Mutation spectrum in the Wnt/β-catenin signaling pathway in gastric fundic

gland-associated neoplasms/polyps

Se-Yong Lee MD a, b, Tsuyoshi Saito MD, PhD a, Hiroyuki Mitomi MD, PhD c, Yasuhiro Hidaka MD,

PhD a, b, Takashi Murakami MD, PhD a, b, Ryosuke Nomura MD a, b, Sumio Watanabe MD, PhD a, b,

Takashi Yao MD, PhD a

a Department of Human Pathology, Juntendo University, School of Medicine, 2-1-1 Hongo,

Bunkyo-ku, Tokyo 113-8421, Japan

b Department of Gastroenterology, Juntendo University School of Medicine

c Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi,

Japan

Corresponding Author

Tsuyoshi Saito, MD, PhD

E-mail: tysaitou@juntendo.ac.jp

Department of Human Pathology, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku,

Tokyo 113-8421, Japan

Keywords: Gastric adenocarcinoma of the fundic gland type; fundic gland polyp; fundic gland polyp

with dysplasia; CTNNB1; Axin; APC; PPP2R1A; mutation

Running title: Wnt/β-catenin signaling pathway in fundic gland neoplasms

Abstract

Frequent activation of the Wnt/β-catenin signaling pathway has recently been demonstrated in

gastric adenocarcinoma/neoplasia of chief cell predominant type (GA-CCP/GN-CCP) with

submucosal involvement. In this study, we examined the activation status of the Wnt/β-catenin

signaling pathway in GN-CCP without submucosal involvement, which is referred to as gastric

dysplasia-CCP (GD-CCP). We also examined β-catenin expression and the mutation spectrum of

PPP2R1A and Wnt pathway genes in 11 cases of GD-CCP, 25 cases of gastric polyps of fundic gland

type (GPs-FG), and 21 cases of GPs-FG with dysplasia (GP-FGD). β-catenin nuclear staining was

observed in 3 cases of GD-CCP, none of GPs-FG, and 6 cases of GPs-FGD. Mutations in Wnt pathway

genes, including PPP2R1A, were observed in 4 cases of GDs-CCP, 10 cases of GPs-FG, and 7 cases of

GPs-FGD. Two of these seven GPs-FGD cases showed β-catenin nuclear staining. However, none of

the 4 GD-CCP cases with mutations or the 10 GPs-FG cases with mutations showed β-catenin

nuclear staining. PPP2R1A mutations were observed in 1 GD-CCP case and 1 GPs-FGD case.

Although the mutation spectra of the Wnt pathway genes in GD-CCP and GP-FG differed, based on

the absence of β-catenin nuclear staining despite the genetic alterations, GD-CCP is more similar to

GP-FG than to GN-CCP, which shows β-catenin nuclear staining and submucosal involvement.

Activation of the Wnt/β-catenin signaling by the β-catenin nuclear transition may be required during

progression from GD-CCP to GN-CCP. Furthermore, this is the first report describing PPP2R1A

mutations in gastric fundic gland-associated neoplasms.

Introduction

Gastric adenocarcinoma of chief cell predominant type (GA-CCP) is a recently described histological

entity of gastric cancer that has not yet been included in the WHO classification. Most of these

tumors present as solitary, small, well-circumscribed lesions in the upper third of the stomach.

Histologically, GA-CCP is composed of tightly packed glands and cords of predominantly chief cells

with anisonucleosis, without chronic gastritis, atrophic changes, or intestinal metaplasia in the

surrounding mucosa [1, 2].

Although GA-CCP with submucosal involvement was noted in previous reports [1-5]. Singhi et al. [6]

described the clinicopathological features of 10 cases of non-invasive (intramucosal) tumors with

similar histological characteristics. They argued that these lesions represent prolapse-type changes

into the submucosa rather than the typical submucosal invasion observed in malignant neoplasms

[6]. Therefore, in this study, we named GA-CCP that is restricted to the mucosa “gastric

dysplasia-chief cell predominant type” (GD-CCP).

