[CANCER RESEARCH 61, 8401– 8404, December 1, 2001]
Advances in Brief
Frequent ␤-Catenin Mutation and Cytoplasmic/Nuclear Accumulation in Pancreatic
Solid-Pseudopapillary Neoplasm
Yukichi Tanaka,1 Keisuke Kato, Kenji Notohara, Hiroshi Hojo, Rieko Ijiri, Tetsumi Miyake, Noriyuki Nagahara,
Fumiakai Sasaki, Norihiko Kitagawa, Yukio Nakatani, and Yasutsugu Kobayashi
Division of Pathology, Kanagawa Children’s Medical Center, Yokohama 232 [Y. T., K. K., R. I., T. M., Y. N.]; Department of Pathology, Okayama University, Okayama [K. N.];
Department of Pathology, Fukushima Prefectural University, Fukushima [H. H.]; Department of Environmental Medicine, Nihon Medical School, Nihon [N. N.]; Department of
Pediatric Surgery, Hokkaido University, Hokkaido [F. S., N. K.]; and Division of Pathology, Osaka City General Medical Center, Osaka [Y. K.], Japan
Abstract
Significance of Wnt signaling with ␤-catenin mutations on solid-pseu-
dopapillary neoplasm (SPN) of the pancreas was studied by immunohis-
tochemistry and molecular analysis. On immunohistochemistry, all 18
SPNs tested showed diffuse cytoplasmic/nuclear positivity for ␤-catenin.
Upon direct DNA sequencing of exon 3 of the ␤-catenin gene, 15 (83%) of
the 18 SPNs showed 1-bp missense mutation in codons 32 (5 cases), 33 (3
cases), 34 (3 cases), 37 (3 cases), and 41 (1 case). Immunoreactivity for
cyclin D1, one of the intranuclear targets of ␤-catenin complexes, was
found in tumor cells of more than half the tumor cells of all of the 18 SPNs.
The present study strongly suggested a significant role of Wnt signaling,
mostly associated with ␤-catenin mutations in the tumorigenesis of SPN.
Introduction
SPN2 of the pancreas is a rare and distinct tumor classified in
tumors of the exocrine pancreas in the current WHO classification (1).
SPN is known for its indolent biological behavior and predilection in
young women (1–3). The cell of origin in SPN remains uncertain
(1–3). Although several studies have referred to molecular/cytoge-
netic characters of pancreatic tumors (4 – 6), few reports have been
concerned with molecular changes of SPNs. Molecular changes often
detected in pancreatic DCs, such as alterations of p53 and K-ras, have
not been detected in SPNs (3, 6 – 8).
The protein ␤-catenin was originally identified as a submembra-
nous element of the cadherin-mediated cell-cell adhesion complex.
␤-Catenin acts as a downstream transcriptional activator of Wnt
signaling, by accumulating in the nucleus and forming complexes
with the DNA-binding proteins such as Tcf and Lef-1 (9). Recent
studies have identified the target genes of these transcriptional factors,
including one of the cell cycle regulators, cyclin D1 (10). During the
systemic immunohistochemical study of ␤-catenin in pediatric tu-
mors, we found a consistent cytoplasmic/nuclear reactivity for ␤-cate-
nin in SPNs. This novel finding prompted us to perform a large-scale
study of ␤-catenin immunoreactivity and mutation analysis of the
␤-catenin gene in SPNs.
Materials and Methods
Case and Tissue Selections. Age and sex of the 18 patients with SPN is
shown in Table 1. Cases 12–14 had been included in the previous immuno-
histochemical study (11). The ages ranged from 11 to 61 years (mean, 33
years; 13 females and 5 males). All tumors were completely resected and have
Received 7/30/01; accepted 10/19/01.
The costs of publication of this article were defrayed in part by the payment of page
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1
To whom requests for reprints should be addressed, at Department of Pathology,
Kanagawa Children’s Medical Center, Mutsukawa 2-138-4, Miami-ku, Yokohama 232,
Japan.
2
The abbreviations used are: SPN, solid-pseudopapillary neoplasm; DC, ductal car-
cinoma; FAP, familial adenomatous polyposis; NET, neuroendocrine tumor.
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not recurred or metastasized without additional therapy. No case had a history
of FAP. The tumor sample was fixed in 10% buffered formalin, processed
routinely, and embedded in paraffin. Four-␮m sections were stained with
H&E. Histopathological diagnosis was confirmed by the authors, including at
least four pathologists.
