Inada et al. page 1

Name of journal: World Journal of Gastroenterology
ESPS Manuscript NO: 12338
Columns: Research Report
ARID1A expression in gastric adenocarcinoma: clinicopathological significanceand correlation with DNA mismatch repair status

MMR and ARID1A in gastric cancer

Ryo Inada, Shigeki Sekine, Hirokazu Taniguchi, Hitoshi Tsuda, Hitoshi Katai,
Toshiyoshi Fujiwara, Ryoji Kushima

Ryo Inada, Hirokazu Taniguchi, Hitoshi Tsuda, Ryoji Kushima, Pathology andClinical Laboratory Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku,
Tokyo, 104-0045, Japan

Ryo Inada, Toshiyoshi Fujiwara, Department of Gastroenterological Surgery,Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical

Inada et al. page 2

Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8558, Japan

Shigeki Sekine, Molecular Pathology Division, National Cancer Center ResearchInstitute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

Hitoshi Katai, Gastric Surgery Division, National Cancer Center Hospital, 5-1-1
Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

Author contributions: Inada R performed experiments and data analysis and wrote
the manuscript, Sekine S designed the study, guided the experiments, and wrote themanuscript, Taniguchi H, Tsuda H, and Kushima R performed experiments andedited the manuscript, Katai H collected clinical samples and edited the manuscript,
and Fujiwara T edited the manuscript.

Correspondence to: Shigeki Sekine, MD, PhD, Molecular Pathology Division,
National Cancer Center Research Institute, Tokyo, Japan, 5-1-1 Tsukiji, Chuo-ku,
Tokyo, 104-0045, Japan. ssekine@ncc.go.jp

Inada et al. page 3

Telephone: +81-3-3542-2511 (ext.4210)

Received:

Revised:Accepted:
Published online:

Fax: +81-3-3248-2463

Inada et al. page 4

Abstract
AIM: To analyze the mismatch repair (MMR) status and the ARID1A expression as
well as their clinicopathological significance in gastric adenocarcinomas.

METHODS: We examined the expressions of MMR proteins and ARID1A by
immunohistochemistry in consecutive 489 primary gastric adenocarcinomas. The
results were further correlated with clinicopathological variables.

RESULTS: The loss of any MMR protein expression, indicative of MMR deficiency,
was observed in 38 cases (7.8%) and was significantly associated with an older age(68.6 ± 9.2 vs 60.4 ± 11.7, P < 0.001), a female sex (55.3% vs 31.3%, P = 0.004), an antral
location (44.7% vs 25.7%, P = 0.021), and a differentiated histology (57.9% vs 39.7%, P =0.023). Abnormal ARID1A expression, including reduced or loss of ARID1A
expression, was observed in 109 cases (22.3%) and was significantly correlated withlymphatic invasion (80.7% vs 69.5%, P = 0.022) and lymph node metastasis (83.5% vs73.7%, P = 0.042). The tumors with abnormal ARID1A expression more frequently
indicated MMR deficiency (47.4% vs 20.2%, P < 0.001). A multivariate analysis

Inada et al. page 5

identified abnormal ARID1A expression as an independent poor prognostic factor(HR: 1.36, 95%CI: 1.01-1.84, P = 0.040).

CONCLUSION: Our observations suggest that the AIRD1A inactivation is associatedwith lymphatic invasion, lymph node metastasis, poor prognosis, and MMR
deficiency in gastric adenocarcinomas.

© 2014 Baishideng Publishing Group Inc. All rights reserved.

Keywords:

Adenocarcinoma,

ARID1A,

Mismatch

Repair,Stomach,ImmunohistochemistryCore tip: Alterations of ARID1A, a key component of the chromatin remodelingcomplex, have been recently reported in several tumors, including gastric cancer.
Previous studies showed a significant relationship between ARID1A mutations and
MMR deficiency in gastric cancers. On the other hand, there have been inconsistentreports on the clinicopathological significance of altered ARID1A expression. In theInada et al. page 6present study, we examined expressions of ARID1A and MMR proteins in a large
series of primary gastric adenocarcinomas, and showed their clinicopathologicalsignificance.

Please provide all authors abbreviation names and manuscript title here. The
abbeviation names should be the same as the copyright.
World J Gastroenterol 2014; In press
Available from:
DOI:

Inada et al. page 7

INTRODUCTION
The incidence of gastric cancer has been declining, but it remains one of the leading
causes of death from cancer worldwide[1]. Multiple genetic and epigenetic alterations
in oncogenes and tumor suppressor genes are involved in the process of gastriccarcinogenesis[2,3]. Defects in the DNA mismatch repair (MMR) system are involvedin the development of some tumors including gastric cancers[4,5]. During DNA
replication, DNA polymerase makes base pairing errors at a certain rate[4,5]. The MMR
system is critical for correcting these errors, and defects in the system lead to an
accelerated accumulation of mutations and a predisposition to certain types ofcancers[4,5]. For instance, the loss of MLH1 because of promoter hypermethylation is
known to be a major cause of MMR defects in sporadic gastrointestinal cancers[6].
Patients

with

MMR-deficient

gastric

cancers

reportedly

exhibitsomeclinicopathological features, including an older age, a female sex, an antral location
and a differentiated histology[2,7-14].
ARID1A, also known as BAF250a, is a key component of the multi-protein
SWI/SNF chromatin remodeling complex, and is involved in the regulation of diverse
cellular processes, from development and differentiation to proliferation[15-17]. The

Inada et al. page 8

SWI/SNF complex interacts directly or indirectly with p53 and regulates thetranscription of target genes downstream of p53, thereby suggesting that ARID1A
plays important roles in tumor suppression[15-18]. Somatic mutations in ARID1A are
reportedly present in a nearly half of all ovarian clear cell carcinomas and about 30%
of endometrioid carcinomas[19,20]. The prevalence of ARID1A mutations has beenreported to be variable among tumor types, and recent studies have reported thefrequent presence of mutations in tumors of several organs, including gastriccancer[7,21-27]. Some studies have examined clinicopathological significance of ARID1A
inactivations[7,23,26,27]; interestingly, a significant relationship between ARID1A
mutations and MMR deficiency have been suggested in gastric cancers[7,23,26,27].
The purpose of the present study was to examine the clinicopathologicalsignificance and correlation between MMR deficiency and ARID1A abnormality in alarge consecutive series of advanced gastric cancers using immunohistochemistry.

