Clinical Significance of Cellular Distribution of Moesin in Patientswith Oral Squamous Cell Carcinoma

Hiroichi Kobayashi,1 Junji Sagara,2

Hiroshi Kurita,1 Masayo Morifuji,3

Masamichi Ohishi,3 Kenji Kurashina,1 andShun’ichiro Taniguchi2

Departments of 1Dentistry and Oral Surgery and 2MolecularOncology and Angiology, Aging and Adaptation, Shinshu UniversitySchool of Medicine, Matsumoto, Japan and 3First Department of Oraland Maxillofacial Surgery, Faculty of Dentistry, Kyushu University,Fukuoka, Japan

ABSTRACTPurpose: Moesin is a linking protein of the submembra-

neous cytoskeleton and plays a key role in the control of cellmorphology, adhesion, and motility. The aim of the presentstudy was to elucidate the clinical significance of expressionpatterns of moesin in patients with oral squamous cell car-cinoma (OSCC).

Experimental Design: Immunohistochemistry for moe-sin monoclonal antibody was performed on 103 paraffin-embedded specimens from patients with primary OSCC,including 30 patients with locoregional lymph node metas-tasis, and in the sections from nude mice transplanted withtwo cell lines derived from a single human tongue cancer(SQUU-A and SQUU-B).

Results: Expression patterns of moesin in OSCCs weredivided into three groups: membranous pattern; mixed pat-tern; and cytoplasmic pattern. These expression patternscorrelated with tumor size, lymph node metastasis, mode ofinvasion, differentiation, and lymphocytic infiltration. Inabout two-thirds of the patients with metastatic lymph node,homogeneous cytoplasmic expression was detected in themetastatic lymph nodes. In addition, SQUU-B with highmetastatic potential showed more reduced levels of mem-brane-bound moesin than SQUU-A with low metastatic po-tential. A multivariate analysis demonstrated that expres-sion patterns of moesin can be an independent prognosticfactor.

Conclusions: Our results suggest that moesin expres-sion contributed to discriminating between patients with thepotentiality for locoregional lymph node metastasis and

those with a better prognosis and might improve the defini-tion of suitable therapy for each.

INTRODUCTIONOral cancer, 1 of the 10 most common cancers in the world,

remains a morbid and often fatal disease. Despite marked advancesof management and diagnosis of oral squamous cell carcinoma(OSCC), the overall survival ratio has showed only a modestincrease in recent years. Therefore, the development of molecularmarkers is needed to improve the diagnosis and assessment oftumor progression and metastasis in OSCC patients.

Moesin is a member of the ERM (ezrin/radixin/moesin)family, which shares �78% amino acid sequence identity witheach other. ERM proteins, part of the band 4.1 superfamily, actas a membrane-cytoskeleton linker in actin-enriched specializedplasma membrane structures, especially microvilli, rufflingmembranes, and cleavage furrows and thus play a key role in thecontrol of cell morphology, adhesion, and motility (1–6). Theintegral membrane proteins such as CD44, CD43, intercellularadhesion molecules 1 and 2, and actin are identified as ligandsfor ERM proteins (7–9). Merlin, encoded by the neurofibroma-tosis type 2, is classified as a tumor suppressor protein (10).Because moesin shares high homology with Merlin and colo-calizes with it beneath the plasma membrane, it has been spec-ulated that moesin may also be a tumor suppressor (11, 12).However, recent studies have indicated that ERM proteins areup-regulated in various kinds of tumors (13–17). Whether moe-sin is functional as a tumor suppressor in carcinogenesis andtumor development remains unclear.

Because little is known about the role of moesin in the oralnormal mucosa and oral lesions, including leukoplakia, verru-cous carcinoma, and small cell carcinoma, we initiated a seriesof studies aimed at characterizing expression of moesin (18). Inthis particular study, we report that OSCC cells constitutivelydisplay several expression patterns of moesin, thereby providinga new biomolecular marker for use in prediction of metastasisand poor prognosis.