Immunohistochemistry. Immunohistochemical studies were performed
on paraffin sections, which were deparaffinized, hydrated, and immersed in
0.3% hydrogen peroxide in methanol for 30 min to block the endogenous
peroxidase activity. For the detection of ␤-catenin and cyclin D1, mono-
clonal antibodies against each protein (clone 14, Transduction Laborato-
ries, Lexington, KY; and clone P2D11F11, Novocastra, Newcastle, United
Kingdom, respectively) were applied at a dilution of 1:100 and 1:200,
respectively, followed by peroxidase-conjugated antimouse immunoglob-
ulin antibody (MBL, Nagoya, Japan; 1:50). Antigen retrieval was per-
formed by microwave treatment for two 10-min cycles with 180 W of
power in 10 mM citrate buffer (pH 6.0) and in 1 mM EDTA (pH 8.0) for
␤-catenin and cyclin D1, respectively. Final visualization was carried out
by development in PBS (pH 7.6) containing 20 mg/100 ml of diaminoben-
zidine tetrachloride and 0.1% H2O2 for 10 min. Sections incubated with
PBS instead of the primary antibodies served as negative controls. The
sections were counterstained by Mayer’s hematoxylin.
Genomic DNA Preparation. Sufficient numbers of formalin-fixed, paraf-
fin-embedded sections were available in 17 cases (all cases other than case 9).
Tumor areas were dissected from 5 to 10 4-␮m sections, which were depar-
affinized in xylene, rehydrated in ethanol, and air dried. Adjacent nontumorous
tissue was found in 8 cases and was separately dissected. Fresh frozen tumor
samples were available in 2 cases (cases 6 and 9). DNA extraction was
performed according to the methods described previously (12, 13). The DNA
was dissolved in 20 ␮l of 10 mM Tris-HCl (pH 8.0).
Mutational Analysis. Genomic DNAs from the sections were amplified by
seminested PCR, using 5⬘ BCTN (5⬘-TGATTTGATGGAGTTGGACATG-3⬘)
and 3⬘ BCTN (5⬘-CTCTTCCTCAGGATTGCCTTTA-3⬘) primers for the first
PCR and using the same 5⬘ BCTN and 3⬘ (2nd) BCTN (5⬘-TCAGGATTGC-
CTTTACCACTCA-3⬘) primers for the second PCR. These primers were
designed to amplify a 144-bp DNA fragment of ␤-catenin gene exon 3,
containing the sequence for phosphorylation by glycogen synthase kinase-3␤.
The thermal cycler (Perkin-Elmer Cetus 480; Applied Biosystems, Foster City,
CA) was programmed as follows: initial denaturation at 94°C for 10 min and
35 amplification cycles for the first PCR; and initial denaturation at 94°C for
10 min and 30 amplification cycles for the second PCR. One amplification
cycle comprised denaturation at 94°C for 30 s, annealing at 66°C for 30 s, and
elongation at 72°C for 1 min. Because DNAs from 4 tumors (cases 13, 14, 16,
and 17) were not amplified by this procedure, additional experiments were
done to amplify shorter fragments (95 bp) using 5⬘ BCTN (short) (5⬘-TGT-
TAGTCACTGGCAGCAACAG-3⬘) and 3⬘ BCTN (short) (5⬘-CCTCAGGAT-
TGCCTTTACCACT-3⬘) primers for the first PCR and, for the second PCR,
the same 5⬘ BCTN (short) and 3⬘ BCTN (2nd short) (5⬘-CAGGATTGCCTT-
TACCACTCA-3⬘) primers under the same PCR schedule. Genomic DNAs
from frozen samples were amplified using 5⬘ BCTN exon 3 (5⬘-CTGATTT-
GATGGAGTTGGACAT-3⬘) and 3⬘ BCTN exon 3 (5⬘-CTTGTTCTTGAGT-
GAAGGACTGA-3⬘) primers to amplify a 222-bp fragment. The PCR was
carried out according to the same procedure of the first PCR for the paraffin
sections. The PCR products were confirmed by agarose gel electrophoresis,
purified using GFX Gel Band Purification kit (Amersham Pharmacia), and
 CATENIN MUTATION IN SOLID-PSEUDOPAPILLARY NEOPLASM

Table 1 Clinical abstract and mutations in the ␤-catenin gene in the solid- gene in any of the 78 DCs and the 33 NETs nor in the 14 pancreatic
pseudopapillary neoplasms
cancer cell lines analyzed, and intracellular ␤-catenin accumulation
␤-Catenin gene mutation was not identified in any of the 40 DCs examined. They concluded
Amino acid that Wnt signaling linked with ␤-catenin mutation does not play a
Casea Age Sex Mutated Codon Base change substitution significant role in tumorigenesis of those tumors (14). No attention,
1 15 F 33 TCT 3 CCT Ser 3 Pro however, has been paid to the relationship between ␤-catenin and
2 44 M 33 TCT 3 TGT Ser 3 Cys SPNs.