Inada et al. page 9

MATERIALS AND METHODS
Study population
This study was approved by the ethical committee of the National Cancer Center,
Tokyo, Japan. The present study involved a consecutive series of 489 primary gastriccancers with invasion to the muscularis propria or deeper that were treated by
gastrectomy at the National Cancer Center Hospital, Tokyo, Japan, between 1999 and2001. All the cases had been histologically confirmed as adenocarcinoma. Of the 489
cases, 327 were men and 162 were women. The mean age was 61 years. Six patients
received adjuvant chemotherapy. Tumors were classified into differentiated type(papillary and tubular adenocarcinoma) and undifferentiated type (poorly
differentiatedadenocarcinoma

andsignetring

cellcarcinoma).

Mucinous

adenocarcinomas were subclassified into differentiated type and undifferentiatedtype, depending on their histology. The pathological stage was determined according
to the UICC TNM classification (the 7th edition)[28].

Immunohistochemical staining
Representative formalin-fixed and paraffin-embedded specimens from each case

Inada et al. page 10

were cut into 4 μm-thick sections. Antibodies against MLH1 (clone G168-15; diluted
1:100; BD Pharmingen, San Diego, CA, USA), PMS2 (clone A16-9; diluted 1:100; BD
Pharmingen, San Diego, CA, USA), MSH2 (clone FE11; diluted 1:200; Caibiochem, La
Jolla, CA, USA), MSH6 (clone 44; diluted 1:500; BD Pharmingen, San Diego, CA, USA),
and ARID1A (polyclonal, HPA005456; diluted 1:200; Sigma-Aldrich, St Louis, MO,
USA) were used as primary antibodies. The sections were deparaffinized andautoclaved at 121°C for 15 min in Target retrieval solution with a high pH of 9 (Dako,
Glostrup, Denmark) and then allowed to cool at room temperature. Endogenous
peroxidase was blocked using 0.3% hydrogen peroxide. The slides were incubated forthree hours with the primary antibodies and then were reacted for one hour with
HRP conjugated secondary antibodies (Dako, Glostrup, Denmark) at room
temperature. The signals were visualized using substrate chromogen (Dako liquid
DAB chromogen; Dako, Glostrup, Denmark), and counterstaining was performed
using Mayer’s hematoxylin.
Non-neoplastic cells, including endothelial cells, fibroblasts, and lymphocytes,
typically showed nuclear expression for all five of the antibodies that were used andserved as positive controls.

Inada et al. page 11

Evaluation of immunohistochemical staining
The tumors were classified into two categories according to the MMR protein
expression status as follows: MMR deficient, negative staining for one or more MMR
proteins; or MMR intact, positive nuclear staining for all four MMR proteins.
The expression of ARID1A was evaluated based on the intensity and pattern ofstaining. The staining intensity was classified as loss, weak, and retained. Weakstaining was defined by comparison with the staining intensities of the internal
controls. The staining patterns were classified into either homogenous orheterogeneous. Heterogeneous expression was defined as a reduced or loss of
staining in 10%-90% of the tumor cells. Two observers independently evaluated thestaining results. Discrepant cases were reviewed using a multiheaded microscope toachieve consensus.

Statistical analysis
Categorical variables were compared using the Fisher’s exact test. Continuous
variables were presented as mean ± SD and compared using the Mann-Whitney U test.

Inada et al. page 12

Disease specific survival curves were calculated using the Kaplan–Meier method, andthe differences in survival times among subgroups were compared using the log–rank
test. Univariate and multivariate analyses were performed using the Cox proportional
hazard regression model to determine the associations between clinicopathologicalvariables and cancer-related mortality. The factors with P-values of < 0.1 in theunivariate analyses were included in a multivariate analysis to determine
independent prognostic factors. P-values of < 0.05 were considered significant.

Inada et al. page 13

RESULTS
MMR protein expression and its clinicopathological significanceOf the 489 cases that were analyzed, 33 cases showed the concurrent loss of MLH1
and PMS2, three cases showed the isolated loss of PMS2, one case showed theconcurrent loss of MSH2 and MSH6, and one case showed the loss of all four proteins(Figure 1, Table 1). The remaining 451 cases retained the expressions of all four
proteins. Overall, 38 cases (7.8%) were regarded as MMR-deficient. All but one
MMR-deficient case showed the homogeneous loss of MMR protein expression ininvasive components. Eighteen MMR-deficient lesions were associated withintramucosal components. Among them, 12 cases showed homogeneous loss,
whereas three showed heterogeneous loss and three other cases retained theexpressions of the MMR proteins in the intramucosal components.
The clinicopathological features according to the MMR status are shown in Table 2.
MMR deficiency was significantly associated with an older age (P < 0.001), a femalesex (P = 0.004), an antral location (P = 0.021), and a differentiated histology (P = 0.023).