MATERIALS AND METHODSPatients and Tumor Sample. The study group consisted

of 103 patients with OSCC who were diagnosed at the Departmentof Dentistry and Oral Surgery, Shinshu University School of Med-icine. Tissues of primary (n � 103) and metastatic (n � 30) lesionsof OSCCs were collected during biopsy or operation after patientssigned the informed consent form approved by the InstitutionReview Committee. Permission to perform this study was obtainedfrom the committee. Thirty-one patients (T1–T2) without lymphnode metastasis underwent radiotherapy alone while 72 patientsunderwent surgery. The grade of tumor differentiation was deter-mined according to the criteria proposed by the WHO (19). Modeof invasion was classified according to Jakobsson’s classification(20). Median follow-up time was 32.0 months (range, 2–115

Received 11/1/02; revised 10/14/03; accepted 10/20/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Requests for reprints: Hiroichi Kobayashi, Department of Dentistryand Oral Surgery, Shinshu University School of Medicine, Asahi 3-1-1,Matsumoto 390-8621, Japan. Phone: 81-263-37-2677; Fax: 81-263-37-2676; E-mail: h-koba@hsp.md.shinshu-u.ac.jp.

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months). The study population consisted of 59 men and 44 womenaveraging 65.0 years of age (range, 27–88 years). For controls,normal oral mucosa were obtained from consenting patients duringremoval of a lower wisdom tooth.

Cell Lines and Culture. Human oral cancer cell lines,SQUU-A and SQUU-B, were established as reported previously(21). These cell lines were cultured in Eagle’s MEM (Nissui,Tokyo, Japan) supplemented with 10% fetal bovine serum (Life

Fig. 1 Staining of normal oral epithelium with monoclonal antibody against moesin. A, expression level was gradually reduced from the parabasallayer toward the top layer. B, reactivity for moesin monoclonal antibody was prominent in the cell membrane of basal layer and parabasal layer cells.Bar, 100 �m in A; 10 �m in B.

Fig. 2 Three staining patterns in oral squamous cell carcinomas withmonoclonal antibody against moesin. A, membranous expression pat-tern, predominant expression in the cell membrane. B, mixed expressionpattern, reactivity in the cell membrane about equal to that in thecytoplasm. C, cytoplasmic expression pattern, predominant cytoplasmiclabeling. Bar, 10 �m.

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Technologies, Inc., Grand Island, NY), penicillin (100 IU/ml),streptomycin (100 mg/ml), and fungizone (1 mg/ml) at 37°C inan atmosphere of 5% CO2.

Animals and Experimental Treatment. FemaleBALB/c mice (6 weeks old) were purchased from SLC (Shi-

zuoka, Japan) and were housed under conventional conditionswith free access to animal chow and water. Under generalanesthesia with diethylether, �3.5 � 105/0.035 ml viable cellswere injected in the s.c. tissue of the right side of the tongue.The mice were sacrificed 5 weeks after the infection, and

Fig. 3 Immunohistochemical localization of moesin in primary tissue and metastatic lymph nodes of the same oral squamous cell carcinoma patient.A and B, membranous expression of moesin in primary tissue. C and D, cytoplasmic expression in front of an invasive margin of primary tumor cells.E and F, cytoplasmic expression homogeneously observed. Bar, 100 �m in A, C, and E; 10 �m in B, D, and F.

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tongues were resected. The care and use of these experimentalanimals were in accordance with institutional guidelines.

Antibodies and Immunohistochemical Staining.Mouse monoclonal antibody (CR-22) was kindly provided byDrs. Shoichiro Tsukita and Sachiko Tsukita, Kyoto University(Kyoto, Japan); this antibody has a higher affinity for moesin(22). The specificity of antibody against moesin was confirmedwith Western blotting and immunoprecipitation of the cell ly-sates from human peripheral leukocyte (23) and human malig-nant melanoma cells (11). In addition, the samples were re-ported to show similar staining in frozen sections as well asformalin-fixed, paraffin-embedded section (11, 23). Further-more, our previous study also validated the specificity of thisantigen by Western blot analysis from OSCC tissues and im-munohistochemical staining in frozen OSCC tissues (18).Horseradish peroxidase-conjugated goat antimouse polyclonalantibody (Dako, Copenhagen, Denmark) and anti-actin (�)monoclonal antibody (Abcam, Cambridge, United Kingdom)were purchased, respectively.