3 20 F — —
4 45 M 32 GAC 3 GGC Asp 3 Gly We identified cytoplasmic/nuclear immunoreactivity for ␤-catenin
5 61 F — — and mutations of exon 3 of the ␤-catenin gene in 18 (100%) and 15
6 12 F 37 TCT 3 TTT Ser 3 Phe
7 17 F 32 GAC 3 GGC Asp 3 Gly
(83%) of 18 SPNs, respectively. These mutations were all 1-bp
8 32 F 32 GAC 3 AAC Asp 3 Asn missense mutations that lead to loss of serine/threonine sites for
9 11 F 33 TCT 3 TGT Ser 3 Cys glycogen synthase kinase-3␤ phosphorylation or may interfere with
10 46 M 33 TCT 3 CCT Ser 3 Pro
11 48 F 34 GGA 3 AGA Gly 3 Arg degradation of the ␤-catenin gene product. These results suggested
12 14 F — — that Wnt signaling activated mostly by mutations of the ␤-catenin
13 35 M 41 AAC 3 ATC Thr 3 Ile gene exon 3 has an important role for the development of SPN.
14 50 M 37 TCT 3 CCT Ser 3 Pro
15 14 F 32 GAC 3 AAC Asp 3 Asn Among patients with FAP, neoplasms typically arise through bial-
16 54 F 34 GGA 3 GTA Gly 3 Val lelic (germ-line then somatic) inactivation of adenomatous polyposis
17 45 F 34 GGA 3 AGA Gly 3 Arg
18 36 F 32 GAC 3 AAC Asp 3 Asn
coli gene. The present series, however, included no case associated
a
In case 6, the mutation was found both in paraffin sections and frozen sample. In case
with FAP, and a literature review showed only 1 SPN case with a
9, the mutation was found in a frozen sample (paraffin sections were not available). In history of FAP (15). Nuclear ␤-catenin accumulation can result from
cases 13, 14, 16, and 17, the mutations were found by direct DNA sequencing after PCR somatic mutations in adenomatous polyposis coli or axis inhibitor 1
for amplifying shorter fragments.
genes (16, 17). Because of the demonstration of cytoplasmic/nuclear
␤-catenin accumulation in all 18 SPNs despite the lack of ␤-catenin
submitted to direct sequencing using the ABI Big Dye Terminator Cycle gene mutations in 17% (3 of 18 tumors) of the SPNs examined, some
Sequencing kit (Applied Biosystems) and the primers used for PCR [5⬘ BCTN of SPNs are presumed to have other molecular changes activating Wnt
and 3⬘ (2nd) BCTN primers, or 5⬘ BCTN (short) and 3⬘ BCTN (2nd short) signaling.
primers for the paraffin sections and 5⬘ BCTN exon 3 and 3⬘ BCTN exon 3 Although few SPNs with malignant course have been reported,
primers for the frozen samples]. Sequencing of each PCR product was per- SPNs usually behave in a benign fashion (1–3). In this study, more
formed with ABI Genetic Analyzer Model 310 (Applied Biosystems). Each
than half the tumor cells of all SPNs conducted showed nuclear
mutation was verified in both sense/antisense directions and in another inde-
overexpression of cyclin D1. Cyclin D1, which participates in the cell
pendent experiments. Strict laboratory work was performed to avoid cross/
carryover contamination. cycle control at the G1-S transition, has been shown to be one of the
target molecules of ␤-catenin/Lef-1 complexes (10). Although over-
Results expression of cyclin D1 generally indicates aggressive behavior of the
tumor, recent studies have shown the contrary observation (18).