ARID1A expression and its clinicopathological significanceInada et al. page 14

Abnormal ARID1A expression was observed in 109 cases (22.3%). These cases
included homogeneous loss (43 cases, 8.8%), heterogeneous loss (29 cases, 5.9%),
homogeneously weak expression (21 cases, 4.3%), and heterogeneously weak
expression (16 cases, 3.3%; Figure 2). Among the 45 cases that showed heterogeneous
ARID1A expression, 34 cases showed heterogeneity within the invasive component.
In remaining 11 cases, ARID1A expression was homogenously lost or weakened in
the invasive component; and in the intramucosal component, the expression washeterogeneous in 8 cases and retained in 3 cases. ARID1A expression was retained in
the remaining 380 cases (77.7%). Among the clinicopathological factors that were
examined, lymphatic invasion (P = 0.022) and lymph node metastasis (P = 0.042) weresignificantly correlated with abnormal ARID1A expression (Table 3).

Survival analysis
The median follow-up period of the patients was 44 months. The disease specificsurvival curves according to the MMR and ARID1A expression statuses did not show
any significant differences (Figure 3). A multivariate analysis revealed several factors
to be associated with a poorer prognosis, including a female sex, a higher serum CEA

Inada et al. page 15

level, a larger tumor size, an undifferentiated-type histology, a higher pathologicalstage, a positive residual disease status and abnormal ARID1A expression (Table 4).

Relationship between the MMR status and ARID1A expression
Among the 38 MMR-deficient cases, 18 cases (47.4%) showed abnormal ARID1A
expression. On the other hand, among the 451 cases with intact MMR protein
expression, only 91 cases (20.2%) indicated abnormal ARID1A expression (Table 5). A
statistical analysis showed a significant correlation between the ARID1A expression
and the MMR statuses (P < 0.001).

Inada et al. page 16

Discussion
In the present study, we used immunohistochemistry for four MMR proteins toanalyze the MMR status. While microsatellite instability (MSI) testing has been
widely used to examine the MMR status[29,30], the immunohistochemical detection of
MMR proteins has been proved to be as sensitive and specific as MSI testing and isbeing increasingly used to screen for colorectal cancer with MMR deficiency[31-33]. An
excellent correlation between the results of MSI testing and immunohistochemistry
has also been reported for gastric cancer[6,8,34]. The majority of MMR deficiencies ingastric cancer is thought to arise from the hypermethylation of the MLH1 promoter[6].
In our study, 33 cases showed the concurrent loss of MLH1 and PMS2 expression,consistent with the consequences of defects in MLH1[31,32,35]. The previously reported
prevalence of MMR deficiency in gastric cancers has been variable, ranging from
7.7%-25.2%[2,7-14,34]. There seems geographical difference in the prevalence of
MMR-deficient gastric cancers. In general, studies from Western countries reported
higher frequencies of MMR deficiencies in gastric cancer, whereas those in Asian
countries are usually less than 10%, similar to the present result. This may be due toepidemiological differences, such as the prevalence of Helicobacter pylori infection [36].

Inada et al. page 17

In tumors defined as MMR-deficient, the loss of MMR protein was mostly
homogeneous within the respective tumors, including the majority of intramucosal
components. This suggests that MMR deficiency occurs at an early stage of gastriccarcinogenesis. Some previous studies have similarly reported that defects in MMR
are an early event during gastric carcinogenesis[3,37,38].
MMR deficiency was significantly associated with several clinicopathologicalfeatures, including an older age, a female sex, an antral location, and a differentiatedhistology; however, no prognostic significance was observed. These observations aremostlyinagreement

with

some

previous

large-scalestudies[2,7-14].

The

clinicopathological features of MMR-deficient colorectal cancers are well recognized:
an older age, a female sex, a proximal location, an undifferentiated histology, a lower
clinical stage, and a better prognosis[39,40]. Among these, an older age and a female sex
are common to the clinicopathological characteristics of gastric cancer with MMR
deficiency, whereas the histology associated with the MMR status differed between
gastric and colorectal cancers.
We examined ARID1A expression using immunohistochemistry. Of note, previous
studies demonstrated a good correlation between genetic defects in ARID1A andInada et al. page 18

immunohistochemically detected ARID1A expression[19,41]. A previous study showedthat either the loss of or the weak expression of ARID1A was indicative of thepresence of ARID1A mutations in gastric cancers[23]. In the present study, a loss of
ARID1A expression was observed in 14.7% and weak ARID1A expression wasobserved in 7.6% of the cases that were examined. Among the previous
immunohistochemical studies of ARID1A expression, five studies defined only theloss of expression as an abnormal pattern and reported prevalence of 11%[7], 11%[22],14%[21],21.7%[26] and 51%[27], respectively. Another study reported the loss of and theweak expression of ARID1A as 20.2% and 7.3%, respectively[23]. While somevariability exists, the prevalence of abnormal ARID1A expression seems to agree
roughly among the studies excluding one study[27].
In ovarian clear cell carcinomas, ARID1A mutations are thought to occur during
the early stage of tumorigenesis, since the loss of ARID1A expression is consistently
homogeneous

and

is

also

observed

in

their

precursor

lesion,atypicalendometriosis[19,42]. In contrast, the loss of or the weak expression of ARID1A was
more commonly heterogeneous within the respective tumors in our study. Even
though several immunohistochemical studies examining ARID1A expression inInada et al. page 19