All samples were fixed in 10% formalin and embeddedin paraffin to prepare serial sections. Expression of moesinwas examined by the indirect peroxidase technique as de-scribed previously (23). Tissues embedded in paraffin werecut into 3-�m sections and mounted onto silane-coated glassslides. The slides were dewaxed in xylene, dehydrated indescending dilutions of ethanol, and preincubated with asolution of 1% hydrogen peroxide in methanol to suppressthe endogenous peroxidase for 30 min at room temperature.After being rinsed in distilled water, the sections were mi-

crowaved (500 w, 25 min) for antigen retrieval in 0.01 M

citric acid (pH 6.0). After being washed with distilled waterand 0.05 M Tris-buffered saline (TBS; pH 7.6), the specimenswere treated with mouse anti-moesin antibody diluted byTBS containing 1% BSA at 4°C overnight, washed threetimes with TBS, and then incubated with goat antimouseimmunoglobulin polyclonal antibody diluted by TBS con-taining 1% BSA for 60 min at room temperature. After beingwashed three times with TBS, the sections were developed in0.05 M Tris-buffer (pH 7.6) containing 25 mg/125 ml 3,3�-diaminobenzidine and 0.0015% hydrogen peroxide for 7 min.The sections were then washed in water, counterstained withMayer’s hematoxylin, dehydrated, cleaned, and coverslipped.Negative controls were treated by replacing the primaryantibody with TBS 1% BSA.

Scoring of Results. Sections were examined by two in-dependent researchers (H. Ko., H. Ku.) in an effort to provide aconsensus of staining pattern. Moesin or �-actin expression ofneoplastic cell in primary lesions was classified as follows:membranous pattern—membranous expression of moesin or�-actin was more dominant than cytoplasmic expression; mixedpattern—membranous expression of moesin or �-actin was ap-proximately equal to cytoplasmic expression; and cytoplasmicpattern—membranous expression of moesin or �-actin wasweaker than cytoplasmic expression. We used the expressionpattern of moesin used as the predominant pattern on the wholehistological section of the tumor.

Fig. 4 Comparison of percentage of cytoplasmic expression of moesinin primary tissues and metastatic lymph nodes in the same oral squa-mous cell carcinoma patient with cervical lymph node metastasis. In allprimary tissues, cytoplasmic expression type was heterogeneously ob-served (range, 10–92%), whereas in about two-thirds of the metastaticlymph nodes, cytoplasmic expression pattern was homogeneously dis-played.

Table 1 Expression pattern of moesin in oral squamous cellcarcinoma according to clinicopathological features of patients

FeaturesTotalno.

Expression pattern of moesin

PMembranous Mixed Cytoplasmic

Total no. of patients 103 28 38 37Age (yrs) 0.3277

�65 44 8 20 16�65 59 20 18 21

Gender 0.8481Male 59 15 23 21Female 44 13 15 16

T classification 0.0012T1 � 12T2 � 53

65 24 24 17

T3 � 14T4 � 24

38 4 14 20

N classification �0.0001N0 69 26 27 16N1 N2 34 2 11 21

Mode of invasion 0.00171 � 62 � 383 � 38

82 27 31 24

4 � 21 21 1 7 13Differentiation 0.0002

Well � 65 65 25 24 16Moderate � 28Poor � 10

38 3 14 21

Lymphocyticinfiltration

0.0034

Low � 29 29 4 8 17Moderate � 53Marked � 21

74 24 30 20

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Thirty cases with locoregional lymph node metastasis wereevaluated for heterogeneity of tumor cells in the primary lesionsas well as in the metastatic lesions by examination in 10 ran-domized fields of sections at a magnification of �400. Expres-sion percentages of cytoplasmic expression pattern of moesinwas calculated from these.

Statistics. The relationships between expression of moe-sin and clinicopathological indices such as age, gender, tumorsize, lymph node metastasis, differentiation, mode of invasion,and lymphocytic infiltration were evaluated by Mann-Whitney’sU test. Kaplan-Meier survival curves were constructed andlog-rank tests performed to assess whether the expression pat-tern of moesin in neoplastic cells had any effect on overallsurvival of patients with oral cancer. Relative risk of death was

calculated by univariate and multivariate analysis using Coxregression models. The correlation between expression patternsof moesin and �-actin was estimated by Spearman’s rank cor-relation.