Gastric polyps of fundic gland type (GPs-FG) are small, benign mucosal polyps that arise in the

gastric body and fundus [7]. Histologically, they are composed of dilated glands lined by oxyntic

mucosa. GPs-FG were originally believed to be hamartomatous [8]; however, most sporadic GPs-FG

have activating mutations in β-catenin, and syndromic GPs-FG frequently harbor adenomatous

polyposis coli (APC) mutations, suggesting that they are benign and self-limiting neoplasms. In

some GPs-FG, dysplasia of the foveolar epithelium has been observed. GPs-FG with dysplasia

(GPs-FGD) are characterized by surface crowding and the presence of gastric neck mucin cells,

which display enlarged hyperchromatic nuclei and a loss of cytoplasmic mucin [9, 10]. Thefrequency

of GPs-FGD is closely associated with the setting in which they arise. Low-grade dysplasia in familial

adenomatous polyposis (FAP)-associated GPs-FG is relatively common (24–49%), whereas it is rare

in sporadic GPs-FG [9, 11]. GPs-FGD are associated with APC alterations [10, 12] and nuclear

β-catenin accumulation [11, 13].

There are several similarities between GDs-CCP and GPs-FG. However, in contrast to the clustered

glands and anastomosing cords observed in GDs-CCP, GPs-FG contain cystically dilated glands with

irregular budding [6]. In addition, GPs-FG are lined not only by parietal and chief cells but also by

mucinous foveolar cells instead of mucous neck cells [6].

The Wnt/β-catenin pathway is a conserved signaling pathway that plays a major role in

development and homeostatic tissue self-renewal [14]. Wnt binds to transmembrane Frizzled

receptors, which leads to activation of Dishevelled (Dsh) protein in the cytoplasm [15, 16]. Dsh

forms a complex with proteins of the Axin family [17-20], which also binds glycogen synthase

kinase-3β (GSK-3β) [21], APC, serine/threonine protein phosphatase 2A (PP2A), and β-catenin

[22-25]. Regulation of phosphorylation is likely important, because Dsh, Axin, APC, GSK-3β, and

β-catenin are all phosphoproteins. PP2A, encoded by protein phosphatase 2 regulatory protein 1A

(PPP2R1A), has a wide range of substrates and is important in many cellular processes [26]. Catenin

(cadherin-associated protein), beta 1 (encoded by CTNNB1) is associated with the Wnt/β-catenin

signaling pathway. CTNNB1 mutations that are linked to nuclear β-catenin accumulation have been

investigated in gastric cancer, and were reported to be less frequent than expected based on its

nuclear expression [27-30]. As we have noted frequent β-catenin nuclear accumulation in GA-CCP,

we recently examined the mutational status of genes involved in the Wnt/β-catenin pathway, such

as CTNNB1, APC, and AXIN in GA-CCP and conventional gastric adenocarcinoma [2]. Interestingly,

the frequencies of CTNNB1, AXIN1, and AXIN2 mutations were higher in GA-CCP than in

conventional gastric adenocarcinoma, and approximately one-half of the GAs-CCP harbored

mutations in either CTNNB1, AXIN1 or AXIN2 as the suspected cause of constitutive Wnt/β-catenin

pathway activation [2].

In this study, we collected 11 cases of GD-CCP, 25 cases of GP-FG, and 21 cases of GP-FGD and

examined the expression of β-catenin by immunohistochemistry and the genetic alterations in

CTNNB1, APC, AXIN, and PPP2R1A to investigate the mechanism of Wnt signal activation in these

tumors.

Materials and Methods

Patients and materials

We selected 11 cases of non-invasive lesions (GD-CCP) from a previously described series [1, 2],

including newly added cases. The material for our study included 11 GDs-CCP from 11 patients that

were resected endoscopically or surgically at Juntendo University Hospital and our affiliated

hospitals between 2004 and 2012. Without exception, these tumors were positive for MUC6 and

pepsinogen-I immunostaining (some cases were also focally positive for H+/K+-ATPase α subunit)

and negative for MUC2 and CD10.

For comparison, 25 cases of GP-FG, which presented with cystically dilated oxyntic glands and

parietal cells in the epithelium that was usually accompanied by small gland clustering or budding,

and 21 cases of GP-FGD with nuclear enlargement, stratification, and hyperchromatism in the

foveolar or surface epithelium overlying the GPs-FG, were selected from the pathological files of

Juntendo University Hospital and our affiliated hospitals. All samples were independently reviewed

by 2 experienced gastrointestinal pathologists (H.M. and T.Y.), and inter-observer variation was

resolved by re-evaluation and discussion to reach a consensus. Detailed clinicopathological data for

the 11 GDs-CCP, 25 GPs-FG, and 21 GPs-FGD are summarized in Table 1. This study was approved

by the Institutional Review Board and the ethical committee of our hospital (registration #2012008).