gastric cancer have previously been reported, most of the studies have neverdescribed heterogeneous expression[7,21-23]. This circumstance is probably because theprevious studies used tissue microarrays in their analyses. The frequent
heterogeneous expression of ARID1A suggests that defects in ARID1A occur often
during the later stage of tumorigenesis in gastric adenocarcinomas, unlike in ovariancancers.
Our study showed that abnormal ARID1A expression was significantly associatedwith lymphatic invasion and lymph node metastasis. Furthermore, abnormal
ARID1A expression was significantly associated with a poor prognosis in a
multivariate analysis. Three previous studies showed several clinicopathologicalfeatures of abnormal ARID1A expression in gastric cancers[7,26,27], including fundus
and corpus locations[7], an undifferentiated histology[27], a lymphatic invasion[7], avenous invasion[7], a lymph node involvement[26], and a tumor infiltration[7,26,27].
Regarding prognosis, three studies have reported that ARID1A abnormalities wereassociated with a poorer prognosis in multivariate analyses[7,26,27]; however, ARID1A
abnormalities were associated with a better prognosis in a stage-independent mannerin one study[23]. In our study, cases with abnormal ARID1A expression had aInada et al. page 20

significantly worse prognosis in a multivariate analysis. This discrepancy might bedue to the different parameters analyzed in the multivariate analyses. The previous
study reporting ARID1A abnormalities as a better prognostic factor analyzed only
clinical stage, MMR status, and histology in their multivariate analysis[23]. Moreover,this study involved a relatively limited number of cases compared with the otherstudies including ours[23].
The current studies confirmed the previously reported correlation between MMR
deficiency and the loss of ARID1A expression[7,23-26]. ARID1A contains many shortrepeats of 4-7 mononucleotides in its coding region, which is prone toinsertion/deletion mutations in MMR-deficient tumors. Indeed, previous studies
have shown that the majority of ARID1A mutations occur in its repeating sequence,leading to frameshift mutations and the complete loss of ARID1A proteins, in gastriccancers with MMR-deficiency[23,24].
In conclusion, the present study showed the clinicopathological significance of
MMR deficiency and ARID1A abnormalities and the correlation of these two
conditions in gastric cancers. Furthermore abnormal ARID1A expression was
independently associated with an unfavorable prognosis. We also confirmed theInada et al. page 21

previously reported association between MMR deficiency and abnormal ARID1A
expression.

Acknowledgments
We are grateful to Ms. Sachiko Miura and Ms. Chizu Kina for technical assistance.
COMMENTS
Background
ARID1A plays a role in the regulation of diverse cellular processes, fromdevelopment and differentiation to proliferation. Recently, ARID1A mutations havealso been reported in some tumors, including gastric cancer.
Research frontiersSomestudieshave

examinedclinicopathologicalsignificanceof

ARID1A

inactivations; interestingly, a significant relationship between ARID1A mutations and
MMR deficiency have been suggested in gastric cancers.
Innovations and breakthroughs
We showed that abnormal ARID1A expression was independently associated with anunfavorable prognosis in a large consecutive series of advanced gastric cancers using

Inada et al. page 22

immunohistochemistry, and we also confirmed the association between MMR
deficiency and abnormal ARID1A expression.
Applications
The present study suggests that ARID1A inactivation could be a potentially negative
prognostic factor in gastric cancers.
Terminology
ARID1A is a key component of the multi-protein SWI/SNF chromatin remodelingcomplex, and is involved in the regulation of diverse cellular processes, fromdevelopment and differentiation to proliferation.
Peer review

Inada et al. page 23

REFERENCES
1.

Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence andmortality rates and trends. Cancer Epidemiol Biomarkers Prev 2010; 19: 1893-1907[PMID: 20647400 DOI: 10.1158/1055-9965]

2.

Oki E, Kakeji Y, Zhao Y, Yoshida R, Ando K, Masuda T, Ohgaki K, Morita M,
Maehara Y. Chemosensitivity and survival in gastric cancer patients withmicrosatellite instability. Ann Surg Oncol 2009; 16: 2510-2515 [PMID: 19565284 DOI:
10.1245/s10434-009-0580-8]

3.

Lee JH, Abraham SC, Kim HS, Nam JH, Choi C, Lee MC, Park CS, Juhng SW, Rashid
A, Hamilton SR, Wu TT. Inverse relationship between APC gene mutation in gastricadenomas and development of adenocarcinoma. Am J Pathol 2002; 161: 611-618[PMID: 12163385 DOI: 10.1016/S0002-9440(10)64216-2]

4.

Jascur T, Boland CR. Structure and function of the components of the human DNA
mismatch repair system. Int J Cancer 2006; 119: 2030-2035 [PMID 16804905 DOI:
10.1002/ijc.22023]

5.

Maehara Y, Egashira A, Oki E, Kakeji Y, Tsuzuki T. DNA repair dysfunction in
gastrointestinal tract cancers. Cancer Sci 2008; 99: 451-458 [PMID: 18271874 DOI:

Inada et al. page 24

10.1111/j.1349-7006.2007.00671.x.]
6.

Leung SY, Yuen ST, Chung LP, Chu KM, Chan AS, Ho JC. hMLH1 promoter
methylation and lack of hMLH1 expression in sporadic gastric carcinomas withhigh-frequency microsatellite instability. Cancer Res 1999; 59: 159-164 [PMID: 9892201]

7.

Abe H, Maeda D, Hino R, Otake Y, Isogai M, Ushiku AS, Matsusaka K, Kunita A,
Ushiku T, Uozaki H, Tateishi Y, Hishima T, Iwasaki Y, Ishikawa S, Fukayama M.
ARID1A expression loss in gastric cancer: pathway-dependent roles with and without
Epstein-Barr virus infection and microsatellite instability. Virchows Arch 2012; 461:
367-377 [PMID: 22915242 DOI: 10.1007/s00428-012-1303-2]

8.

Beghelli S, de Manzoni G, Barbi S, Tomezzoli A, Roviello F, Di Gregorio C, Vindigni
C, Bortesi L, Parisi A, Saragoni L, Scarpa A, Moore PS. Microsatellite instability ingastric cancer is associated with better prognosis in only stage II cancers. Surgery 2006;
139: 347-356 [PMID: 16546499 DOI: 10.1016/j.surg.2005.08.021]

9.