RESULTSMoesin Expression in Normal Oral Epithelia. In basal

layer cells as well as restricted parabasal layer cells and spinouslayer cells, the membranous expression pattern of moesin isdominant. Weak immunoreactivity for moesin was seen in thecytoplasm of basal layer cells, and no apparent staining withanti-moesin antibody was observed in cornified layer cells (Fig.1, A and B).

Moesin Expression in Primary and Metastatic Tumors.In a previous study, we showed that moesin expression de-creased in the membrane and increased in the cytoplasm inaccordance with the degree of transformation and malignancy oforal lesions, including OSCC, verrucous carcinoma, and dys-plastic lesion. In this study, we focused on moesin expression inprimary tumors and metastatic lymph nodes.

The cellular distribution pattern of moesin differed sub-stantially in primary tumors and metastatic lymph nodes. Wedivided moesin distribution patterns into three types: membra-nous (Fig. 2A); mixed (Fig. 2B); and cytoplasmic (Fig. 2C)patterns. In OSCC patients with locoregional lymph node me-tastasis, primary tumors showed various distribution patterns ofmoesin as in Fig. 2, but most metastatic tumors in lymph nodesshowed the cytoplasmic distribution pattern. A case of OSCCpatient with cervical lymph node metastasis is presented in Fig.3. Membranous or cytoplasmic patterns are seen in the primarytumor of the patient (Fig. 3, A–D), but all of the metastatictumors in the lymph nodes display the cytoplasmic pattern (Fig.

Fig. 5 Kaplan-Meier survival curves of patients with oral squamouscell carcinoma according to expression pattern of moesin. The survivalcurves were analyzed by the log-rank test.

Table 2 Univariate and multivariate analysis of clinicopathological data and expression pattern of moesin in 103 cases of oral cancer

Variable HRa 95% CIb P

Univariate analysisAge (�65/�65 years) 1.837 0.729–4.631 0.1973Gender (male/female) 1.128 0.461–2.761 0.4927T classification (T1 T2/T3 T4) 0.325 0.132–0.797 0.0141N classification (N0/N1 N2) 0.262 0.107–0.643 0.0035Mode of invasion (1 2 3/4) 0.297 0.121–0.729 0.0081Differentiation (well/moderate poor) 0.209 0.080–0.554 0.0013Lymphocytic infiltration (marked moderate/low) 0.274 0.114–0.662 0.0040Moesin 0.0014

Cytoplasmic 1.000Mixed 0.203 0.067–0.614Membranous 0.07 0.009–0.532

Multivariate analysisT classification (T1 T2/T3 T4) 0.765 0.228–2.567 0.6639N classification (N0/N1 N2) 0.691 0.202–2.359 0.5550Mode of invasion (1 2 3/4) 0.712 0.265–1.915 0.5008Differentiation (well/moderate poor) 0.408 0.141–1.183 0.0988Lymphocytic infiltration (marked moderate/low) 0.493 0.190–1.282 0.1470Moesin 0.0470

Cytoplasmic 1.000Mixed 0.305 0.098–0.947Membranous 0.163 0.020–1.361

a Hazard ratio (HR) estimated from Cox proportional hazard regression model.b Confidence interval (CI) of the estimated HR.

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3, E and F). Comparison in moesin expression between primaryand metastatic tumors in the same patients demonstrated thatmetastatic cells predominantly showed cytoplasmic pattern,whereas primary tumors showed heterogeneous expression pat-terns (Fig. 4).

Relationships between Expression Pattern of Moesinand Clinicopathological Parameters and Prognosis in OSCCPatients. We compared moesin expression pattern with clin-icopathological parameters in 103 OSCC patients (Table 1).There was an association of expression pattern of moesin withtumor size, cervical lymph node involvement, mode of invasion,differentiation, and lymphocytic infiltration. However, the ex-pression pattern of moesin did not differ significantly withrespect to age and gender.

By the Kaplan-Meier curves, there was significant differ-ence among three groups, membranous pattern, mixed pattern,and cytoplasmic pattern (2 � 18.841, P � 0.0001 by log-ranktest; Fig. 5).