Immunohistochemistry of β-catenin

A subset of the 11 GDs-CCP, 25 GPs-FG, and 21 GPs-FGD were subjected to immunohistochemistry.

Tissue sections (4-μm thick) were prepared from formalin-fixed paraffin-embedded blocks and

subjected to immunohistochemistry using a β-catenin monoclonal antibody (clone 14, 1:200dilution;

BD Bioscience, San Diego, CA, USA). Antigen retrieval was performed by heating in an autoclave in

Tris-EDTA buffer (pH 6.0). The sections were incubated with the primary antibodies at 4°C overnight.

Immunohistochemical staining was performed by using the Envision Kit (Dako, Glostrup, Denmark)

with substrate-chromogen solution. The percentage of cells with nuclear staining (labeling index, LI)

was evaluated in the most representative areas showing the highest immunoreactivity, by counting

the number of positive cells among at least 500 tumor cells. When the LI was less than 5%,

β-catenin nuclear staining was considered negative. When the LI was more than 5%, β-catenin

nuclear staining was considered positive. Slides were evaluated by 2 independent investigators

(S-Y.L. and H.M.) without prior knowledge of the clinicopathological data. Discrepancies were

resolved by re-evaluation to reach a consensus.

DNA extraction

Tumor DNA was extracted from the manually macro-dissected samples on unstained slide glasses

prepared from routinely processed, formalin-fixed, paraffin-embedded (FFPE) specimens using the

QIAamp DNA FFPE Tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s

instructions.

Mutational analysis of CTNNB1, APC, AXIN, and PPP2R1A

PCR followed by direct sequencing was used to detect mutations in CTNNB1, APC, AXIN1, AXIN2,

and PPP2R1A. The primer sequences to amplify CTNNB1, APC, AXIN1, and AXIN2 have been

previously described [5], and the primer sequences used for PPP2R1A were exon5-F1,

5’-AAACCTGGACCCACACAAC-3’; exon5-R1, 5’-ATCATCCCCATGTTCTCCAA-3’; exon5-F2,

5’-GTACTTCCGGAACCTGTGCTC-3’; exon5-R2, 5’-AGGTGAGTTTTGCTTCCTGG-3’;

exon6-F5’-CTCTCCTCTCCCTAGGACTCG-3’; and exon6-R, 5’-ACTGGTGGGGACACTGACAC-3’.

Sequences were analyzed with a capillary sequencing machine (3730xl Genetic Analyzer; Applied

Biosystems). Samples with mutation peaks whose height was 20% of the height of a normal peak,

were considered to have a mutation. All mutations were verified by sequencing both the sense and

antisense strands. The presence of mutations was evaluated by 5 independent investigators (T.M.,

Y.H., S-Y.L., H.M., and T.S.).

Statistical analysis

All categorical variables were analyzed with the Chi-square test (with Yates’ correction), as

appropriate. Continuous variables were analyzed using the Mann-Whitney test. All P values were

2-sided, and P values less than 0.05 were considered statistically significant. All statistical analyses

were performed using StatView for Windows Version 5.0 (SAS Institute Inc., Cary, NC, USA).

Results

Clinicopathological characteristics

The clinicopathological characteristics of the GDs-CCP, GPs-FG, and GPs-FGD are summarized in

Table 1. In this study, most of the tumors were located in the upper and middle thirds of the

stomach, and the sex distribution in all groups showed no significant difference. The age of the

patients with GD-CCP was significantly older than the age of the patients in the other groups. The

tumor sizes of the GDs-CCP (mean/median: 7.6/6.0 mm; P < 0.0004) and GPs-FGD (6.3/5.0 mm; P

= 0.0037) were significantly larger than that of the GPs-FG (3.6/3.0 mm). Grossly, approximately

one-half of the GDs-CCP (45.5%) presented as depressed type, whereas all of the GPs-FG (P =

0.0012) and GPs-FGD (P = 0.0023) were protruded type.