Kim SH, Ahn BK, Nam YS, Pyo JY, Oh YH, Lee KH. Microsatellite instability is
associated with the clinicopathologic features of gastric cancer in sporadic gastriccancer patients. J Gastric Cancer 2010; 10: 149-154 [PMID: 22076179 DOI:
10.5230/jgc.2010.10.4.149]

Inada et al. page 25

10. An JY, Kim H, Cheong JH, Hyung WJ, Kim H, Noh SH. Microsatellite instability insporadic gastric cancer: its prognostic role and guidance for 5-FU based
chemotherapy after R0 resection. Int J Cancer 2012; 131: 505-511 [PMID: 21898388 DOI:
10.1002/ijc.26399]
11. Seo HM, Chang YS, Joo SH, Kim YW, Park YK, Hong SW, Lee SH. Clinicopathologic
characteristics and outcomes of gastric cancers with the MSI-H phenotype. J Surg
Oncol 2009; 99: 143-147 [PMID: 19117018 DOI: 10.1002/jso.21220]
12. Lee HS, Choi SI, Lee HK, Kim HS, Yang HK, Kang GH, Kim YI, Lee BL, Kim WH.
Distinct clinical features and outcomes of gastric cancers with microsatellite
instability.

Mod

Pathol2002;

15:

632-640[PMID:

12065777

DOI:

10.1038/modpathol.3880578]
13. Falchetti M, Saieva C, Lupi R, Masala G, Rizzolo P, Zanna I, Ceccarelli K, Sera F,Mariani-Costantini R, Nesi G, Palli D, Ottini L. Gastric cancer with high-level
microsatellite instability: target gene mutations, clinicopathologic features, andlong-term survival. Hum Pathol 2008; 39: 925-932 [PMID: 18440592 DOI:
10.1016/j.humpath.2007.10.024]
14. Arai T, Sakurai U, Sawabe M, Honma N, Aida J, Ushio Y, Kanazawa N, Kuroiwa K,

Inada et al. page 26

Takubo K. Frequent microsatellite instability in papillary and solid-type, poorly
differentiated adenocarcinomas of the stomach. Gastric cancer 2013; 16: 505-512[PMID: 23274922 DOI: 10.1007/s10120-012-0226-6]
15. Wilson BG, Roberts CW. SWI/SNF nucleosome remodellers and cancer. Nat Rev
Cancer 2011; 11: 481-492 [PMID: 21654818 DOI: 10.1038/nrc3068]
16. Ho L, Crabtree GR. Chromatin remodelling during development. Nature 2010; 463:
474-484 [PMID: 20110991 DOI: 10.1038/nature08911]
17. Weissman B, Knudsen KE. Hijacking the chromatin remodeling machinery: impact of
SWI/SNF perturbations in cancer. Cancer Res 2009; 69: 8223-8230 [PMID: 19843852
DOI: 10.1158/0008-5472.CAN-09-2166]
18. Guan B, Wang TL, Shih IeM. ARID1A, a factor that promotes formation of
SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic
cancers.

Cancer

Res

2011;

71:

6718-6727[PMID:

21900401

DOI:

10.1158/0008-5472.CAN-11-1562]
19. Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, Senz J, McConechy MK,
Anglesio MS, Kalloger SE, Yang W, Heravi-Moussavi A, Giuliany R, Chow C, Fee J,
Zayed A, Prentice L, Melnyk N, Turashvili G, Delaney AD, Madore J, Yip S,

Inada et al. page 27

McPherson AW, Ha G, Bell L, Fereday S, Tam A, Galletta L, Tonin PN, Provencher D,
Miller D, Jones SJ, Moore RA, Morin GB, Oloumi A, Boyd N, Aparicio SA, Shih IeM,
Mes-Masson AM, Bowtell DD, Hirst M, Gilks B, Marra MA, Huntsman DG. ARID1A
mutations in endometriosis-associated ovarian carcinomas. N Engl J Med 2010; 363:
1532-1543 [PMID: 20942669 DOI: 10.1056/NEJMoa1008433]
20. Jones S, Wang TL, Shih IeM, Mao TL, Nakayama K, Roden R, Glas R, Slamon D, Diaz
LA Jr, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N. Frequent
mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma.
Science 2010; 330: 228-231 [PMID: 20826764 DOI: 10.1126/science.1196333]
21. Wiegand KC, Lee AF, Al-Agha OM, Chow C, Kalloger SE, Scott DW, Steidl C,
Wiseman SM, Gascoyne RD, Gilks B, Huntsman DG. Loss of BAF250a (ARID1A) isfrequent in high-grade endometrial carcinomas. J Pathol 2011; 224: 328-333 [PMID:
21590771 DOI: 10.1002/path.2911]
22. Guan B, Mao TL, Panuganti PK, Kuhn E, Kurman RJ, Maeda D, Chen E, Jeng YM,
Wang TL, Shih IeM. Mutation and loss of expression of ARID1A in uterine low-grade
endometrioid carcinoma. Am J Surg Pathol 2011; 35; 625-632 [PMID: 21412130 DOI:
10.1097/PAS.0b013e318212782a]