Conventional Histopathology. H&E-stained sections of each tu- Cyclin D1 contributes not only to oncogenic transformation but also
mor sample showed typical histopathology of SPN, characterized by to growth arrest, and some investigators speculated that the decision
the growth of uniform polygonal cells arranged around minute fibro- for cell growth or arrest is associated with the concentration of cyclin
vascular stalks, exhibiting pseudo-papillary pattern (Fig. 1a). The D1 (19). A recent report has shown overexpression of cyclin D1 in
tumors also shows a solid monomorphous pattern, sclerosis, and two SPNs (20). Nuclear accumulation of ␤-catenin and possibly
cystic degeneration in variable degree. subsequent overexpression of cyclin D1 may have some relationship
Immunohistochemistry. Intense ␤-catenin immunoreactivity was with mostly favorable clinical behavior of SPNs.
found in the cytoplasm and the nuclei of almost all tumor cells of 18
tumors examined (Fig. 1, b and c). Membranous reactivity was in-
conspicuous. Acinic cells, duct cells, and some islet cells in the
nonneoplastic pancreatic parenchyma showed only membranous re-
activity (Fig. 1, b and d). More than half the tumor cells in each tumor
showed nuclear reactivity for cyclin D1 (Fig. 1e).
Mutational Analysis. Fifteen of 18 tumors (83%) showed 1-bp
missense mutations in the region between codons 32 and 41 of exon
3 of the ␤-catenin gene. The mutations were detected in paraffin
sections of 14 of 17 tumors and in fresh-frozen samples of 2 of 2
tumors. The involved codons are as follows: codon 32 (5 cases),
codon 33 (3 cases), codon 34 (3 cases), codon 37 (3 cases), and codon
41 (1 case; Fig. 2). No mutation was found in any corresponding
nontumorous tissue.

Discussion

Although there have been several molecular studies on pancreatic

tumors, they were mostly focused on DC and NET, each being a
major component of pancreatic tumors (4 – 6, 14). Gerdes et al. (14)
performed a large-scale ␤-catenin mutation analysis for pancreatic
Fig. 2. Direct sequencing showing point mutation of exon 3 of the ␤-catenin gene.
DCs and NETs. In their study, single-strand conformation polymor- Electropherogram indicating a mutation at codon 33 (TCT to CCT) in the tumor sample
phism analysis revealed no variant bands in exon 3 of the ␤-catenin of case 1.
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 CATENIN MUTATION IN SOLID-PSEUDOPAPILLARY NEOPLASM
In summary, the present study revealed the Wnt signaling mostly
associated with ␤-catenin mutations may play an important role in
tumorigenesis of SPN for the first time.
Acknowledgments
We express great thanks to the following pathologists for providing the
tissue specimens analyzed in this study: Dr. Choutatsu Tsukayama, Depart-
ment of Pathology, Kurashiki Central Hospital, Kurashiki, Japan; Dr. Kenji
Kawabata, Department of Pathology, Akashi Municipal Hospital, Akashi,
Japan; Dr. Shu Nakamoto, Department of Laboratory Medicine, Tottori Pre-
fectural Central Hospital, Tottori, Japan; Dr. Masaharu Mori, Faculty of Health
and Welfare Science, Okayama Prefectural University, Sohja, Japan; Dr.
Yoichi Kameda, Division of Pathology, Kanagawa Cancer Center, Yokohama,
Japan; and Dr. Jinyu Sano, Division of Pathology, Yokohama Municipal
Hospital, Yokohama, Japan.
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Fig. 1. a, histopathology of SPN. H&E stain, ⫻100. b, low-power view of ␤-catenin
immunostain in normal pancreatic tissue (P) and SPN (T) separated by fibrous capsule
(C). Hematoxylin counterstain, ⫻40. c, high-power view of ␤-catenin immunostain in
SPN. Note cytoplasmic and nuclear positivity in almost all tumor cells. Membranous
positivity is weak and discontinuous. Hematoxylin counterstain, ⫻125. d, high-power
view of ␤-catenin immunostain in normal pancreatic tissue. Note only membranous
positivity in the acini, ducts (D), and islets (I). Hematoxylin counterstain, X125. e, cyclin
D1 immunostain in SPN. Note nuclear positivity of almost all tumor cells. Hematoxylin
counterstain, ⫻200.
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Frequent β-Catenin Mutation and Cytoplasmic/Nuclear
Accumulation in Pancreatic Solid-Pseudopapillary Neoplasm
Yukichi Tanaka, Keisuke Kato, Kenji Notohara, et al.

Cancer Res 2001;61:8401-8404.

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