β-Catenin expression

In the GDs-CCP, nuclear β-catenin staining was observed in the chief cells with dysplasia (Figure 1).

On the other hand, in GPs-FGD, nuclear β-catenin staining was observed in the superficial/foveolar

atypical epithelium (Figure 2A–C). In GPs-FG, the chief cells, some of which were dilated, showed

membranous/cytoplasmic β-catenin staining, and all cases were classified as negative for nuclear

staining of β-catenin (Figure 2D–F).

The nuclear β-catenin staining results are summarized in Table 2. Nuclear β-catenin labeling indices

were significantly higher in GDs-CCP (mean/median, 8.7/0.7%; interquartile range, 0–22%) than in

GPs-FG (0.3/0.0%; 0–2.3%; P = 0.0231). In GDs-CCP, this index was significantly lower than that

observed in the GNs-CCP with submucosal involvement (mean/median, 22.9/19.3%) [5].

Furthermore, a similar trend was observed between GPs-FGD (5.3/1.0%; 0–30.4%) and GPs-FG

(P= 0.0058). There was no significant difference observed in the β-catenin LI of GDs-CCP and

GPs-FGD.

Among the GD-CCP cases, 3 of 11 (27%) were classified as positive for β-catenin nuclear staining,

and 8 (73%) were classified as negative. Among the GPs-FGD cases, 6 of 21 (29%) were classified

as positive and 15 (71%) were classified as negative. Among the GPs-FGs cases, all 25 were

categorized as negative. The frequencies of nuclear β-catenin expression (positive vs. negative) in

the GD-CCP cases (P = 0.0005) and GPs-FGP case (P = 0.0007) were significantly higher than that

in the GPs-FG cases.

Mutation analyses

We evaluated exon 3 of CTNNB1, exon 15 of APC, exon 5 and 6 of PPP2R1A, and the entire coding

region of both AXIN1 and AXIN2 in 3 GD-CCP cases. Because the mutations detected in these 3

cases were located within restricted regions of AXIN1 and AXIN2, we reduced the regions analyzed

to exon 1 and exon 6 in AXIN1 and exon 1 of AXIN2. The frequencies and spectrum of mutations in

CTNNB1, APC, AXIN1, AXIN2, and PPP2R1A are summarized in Table 3 and Table 4, respectively.

No mutations in CTNNB1 were detected in any case of GD-CCP. The frequency of CTNNB1

mutations tended to be higher in GPs-FG (6/25 cases; 24%) than in GPs-FGD (2/21; 10%). A

missense mutation in the β-catenin binding domain of APC was observed in 1 GD-CCP case (1/11;

9%). In GPs-FG cases, 1 of 25 (4%) and in GPs-FGD cases, 2 of 21 (10%) harbored APC missense

mutations. One case of GD-CCP (9%) harbored an AXIN1 mutation, and loss of function mutations

in AXIN1 were identified in 3 of 25 GPs-FG cases (12%) and 2 of 21 GPs-FGD cases (10%). AXIN2

loss of function mutations were identified in 3 of 11 GD-CCP cases (27%) and in 3 of 25

GPs-FGcases (12%), whereas none of the 21 GPs-FGD cases harbored AXIN2 mutations. The

frequency of AXIN2 mutations was significantly higher in GD-CCP cases than in the GPs-FGD cases

(P = 0.033).

PPP2R1A mutations were rare in this series of cases, and only 1 of 11 GD-CCP cases (9%) and 1 of

21 GPs-FGD cases (4%) harbored mutations in this gene. Among all the genes examined, 4 of 11

GD-CCP cases (36%), 10 of 25 GPs-FG cases (40%), and 7 of 21 GPs-cases FGD (33%) harbored at

least 1 mutation in CTNNB1, APC, AXIN, or PPP2R1A. However, interestingly, none of the GD-CCP

cases with any of these mutations showed nuclear β-catenin staining. Representative cases with

mutations are shown in Figure 3.

Clinicopathological relationships w ith the detected genetic alterations

The impact of these genetic alterations and the significance of nuclear β-catenin staining on the

clinicopathological variables in GD-CCP were also assessed. However, no significant associations

were found between the genetic alterations or nuclear β-catenin staining and clinicopathological

variables.