Inada et al. page 28

23. Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, Chan TL, Kan Z, Chan AS, Tsui WY,
Lee SP, Ho SL, Chan AK, Cheng GH, Roberts PC, Rejto PA, Gibson NW, Pocalyko DJ,
Mao M, Xu J, Leung SY. Exome sequencing identifies frequent mutation of ARID1A inmolecular subtypes of gastric cancer. Nat Genet 2011; 43: 1219-1223 [PMID: 22037554
DOI: 10.1038/ng.982]
24. Jones S, Li M, Parsons DW, Zhang X, Wesseling J, Kristel P, Schmidt MK, Markowitz
S, Yan H, Bigner D, Hruban RH, Eshleman JR, Iacobuzio-Donahue CA, Goggins M,
Maitra A, Malek SN, Powell S, Vogelstein B, Kinzler KW, Velculescu VE,
Papadopoulos N. Somatic mutations in the chromatin remodeling gene ARID1A
occur in several tumor types. Hum Mutat 2012; 33: 100-103 [PMID: 22009941 DOI:
10.1002/humu.21633]
25. Zang ZJ, Cutcutache I, Poon SL, Zhang SL, McPherson JR, Tao J, Rajasegaran V, Heng
HL, Deng N, Gan A, Lim KH, Ong CK, Huang D, Chin SY, Tan IB, Ng CC, Yu W, Wu
Y, Lee M, Wu J, Poh D, Wan WK, Rha SY, So J, Salto-Tellez M, Yeoh KG, Wong WK,Zhu YJ, Futreal PA, Pang B, Ruan Y, Hillmer AM, Bertrand D, Nagarajan N, Rozen S,
Teh BT, Tan P. Exome sequencing of gastric adenocarcinoma identifies recurrent
somatic mutations in cell adhesion and chromatin remodeling genes. Nat Genet 2012;

Inada et al. page 29

44: 570-574 [PMID: 22484628 DOI: 10.1038/ng.2246]
26. Wiegand KC, Sy K, Kalloger SE, Li-Chang H, Woods R, Kumar A, Streutker CJ,
Hafezi-Bakhtiari S, Zhou C, Lim HJ, Huntsman DG, Clarke B, Schaeffer DF.
ARID1A/BAF250a as a prognostic marker for gastric carcinoma: a study of 2 cohorts.
Hum Pathol 2014; 45:1258-1268 [PMID: 24767857 DOI: 10.1016/j.humpath.2014.02.006]
27. Wang DD, Chen YB, Pan K, Wang W, Chen SP, Chen JG, Zhao JJ, Lv L, Pan QZ, Li YQ,
Wang QJ, Huang LX, Ke ML, He J, Xia JC. Decreased expression of the ARID1A geneis associated with poor prognosis in primary gastric cancer. PLoS One 2012; 7: e40364[PMID: 22808142 DOI: 10.1371/journal.pone.0040364]
28. Washington K. 7th edition of the AJCC cancer staging manual: stomach. Ann Surg
Oncol 2010; 17: 3077-3079 [PMID: 20882416 DOI: 10.1245/s10434-010-1362-z]
29. Vasen HF, Watson P, Mechlin JP, Lynch HT. New clinical criteria for hereditary
nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by theInternational Collaborative group on HNPCC. Gastroenterology 1999; 116: 1453-1456[PMID: 10348829 DOI 10.1016/S0016-5085(99)70510-X]
30. Zhang L. Immunohistochemistry versus microsatellite instability testing for screening
colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer

Inada et al. page 30

syndrome. Part II. The utility of microsatellite instability testing. J Mol Diagn 2008; 10:301-307 [PMID: 18556776 DOI: 10.2353/jmoldx.2008.080062]
31. Shia J, Tang LH, Vakiani E, Guillem JG, Stadler ZK, Soslow RA, Katabi N, Weiser MR,
Paty

PB,

Temple

LK,

Nash

GM,

Wong

WD,

Offit

K,

KlimstraDS.

Immunohistochemistry as first-line screening for detecting colorectal cancer patientsat risk for hereditary nonpolyposis colorectal cancer syndrome: a 2-antibody panel
may be as predictive as a 4-antibody panel. Am J Surg Pathol 2009; 33: 1639-1645[PMID: 19701074 DOI: 10.1097/PAS.0b013e3181b15aa2]
32. Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA
mismatch repair gene proteins. Virchows Arch 2004; 445: 431-441 [PMID: 15455227 DOI
10.1007/s00428-004-1090-5]
33. Poynter JN, Siegmund KD, Weisenberger DJ, Long TI, Thibodeau SN, Lindor N,
Young J, Jenkins MA, Hopper JL, Baron JA, Buchanan D, Casey G, Levine AJ, Le
Marchand L, Gallinger S, Bapat B, Potter JD, Newcomb PA, Haile RW, Laird PW;
Colon Cancer Family Registry Investigators. Molecular characterization of MSI-H
colorectal cancer by MLHI promoter methylation, immunohistochemistry, andmismatch repair germline mutation screening. Cancer Epidemiol Biomarkers Prev 2008;

Inada et al. page 31

17: 3208-3215 [PMID: 18990764 DOI: 10.1158/1055-9965]
34. Leite M, Corso G, Sousa S, Milanezi F, Afonso LP, Henrique R, Soares JM, Castedo S,
Carneiro F, Roviello F, Oliveira C, Seruca R. MSI phenotype and MMR alterations infamilial and sporadic gastric cancer. Int J Cancer 2011; 128: 1606-1613 [PMID: 20533283
DOI: 10.1002/ijc.25495]
35. Ward RL, Turner J, Williams R, Pekarsky B, Packham D, Velickovic M, Meagher A,
O'Connor T, Hawkins NJ. Routine testing for mismatch repair deficiency in sporadic
colorectal cancer is justified. J Pathol 2005; 207: 377-384 [PMID: 16175654]
36. Prinz C, Schwendy S, Voland P. H pylori and gastric cancer: shifting the global
burden. World J Gastroenterol 2006; 12: 5458-5464 [PMID: 17006981 DOI:
10.3748/wjg.v12.i34.5458]
37. Lee JH, Park SJ, Abraham SC, Seo JS, Nam JH, Choi C, Juhng SW, Rashid A, Hamilton
SR, Wu TT. Frequent CpG island methylation in precursor lesions and early gastricadenocarcinomas.

