplasmic and nuclear ISs in adenomas
(r = 0.771, P < 0.001).
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Fig. 1. ß-Catenin immunostaining in normal gallbladder mucosa
(A), a gallbladder adenoma (B), a
well-differentiated adenocarcinoma (C), and a poorly
differentiated adenocarcinoma (D). Note clear membranous
staining of the normal mucosa, but strong ß-catenin protein
expression in the nuclei and cytoplasm in the adenoma. (Original
magnification, x400).
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Fig. 2. ISs for ß-catenin among four gallbladder tissue
categories. N, normal gallbladder epithelia;
A, adenoma; W, well-differentiated
adenocarcinoma; MP, moderately and poorly differentiated
adenocarcinoma. *, P < 0.001; #,
P < 0.01; $, P < 0.05.
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DNA Direct Sequencing.
DNA direct sequencing was performed for all cases in which nuclear
expression was detected and also some immuno-negative cases. However,
PCR was not successful with 2 adenomas and 7 carcinomas, so that data
were available for only 16 adenomas and 21 carcinomas. The results are
summarized in Table 1
. Exon 3 point mutations were observed in 62.5% (10 of 16) of adenomas,
but 4.8% (1 of 21) of carcinomas (Table 1
and Fig. 3
), the difference being statistically significant
(P < 0.001). The mutation-positive carcinoma
case (C18) histologically consisted of both well-differentiated and
poorly differentiated adenocarcinoma areas without transition. No
adenomatous component was observed. These areas were examined
separately, and diffuse nuclear ß-catenin accumulation and exon 3
point mutation were detected only in the latter area.
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Fig. 3. Sequencing chromatograms demonstrating
ß-catenin mutations in gallbladder
adenomas. A, codon 37 point mutation (TCT to TTT) in
case A08. B and D, no mutations in
carcinoma cases C05 and C06. C, codon 33 point mutation
(TCT to TGT) in case A02.
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Discussion
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In this study, loss of membranous ß-catenin expression was
observed in gallbladder tumors, particularly in poorly differentiated
adenocarcinoma cases, indicating the loss of adhesional function of
E-cadherin system, being in agreement with previous reports such as for
colonic cancer (9)
. On the other hand, prominent nuclear
and cytoplasmic expression was clearly shown in gallbladder adenomas
(Figs. 1
and 2)
. It is considered that ß-catenin migrating into the
nucleus binds to Tcf/Lef family and activates transcription. Recently,
defined target genes of the ß-catenin/Tcf system include
c-myc, cyclin D1, and matrilysin
(10, 11, 12)
. Taking into consideration the fact that cell
turnover in gallbladder adenomas is lower than in gallbladder
carcinomas (13)
, these results indicate that the
ß-catenin migration to the nucleus is not always related to the
transcriptional activation of proliferation-related genes. Similarly,
Brabletz et al. (10)
reported that nuclear
ß-catenin is not correlated with proliferative activity in colorectal
adenomas, suggesting the existence of an unknown regulatory mechanism.
In the present study, we analyzed mutations in exon 3 of the
ß-catenin gene, because this is where most with
physiological significance occurs, corresponding to the GSK-3ß
phosphorylation region (4)
. In addition, the codons for
serines or threonins of the exon 3 (such as codon 33, 37, 41, and 45)
are well known as hot spots of ß-catenin
mutations in human cancers (14)
. In the present study, we
found 9 of 11 ß-catenin missense mutations to
involve serines or threonins, suggesting ß-catenin stabilization and
accumulation. Considering the correlation between immunostaining and
DNA direct sequencing, tumor cells in the exon 3 mutation-positive
cases always expressed nuclear ß-catenin protein
immunohistochemically. However, exon 3 mutations were not detected in
all nuclear ß-catenin-positive cases. It has been reported that the
incidence of APC mutations is low (range, 0–22%) in
gallbladder tumors, including adenomas (15, 16, 17)
. Thus, the
reason for the accumulation in the exceptional cases might be due to
some other alterations in ß-catenin metabolism.
To date, two major pathways, de novo development and
adenoma-carcinoma sequence, have been proposed to account for
gallbladder carcinogenesis. Kozuka et al. (18)
considered that the adenoma-carcinoma sequence is the usual route for
the development of invasive carcinomas, based on the observation that
adenomatous lesions often coexist with invasive carcinomas. However,
Albores-Saavedra et al. (19)
disagreed with
this interpretation, because they considered adenomatous lesions in
adenocarcinomas as malignant tumors with minimal nuclear atypia.
Subsequently, Wee et al. (20)
reported p53
overexpression to be detectable immunohistochemically in 92% of
gallbladder carcinomas, 86% of carcinomas in situ, and even
26% of dysplasia cases, but not in gallbladder adenomas, suggesting
that dysplasia and carcinoma in situ are the precursor
lesions for invasive carcinomas. Several authors have reported gene
mutations in gallbladder tumors, but their results were not
sufficiently comprehensive for all to allow firm conclusions regarding
carcinogenesis (15, 16, 17)
. For example, Itoi et
al. (15)
found K-ras mutations in
gallbladder carcinomas (4 of 40, 10%), but not in adenomas or
carcinomas in adenomas (0 of 16 and 0 of 6, respectively), concluding
that their results indicated an adenoma-carcinoma sequence. However,
their data can be interpreted as supporting either of the two theories.
Our results using ß-catenin as a molecular marker indicate that the
gallbladder adenomas often feature ß-catenin abnormalities, in this
sense clearly differing from carcinomas. Regarding tumorigenesis, there
may not be a direct histogenetic relation between gallbladder adenoma
and carcinoma, as is often suggested for colorectal neoplasms.
In conclusion, this is the first study that nuclear ß-catenin
expression and exon 3 mutations are more frequent in gallbladder
adenomas than in carcinomas, indicating that the adenoma-carcinoma
sequence might be a minor pathway in gallbladder tumorigenesis.
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ACKNOWLEDGMENTS
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We thank Drs. M. Koike and T. Kayano for providing histological
materials.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by the Parents’ Association
Grant of Kitasato University School of Medicine and by grants from
Kitasato University Graduate School of Medical Sciences (9901 and
2020). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Kitasato University, School of
Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan. Phone:
81-42-778-8996; Fax: 81-42-778-8441.
3 The abbreviations used are: APC, adenomatous
polyposis coli; GSK-3ß, glycogen synthase kinase-3ß; IS,
immunoreactive score.
Received 9/21/00.
Accepted 11/14/00.
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ABSTRACT
Introduction