Discussion

It has been shown that β-catenin mutations are frequent in sporadic GPs-FG, and they have been

detected in 64–91% of cases [31, 32]. In this study, β-catenin missense mutations were detected in

24% of sporadic GPs-FG cases. This frequency seems to be lower than those reported previously,

although mutations in APC, AXIN1, and AXIN2 were also detected in 1, 3, and 3 cases, respectively.

In addition, these mutations were almost entirely exclusive. As a result, 10 of 25 GP-FG cases (40%)

harbored a mutation in at least 1 Wnt pathway gene.

It has been reported that alterations in APC were frequent in sporadic GPs-FGD, whereas β-catenin

mutations were rare [12]. In this study, alterations were detected in APC, β-catenin, AXIN1, and

PPP2R1A, and these mutations were mutually exclusive. This finding suggests that mutations in

many genes in the Wnt pathway besides APC can cause epithelial dysplasia of sporadic fundic gland

polyps.

In our previous study of 27 gastric neoplasias of chief cell-predominant type (GN-CCP), all of which

showed submucosal involvement, 14 of 27 cases harbored mutations in Wnt pathway genes, and 12

of the 14 cases showed β-catenin nuclear staining [2]. In contrast, in the present study, none of the

4 GD-CCP cases with missense mutations in the Wnt pathway genes showed nuclear accumulation

of β-catenin. When considering GD-CCP as a precursor lesion of GN-CCP, activation of

Wnt/β-catenin signaling by β-catenin nuclear transition exerted by other mechanisms, such as

epigenetic silencing of Wnt pathway genes may be required for progression from GD-CCP to GN-CCP.

In addition, we recently demonstrated that Axin2 mutations are more frequently seen in GN-CCP

than in conventional gastric adenocarcinoma with submucosal invasion [2]. In this study,

GD-CCPalso frequently harbored Axin2 mutations, demonstrating the close relationship between

GD-CCP and GN-CCP. Furthermore, in familial and sporadic GPs-FG, it has been shown that

membranous/cytoplasmic β-catenin staining without nuclear localization can be observed despite

the presence of APC dysfunction and β-catenin mutations [31, 33], which is in agreement with the

findings in this study. These findings also argue a close relationship between GD-CCP and GP-FG,

although the mutation spectrums observed in the Wnt pathway genes differed. These findings may

explain in part why GDs-CCP maintain their nature without showing submucosal involvement,

though no evidence has been shown regarding GP-FG as a precursor lesion of GD-CCP.

Recently, somatic mutations in PPP2R1A have been reported in certain types of ovarian and uterine

carcinomas [34-36]. However, the biological function of PP2A (encoded by PPP2R1A) remains

unclear. PP2A plays an important role in development, cell proliferation and death, cell mobility, the

cell cycle, and the regulation of numerous signaling pathways [37], and it is expected to be an

important tumor suppressor [38, 39]. However, it has been shown that PP2A dephosphorylates

β-catenin, and that treatment of colon cancer cells with the classical PP2A inhibitor okadaic acid

increased β-catenin phosphorylation [40]. In addition, a recent study demonstrated that PP2A

inhibitors suppressed the Wnt/β-catenin pathway through the phosphorylation and degradation of

β-catenin [41]. These findings suggest an oncogenic role for PP2A in tumor progression. We

detected 2 cases with PPP2R1A mutations in this series of gastric fundic gland-associated

neoplasms; 1 each in GD-CCP and GP-FGD. This is the first report showing PPP2R1A mutations in

gastric fundic gland-associated neoplasia. From the perspective of cancer genetics, the mutual

exclusivity of PPP2R1A mutations and mutations in Wnt pathway genes in GD-CCP and

GP-FGDsuggests that PPP2R1A mutations likely affect the Wnt signaling pathway. Furthermore, all

PPP2R1A mutations reported in other carcinomas and in this study were heterozygous missense

mutations.

Based on these findings and the finding that both lesions with PPP2R1A mutations did not show

β-catenin nuclear staining (probably PPP2R1A on another allele would be expected to be intact),

PP2A might have a tumor suppressor function in these gastric fundic gland-associated neoplasms.

However, further studies are needed to clarify the biological role of mutant PP2A in these

neoplasms.