Oncogene

2004;

23:

4646-4654[PMID:

15064707

DOI:

10.1038/sj.onc.1207588]
38. Isogaki J, Shinmura K, Yin W, Arai T, Koda K, Kimura T, Kino I, Sugimura H.
Microsatellite instability and K-ras mutations in gastric adenomas, with reference toInada et al. page 32

associated gastric cancers. Cancer Detect Prev 1999; 23: 204-214 [PMID: 10336999 DOI:
10.1046/j.1525-1500.1999.99020.x]
39. Gryfe R, Kim H, Hsieh ET, Aronson MD, Holowaty EJ, Bull SB, Redston M, Gallinger
S. Tumor microsatellite instability and clinical outcome in young patients withcolorectal cancer. N Engl J Med 2000; 342: 69-77 [PMID: 10631274 DOI:
10.1056/NEJM200001133420201]
40. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW,
Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S. A National
Cancer Institute Workshop on Microsatellite Instability for cancer detection andfamilial predisposition: development of international criteria for the determination of
microsatellite instability in colorectal cancer. Cancer Res 1998; 58: 5248-5257 [PMID:
9823339]
41. Maeda D, Mao TL, Fukayama M, Nakagawa S, Yano T, Taketani Y, Shih IeM.
Clinicopathological significance of loss of ARID1A immunoreactivity in ovarian clear
cell carcinoma. Int J Mol Sci 2010; 11: 5120-5128 [PMID: 21614196 DOI:
10.3390/ijms11125120]
42. Yamamoto S, Tsuda H, Takano M, Tamai S, Matsubara O. Loss of ARID1A protein

Inada et al. page 33

expression occurs as an early event in ovarian clear-cell carcinoma development andfrequently coexists with PIK3CA mutations. Mod Pathol 2012; 25: 615-624 [PMID:
22157930 DOI: 10.1038/modpathol.2011.189]
P-Reviewer:

S-Editor: Qi Y L-Editor: E-Editor:

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TablesTable 1. Immunohistochemical expression of MMR† proteins
Immunohistochemical expressionn = 489

Loss of MLH1 and PMS2

33

Loss of PMS2

3

Loss of MSH2 and MSH6

1

Loss of all four proteins1

Retention of all four proteins

451†MMR, Mismatch repair

Inada et al. page 35

Table 2. Clinicopathologic features of the 489 patients with gastric cancers
according to MMR† status
Total(n = 489)

MMR†
Deficient

Intact(n = 38)(n = 451)

Age (years)
Mean ± SD<0.001*61.0 ± 11.8

68.6 ± 9.2

60.4 ± 11.7

Gender

0.004**

Male

327 (66.9%)

17 (44.7%)

310 (68.7%)

Female162 (33.1%)

21 (55.3%)

141 (31.3%)

Serum CEA‡ (ng/ml)
Mean ± SD

0.910*
36.3 ± 315.7

4.4 ± 6.0

39.0 ±328.7

Tumor size (mm)Mean ± SD

0.831*
80.3 ± 45.1

79.0 ± 38.9

80.4 ± 45.6

Tumor location

0.021**

Fundus & Corpus

356 (72.8%)

21 (55.3%)

335 (74.3%)

Antrum133 (27.2%)

17 (44.7%)

116 (25.7%)

Histology

0.023**

Differentiated type

193 (39.5%)

22 (57.9%)

171 (39.7%)

Undifferentiated type

296 (60.5%)

16 (42.1%)

280 (62.1%)

Lymphatic invasion

1.000**

Absent137 (28.0%)

10 (26.3%)

127 (28.2%)

Present

352 (72.0%)

28 (73.7%)

324 (71.8%)

Venous invasion

0.609**

Absent281 (57.5%)

20 (52.6%)

261 (57.9%)

Present

208 (42.5%)

18 (47.4%)

190 (42.1%)

Primary tumor
T2, 3

P-value

0.612**
241 (49.3%)

22 (57.9%)

219 (48.6%)

Inada et al. page 36
T4

248 (50.7%)

16 (42.1%)

232 (51.4%)

Lymph node involvement

0.438**

N0

118 (24.1%)

11 (28.9%)

107 (23.7%)

N1, 2, 3

371 (75.9%)

27 (71.1%)

344 (76.3%)

Distant metastasis

0.202**

M0

336 (68.7%)

30 (78.9%)306 (67.8%)

M1

153 (31.3%)

8 (21.1%)

145 (32.2%)Stage

0.860**

Stage I, II

172 (35.2%)

14 (36.8%)

158 (35.0%)

Stage III, IV

317 (64.8%)

24 (63.2%)

293 (65.0%)

Residual disease

0.161**

Negative375 (76.7%)

33 (86.8%)

342 (74.8%)

Positive

114 (23.3%)

5 (13.2%)

109 (24.2%)†MMR, Mismatch repair; ‡CEA, Carcinoembryonic antigen; *, Mann-Whitney’U
test; **, Fisher’s exact test

Inada et al. page 37

Table 3. Clinicopathological features of 489 patients with gastric cancer
according to ARID1A expression
ARID1A expression
Loss / Weak

Retained(n = 109)(n = 380)

Age (years)
Mean±SD

0.212*
62.1 ± 10.9

60.7 ± 12.0

Gender

0.419**

Male

69 (63.3%)

258 (67.9%)

Female40 (36.7%)

122 (32.1%)