Univariate regression analysis showed that overallsurvival correlated with tumor size, cervical lymph nodemetastasis, mode of invasion, differentiation, lymphocyticinfiltration, and expression pattern of moesin. Furthermore, amultivariate analysis using the Cox proportional hazardsmodel also showed that the expression pattern of moesin(P � 0.0470) is significantly associated with overall survival(Table 2).

Moesin Level and Cellular Localization in Oral Can-cers. In the next experiments, we investigated the expressionof moesin in established cell lines of OSCC. Mixed or predom-inantly cytoplasmic expression patterns of moesin were detectedin a large number of SQUU-A cells with low metastatic poten-tial. These cells showed predominantly membranous expressionof moesin in some part (Fig. 6, A and B), whereas the whole ofSQUU-B cells with high metastatic activity exhibited a down-regulation of membranous expression and an increase in cyto-plasmic expression (Fig. 6, C and D).

Fig. 6 Moesin localization in sections from nude mice transplanted with two cell lines. Tumor cell, at least in part, showed membranous expressionof moesin in SQUU-A with low metastatic ability (A and B), but not in the SQUU-B with high metastatic ability (C and D). Bar, 50 �m in A andC; 10 �m in B and D.

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Correlation between Expression Patterns of Moesinand �-Actin in Primary Tumors. In normal oral epithelia,basal layer and parabasal layer cells showed homogeneouscytoplasmic staining against anti-actin (�) monoclonal antibodyand spinous layer cells showed juxtamembranous staining to-ward the top layer (Fig. 7A). Moreover, in the cytoplasm of allof the tumor cells, �-actin was stained. Especially, in welldifferentiated tumor cells without lymph node metastasis, �-actin was strongly stained beneath the cell membrane (Fig. 7B),whereas �-actin was homogeneously observed in poorly differ-entiated tumor cells with lymph node metastasis (Fig. 7C).Twenty-six (92.8%) of 28 patients whose tumors showed mem-branous expression pattern of moesin showed membranous ex-pression of �-actin, and 31 (83.8%) of 37 patients whose tumorsshowed cytoplasmic expression pattern of moesin showedmixed expression pattern of �-actin. However, no patientsshowed cytoplasmic expression pattern of �-actin. Significantcorrelation between expression patterns of moesin and �-actinwas observed (P � 0.0001; Table 3).

DISCUSSIONIn this study, we noted that expression pattern of moesin

correlated with tumor size, cervical lymph node metastasis,

mode of invasion, differentiation, and lymphocytic infiltration.Furthermore, our results demonstrated that tumor cells withcytoplasmic expression of moesin showed higher incidence oflymph node metastasis than tumor cells with membranous ex-pression of moesin. This is consistent with the observation thatusing a murine model in which almost all the whole cells in ahighly metastatic cell line (SQUU-B) showed cytoplasmic ex-pression of moesin, whereas a small number of cells in a lessmetastatic cell line (SQUU-A) showed membranous expression.Although the biological significance of cellular translocation ofmoesin is unclear, there are several explanations for these find-ings. Firstly, conformational and functional change of moesinresults in redistribution of this molecule in tumor cells. Inactivemoesin is self-associated between the COOH-terminal domainand NH2-terminal domain existing in the cytoplasm (24). Uponreceipt of appropriate activators, phosphatidylinositol 4,5-bisphosphate (25) or phosphorylation of Thr558 (26), moesintranslocates from the cytoplasm to the juxtamembrane by dis-ruption of its intramolecular binding. Thus, the change of bal-ance of activator and/or inactivator for moesin may bring aboutcellular translocation of the molecule. It has been suggested thatphosphatidylinositol 4,5-bisphosphate production is activatedby oncogenic Ras through phosphatidylinositol 3-kinase and Gprotein Rac-induced malignant transformation (27). Secondly,CD44, a cell surface receptor involved in cell adhesion, tumorinvasion, and metastasis, has been cleaved by membrane-type 1matrix metalloproteinase in carcinoma cells at a membrane-proximal domain, thereby suggesting that functional moesinmigrates with CD44 degraded from the cell surface to thecytoplasm (28). Thirdly, because significant correlation betweenexpressions pattern of moesin and �-actin was observed inprimary carcinoma tissues, change of moesin distribution intumor cells may reflect organization of the actin cytoskeletonand an altered cellular environment. Fourthly, according tocarcinogenesis, it is possible that mutant of moesin cannot