In conclusion, we demonstrated, for the first time, PPP2R1A mutations in gastric fundic

gland-associated neoplasms. Although the mutation spectrum in Wnt pathway genes differed in

GD-CCP and GP-FG, from the viewpoint of β-catenin nuclear staining despite genetic alterations,

GD-CCP is more similar to GP-FG than GN-CCP, which shows submucosal involvement, whilst also

GD-CCP and GP-FG are the distinct lesions each other.

Acknow ledgments

The authors thank Dr. Minako Hirahashi (Department of Anatomic Pathology, Pathological Sciences,

Graduate School of Medical Sciences, Kyushu University), Dr. Yumi Oshiro (Department of Pathology,

Matsuyama Red Cross Hospital), Dr. Takehiro Tanaka (Department of Pathology, Okayama

University Graduate School of Medicine), Dr. Yutaka Nakashima (Division of Pathology, Japanese

Red Cross Fukuoka Hospital), Dr. Tetsumi Yamane (Department of Pathology, Tottori Red Cross

Hospital), Dr. Fumiyoshi Fushimi (Department of Pathology, National Kyushu Cancer Center), Dr.

Shinji Kono (Division of Clinical Pathology, Harasanshin Hospital), Dr. Shuichi Ohara (Department of

Gastroenterology, Tohoku Rosai Hospital), Dr. Koyu Suzuki (Department of Pathology, St Luke's

International Hospital), and Dr. Takeshi Yano (Department of Surgery, Asoka Hospital) for kindly

providing samples and clinical information, Dr. Ayumi Osako (Department of Gastroenterology,

Tottori Seikyo Hospital). We also wish to thank Mrs. Keiko Mitani (Department of Human Pathology,

Juntendo University School of Medicine) for her expert technical assistance. We also thank the

Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University

Graduate School of Medicine, Tokyo, Japan, for technical assistance.

Funding

This work was supported, in part, by a Grant-in-Aid for General Scientific Research from the

Ministry of Education, Science, Sports, and Culture (#26670286 to Tsuyoshi Saito, #24590429 to

Hiroyuki Mitomi and #26460428 to Takashi Yao), Tokyo, Japan.

Conflict of interest

The authors declare that there are NO conflicts of interest to disclose.

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Figure legends

Figure 1.

Low-power (A) and high-power (B) views of a case of GD-CCP (#6). The tumor glands are

predominantly composed of primitive chief cells that have tightly proliferated beneath the surface

epithelium. Low-power (C) and high-power (D) views of β-catenin immunohistochemistry. Tumor

cells in the atypical glands show diffuse β-catenin nuclear staining. Low-power (E) and high-power

(F) views of a case of GD-CCP (#2). Low-power (G) and high-power (H) views of β-catenin

immunohistochemistry. Tumor cells in this case did not show β-catenin nuclear staining despite APC

mutation at codon 1305. Original magnification: A x40, B x200, C x100, D x200, E x40, F x200, G

x100, H x400.

Figure 2.

Low-power (A) and high-power (B) views of a case of GP-FGD (#14). Under low-power

magnification, the characteristic architecture of a fundic gland polyp is seen. However, the surface

epithelium shows hyperchromatism. The high-power magnification image shows that the surface

epithelial cells are enlarged, hyperchromatic, and have crowded nuclei, and the cytoplasmic mucin

content is decreased in the atypical surface epithelium (B). A small number of cells in the atypical

surface epithelium show β-catenin nuclear expression (C). Low-power (D) and high-power (E) views

of a case of GP-FG (#20). Under low-power magnification, the fundic gland polyp shows

characteristic cystically dilated, budded, oxyntic glands (D). The high-power magnification image

shows that the surface and foveolar epithelium are composed of non-dysplastic gastric mucin cells

with abundant apical mucin (E). The budded oxyntic glands show membranous β-catenin

staininginstead of nuclear staining (F). A x40, B x200, C x400, D x40, E x200, F x400

Figure 3.

(A) A point mutation (GCA to GTA) that leads to an amino acid substitution (A to V) was observed at

codon 1305 of APC in a case of GD-CCP (#2). (B) A point mutation (CCT to TCT) that leads to an

amino acid substitution (P to S) was detected at codon 50 of AXIN2 in a case of GD-CCP (#3). (C) A

point mutation (CCC to TCC) that leads to an amino acid change (P to S) was detected at codon 206

of PPP2R1A in a case of GP-FGD (#3).