Serum CEA† (ng/ml)
Mean ± SD

0.937*
39.7 ± 363.4

35.3 ± 301.3

Tumor size (mm)Mean ± SD

0.336*
83.9 ± 46.1

79.2 ± 44.8

Tumor location

0.067**

Fundus & Corpus

87 (79.8%)

269 (70.8%)

Antrum22 (20.2%)

111 (29.2%)

Histology

0.739**

Differentiated type

41 (37.6%)

152 (40.0%)

Undifferentiated type

68 (62.4%)

228 (60.0%)

Lymphatic invasion

0.022**

Absent21 (19.3%)

116 (30.5%)

Present

88 (80.7%)264 (69.5%)

Venous invasion

0.443**

Absent59 (54.1%)

222 (58.4%)

Present

50 (45.9%)

158 (41.6%)

Primary tumor
T2, 3

P-value

0.065**
45 (41.3%)

184 (48.4%)

Inada et al. page 38
T4

64 (58.7%)

196 (51.6%)

Lymph node involvement

0.042**N0

18 (16.5%)

100 (26.3%)

N1, 2, 3

91 (83.5%)280 (73.7%)

Distant metastasis0.725**M0

73 (67.0%)

263 (69.2%)

M1

36 (33.0%)

117 (30.8%)

Stage

0.111**

Stage I, II

31 (28.4%)

141 (37.1%)

Stage III, IV

78 (71.6%)

239 (62.9%)

Residual disease

0.798**

Negative

85 (78.0%)

290 (76.3%)

Positive24 (22.0%)

90 (23.7%)†CEA, Carcinoembryonic antigen; *, Mann-Whitney U test; **, Fisher’s exact test

Inada et al. page 39

Table 4. Cox’s proportional hazard model analysis of prognostic factors in 489
patients with gastric cancers
VariablesHR†

95% CI‡

P-Value

Univariate analysis

HR†

95% CI‡

P-Value

Multivariate analysis

Age (years)≧ 60 / ≦ 59

1.22

0.94-1.59

0.123

0.72

0.56-0.94

0.015

0.72

0.55-0.95

0.020

1.75

1.33-2.33<0.001

1.54

1.15-2.06

0.004

3.70

2.50-5.26<0.001

1.88

1.25-2.83

0.002

1.54

1.12-2.01

0.002

1.64

1.24-2.16

0.001

3.23

2.22-4.55<0.001

1.48

0.98-2.22

0.062

1.89

1.45-2.44<0.001

1.21

0.92-1.60

0.171

9.09

5.56-14.29 <0.001

3.77

2.30-6.17

0.023

6.25

5.00-8.33<0.001

3.79

2.85-5.03<0.0010.74

0.44-1.25

0.264

1.30

0.98-1.75

0.070

1.36

1.01-1.84

0.040

Sex
Male / Female
Serum CEA§ (ng/ml)≧ 5.0 / < 5.0
Tumor size (mm)≧ 50 / < 50
Histology
Differentiated/
Undifferentiated type
Lymphatic invasion
Present / Absent
Venous invasion
Present / Absent
Stage
Stage III, IV / I, II
Residual disease
Positive / Negative
MMR status
Deficient / Intact
ARID1A status
Abnormal / Retained†HR, Hazards ratio; ‡CI, confidence interval; §CEA, carcinoembryonic antigen

Inada et al. page 40

Table 5. Relationship between MMR† protein and ARID1A expression
ARID1A expression
Loss

Weak

Homo‡

Hetero§ Homo‡

Hetero§(n = 43)(n = 29) (n = 21)(n = 16)

Retained

P-value(n = 380)

MMR† status<0.001*

Deficient10

4(n=38)(26.3%)

Intact(n=451)2

2

20(10.5%) (5.3%)(5.3%)(52.6%)

33

25

19

14

360

(7.3%)(5.6%)(4.2%)(3.1%)(79.8%)†MMR, Mismatch repair; ‡Homo, Homogeneous; §Hetero, Heterogeneous; *,Extended Fisher’s exact test

Inada et al. page 41

Figure legends
Figure 1. Immunohistochemistry for MMR proteins in a case of gastric cancer witha MMR-deficient phenotype.
Immunohistochemistry for MLH1 (A), PMS2 (B), MSH2 (C), and MSH6 (D). MLH1
and PMS2 expression are absent in tumor cells, whereas stromal cells show nuclear
expression (A, B). On the other hand, the tumor cells retain MSH2 and MSH6
expression (C, D). These staining patterns were consistent with those caused by
MLH1 deficiency.

Figure 2. Immunohistochemistry for ARID1A
A. Homogeneous loss of expression. All the tumor cells show no expression,
whereas the stromal cells retain the nuclear expression of ARID1A. B. Heterogeneous
loss of expression. Most of the tumor cells show no expression, whereas some of thegland-forming tumor cells retain nuclear expression (arrows). C. Homogeneously
weak expression. The tumor cells show the reduced expression of ARID1A.
Non-neoplastic gastric glandular cells retain the expression (arrowheads). D.
Heterogeneously weak expression. Most of the tumor cells exhibit reduced expression,Inada et al. page 42

but a subset of tumor cells retain nuclear expression (arrow). E. Retained expression:
Tumor cells (arrow) show strong nuclear ARID1A expression, similar to
non-neoplastic glandular cells (arrowheads).

Figure 3. Kaplan–Meier estimates of disease specific survival for patients withgastric cancer according to the MMR (A) and ARID1A expression (B) statuses
The disease specific survival curves according to the MMR and ARID1A expression
statuses did not show significant differences. †MST, median survival time; *, thelog–rank test.

Inada et al. page 43

Figure 1

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Figure 2Inada et al. page 45

Figure 3