Table 3 Correlation between expression patterns of moesin and�-actin in primary oral squamous cell carcinomaa

Expression pattern of �-actin

Membranous Mixed Cytoplasmic Total

Expression pattern of moesinMembranous 26 2 0 28Mixed 24 14 0 38Cytoplasmic 6 31 0 37Total 56 47 0 103

a P � 0.0001 by Spearman’s rank correlation.

Fig. 7 Distribution of �-actin in normal oral epithelia and primary oral squamous cell carcinoma tissues. A, normal oral epithelia. B, well-differentiated carcinoma tissue without lymph node metastasis. C, poorly differentiated carcinoma tissue with lymph node metastasis. Bar, 25 �m.

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cross-link between plasma membrane and actin filament, whichshows an increase in the cytoplasm of neoplastic cells.

Of interest, our previous studies found that OSCC dis-played higher rate of cytoplasmic expression pattern when com-pared with the pattern in normal oral epithelium, oral epithelialdysplasia, and verrucous carcinoma (18). Furthermore, it hasbeen reported that squamous cell carcinoma in the skin showedcytoplasmic expression of moesin (29). However, another studydemonstrated that membranous labeling of ezrin was highlyobserved in metastatic tissues as compared with the primarytissues or hyperplastic specimens (30). A recent study indicatedthat ezrin and moesin might be regulated differently because thebinding activity of ezrin and moesin to L-selectin differed whenphorbol myristate acetate stimulation and protein kinase C in-hibitor were used (31). Thus, the discrepancy between the studyand our series may suggest different regulation among ERMproteins and a distinct role for moesin, depending on the type ofcancer.

Interestingly, all specimens in the primary tumors revealedheterogeneous expression pattern of moesin, but most speci-mens in the metastatic lymph nodes homogeneously showedcytoplasmic expression of moesin. We detected cytoplasmicexpression of moesin in the marginal carcinoma cells of thenests, which seem to constitute the advancing front of cancerinvasion. We speculate that because carcinoma cells at theinvasive front in primary tumors show cytoplasmic expressionof moesin, which greatly degrade the extracellular matrix aswell as CD44, these cells probably are involved in pathologicalprocesses of tumor invasion and metastasis.

The tumor size and regional lymph node involvementare known as indicators for tumor aggressiveness and pooroutcome in OSCC patients. In this study, we observed thesame association using univariate analysis. Furthermore, ourdata indicated that expression pattern of moesin has a prog-nostic value: patients whose tumors showed cytoplasmicexpression had a poorer overall survival compared with pa-tients whose tumors showed cell membranous expression. Inthis study, multivariate analysis demonstrated dramaticallythat the expression pattern of moesin is the only independentprognostic factor in patients with OSCC. In view of itsclinical usefulness, early detection methods clearly predictlocoregional lymph node metastasis as well as poor clinicaloutcome may improve strategic planning. We therefore rec-ommend assessing the expression pattern of moesin usingpretreatment specimens in OSCC clinical trials in an effort tovalidate the most appropriate treatments.

This is the first study to state that the expression pattern ofmoesin is an independent prognostic indicator for patients withOSCC. The expression pattern of moesin may be a clinicallyuseful marker for selection of patients for specific treatments;moreover, modulation of the expression pattern of moesin is apotential therapeutic strategy for improving clinical outcome inOSCC patients.

ACKNOWLEDGMENTSWe thank Drs. Shoichiro Tsukita and Sachiko Tsukita for their kind

gift of anti-moesin monoclonal antibody (CR-22).

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580 Moesin Expression in Oral Squamous Carcinoma

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2004;10:572-580. Clin Cancer Res   Hiroichi Kobayashi, Junji Sagara, Hiroshi Kurita, et al.   Patients with Oral Squamous Cell CarcinomaClinical Significance of Cellular Distribution of Moesin in

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