VariableGastric dysplasia-chief

cell predominant type (n= 11)

Gastric polyp-fundic glandtype (n = 25)

Gastric polyp-fundic glandtype with dysplasia (n = 21)

Age (years) *a 68.5 / 67.0 ± 7.2 (55 - 82) 55.5 / 54.0 ± 12.7 (29 - 85) 58.5 /60.0 ± 15.2 (27 - 87)Sex Male 8 11 11 Female 3 14 10Location Upper third 8 9 10 Middle third 3 14 10 Lower third 0 2 1Size of tumor (mm) * 7.6 / 6.0 ± 4.1 (3 - 18) 3.6 / 3.0 ± 1.7 (2 - 8) 6.3 / 5.0 ± 4.1 (2 - 20)Macroscopic typeb

Protruded 6 25 21 Depressed 5 0 0 Mixed 0 0 0

*Data are represented as mean / median ± SD (range)a : GD-CCP vs GP-FG, P＝0.0025; GD-CCP vs GP-FGD, P＝0.0495b : GD-CCP vs GP-FG, P＝0.0002; GD-CCP vs GP-FGD, P＝0.0005

Table 1. Clinicopathological characteristics of the GDs-CCP, GPs-FG and GPs-FGD

Table 2 . Nuclear β-catenin expression

Gastric dysplasia-chief cellpredominant type (n =

11)

Gastric polyp-fundicgland type (n = 25)

Gastric polyp-fundic gland typewith dysplasia (n = 21)

Nuclear β-catenin LI (%)a 5.9 / 0.7 ± 8.7 (0.0 - 22.2) 0.3 / 0.0 ± 0.5 (0.0 - 2.3) 5.3 / 1.0 ± 8.7 (0.0 - 30.4)Nuclear β-catenin expressionb

Positive 3 0 6Negative 8 25 15

*Data are represented as mean / median ± SD

b : GD-CCP vs. GP-FG, P = 0.0005; GP-FG vs. GP-FGD, P = 0.0007a : GD-CCP vs. GP-FG, P = 0.0231; GP-FG vs. GP-FGD, P = 0.0058

Gastric dysplasia-chiefcell predominant type

Gastric polyp-fundicgland type

Gastric polyp-fundicgland type with

dysplasia(n = 11 ) (n = 25 ) (n = 21 )

β-catenin 0 (0 %) 6 (24 %) 2 (10 %)

APC 1 (9 %) 1 ( 4 %) 2 (10 %)

AXIN1 1 (9 %) 3 (12 %) 2 (10 %)

AXIN2 a 3 (27 %) 3 (12 %) 0 (0 %)

PPP2R1A 1 (9 %) 0 (0 %) 1 (5 %)

Total cases 4 (36 %) 10 (40 %) 7 (33 %)a: GD-CCP vs. GP-FGD, P = 0.033

Table 3 .The missense and nonsense mutation of APC, AXIN1, AXIN2 and PP2A.

Table 4. Mutation spectrum in the Wnt pathway genes and PPP2R1A

β-catenin APC AXIN1 AXIN2 PPP2R1A

#1 T1493T P50S#2 A1305V E627G D238N#3 T1493T P50S#4 (+)#5 E196K#6 (+) F105F#7#8#9#10#11 (+)

#1 N1399N T60I N229D#2 V57M S41S

#3 Y42H, L83SD95N

#4 S23I A601T#5 G74G#6 V248D P178P#7 A43T#8 S37F#9 S1426C A237T#10#11 S33Y#12#13#14#15 G74G#16 G74G#17#18 E77K#19#20#21#22 A609A#23#24#25

#1#2 K201K, P206S

#3P1381S,S1426C

P178P

#4#5 D32D#6#7 K49E R1385R R267R#8 L115L#9 (+) E55V, V62I#10 (+) A609A#11 T617S#12#13 (+)

#14 (+) P1422P,Q1444E

#15 (+)#16#17 G621D#18#19#20#21 (+)

(+) : β-catenin nuclear stain Mutation Analysis

　: Missense mutation　: Silent mutation　: Single nucleotide polymorphism

Mutation Analysis

GD-CCP

GP-FG

GP-FGD

histologyNuclear

β-cateninexpression