DF3/MUC1 mucin is expressed in various cancer tissues, and many in vitro studies have suggested that it may play a role in the aggressive behavior of malignant tumors. However, to the best of the authors' knowledge, the relation between DF3/MUC1 expression and outcome has not yet been investigated in patients with oral squamous cell carcinoma (OSCC). The objective of the current study was to evaluate the prognostic significance of DF3/MUC1 expression in patients with OSCC.
The expression profile of DF3/MUC1 in OSCC tissues from 206 patients was examined using immunohistochemistry. Its prognostic significance in OSCC was statistically analyzed on the basis of detailed clinicopathologic factors.
DF3/MUC1 expression was found to be significantly correlated with tumor aggressiveness, such as pathologic lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01). Respectively, the overall survival (OS)and disease-free survival (DFS) rates were significantly worse for patients with DF3/MUC1 expression compared with those without DF3/MUC1 expression (P = .001 and P = .0003, respectively). Multivariate analysis demonstrated that DF3/MUC1 expression was an independent prognostic factor for both OS and DFS (P = .04 for both). In addition, DF3/MUC1 expression was found to be an independent risk factor for subsequent regional lymph node metastasis (P = .03).
Oral cancer is the most common head and neck neoplasm, having affected approximately 270,000 people worldwide in 2002, and oral squamous cell carcinomas (OSCCs) comprise > 90% of these cases.1 Generally, invasion to normal adjacent tissue and regional lymph node metastasis can occur at an early stage in patients with OSCC, which translate into worse outcomes. Regional lymph node metastasis is one of the main prognosticators of survival in OSCC but is quite often subclinical or occult at the time of diagnosis/treatment.2, 3 In fact, patients treated with putative curative resection often have poor outcomes because of unexpected events, such as subsequent lymph node metastasis.2, 4 Thus, prognostic factors that predict poor outcome and subsequent lymph node metastasis may be helpful in guiding treatment decisions in patients with OSCC.
MUC1, a transmembrane glycoprotein that has been mapped to a gene cluster on chromosome 1q21, is expressed at the basal level in human epithelial cells.5, 6 In normal cells, MUC1 is believed to lubricate epithelial cell surfaces and provides a protective barrier for the outer surface of epithelial cells.5-7 In tumor cells, many fundamental studies have demonstrated that MUC1 stimulates cell proliferation and also suppresses apoptosis, which supports a role for MUC1 in tumor progression.5-7 In addition, our previous immunohistochemical studies with the use of the DF3 antibody have indicated that MUC1 aberrant expression/overexpression is associated with poor outcomes.8-15 Among > 50 antibodies that can be used to detect several glycoforms of MUC1, only some, including DF3, have been found to be biomarkers of poor prognosis.8-15 It has been reported that the human DF3/MUC1 protein functions as an oncogene through its interaction with β-catenin.16 However, these in vitro and in vivo studies have generally studied adenocarcinoma, such as pancreatic cancer. Although one of our previous studies demonstrated that DF3/MUC1 was associated with the poor prognosis of patients with esophageal squamous cell carcinoma,17 to our knowledge no investigation of the relation between MUC1 expression and outcomes in patients with OSCC has been performed to date.
Considering the previous study results regarding the impact of DF3/MUC1 on other malignancies, our hypothesis is that the expression of DF3/MUC1 may have prognostic significance in patients with OSCC. Thus, the goal of the current study was to examine the expression profile of DF3/MUC1 in patients with OSCC and analyze the correlation between DF3/MUC1 expression, clinicopathologic factors, and prognosis in patients with OSCC. To the best of our knowledge, the current study provides the first clinical evidence that aberrant DF3/MUC1 expression has an impact on survival and subsequent lymph node metastasis in patients with OSCC.
MATERIALS AND METHODS
After ethics approval from the Human Ethics Committee of Kagoshima University Graduate School of Medical and Dental Sciences, a retrospective search was performed to identify patients who were diagnosed pathologically with OSCC. This study included 206 patients who underwent surgical resection for primary oral cancer at the Department of Oral Surgery at Kagoshima University Hospital in Kagoshima, Japan between 1992 and 2008. Patients consented to the use of their tissue for research. Patients were excluded if they had received previous treatment for oral cancer, had distant metastases at the time of their first visit, or did not undergo surgical treatment. Patient characteristics and outcomes were recorded for subsequent analysis (Table 1). Anatomical sites, TNM classification, and stage grouping of tumors were classified according to the TNM classification of malignant tumors.18 For anatomical sites, the maxilla was considered to include the upper alveolus, gingiva, and hard palate.
Table 1. Patient and Tumor Characteristics (N = 206)
No. of Patients
Abbreviation: OSCC, oral squamous cell carcinoma.
Floor of mouth
Pathologic T classification
Pathologic lymph node metastasis
TNM tumor stage
Mode of invasion
Vital status at follow-up
Death from OSCC
Death from other than OSCC
All the biopsy specimens and surgically resected specimens were fixed in formalin, embedded in paraffin, and cut into 4-μm thick sections for immunohistochemistry, in addition to the usual hematoxylin and eosin staining for histopathological analysis. Detailed tumor characteristics including pathologic TNM classification, differentiation, mode of invasion, vascular invasion, lymphatic permeation, and perineural infiltration were histologically evaluated. Using biopsy specimens, the aggressiveness of invasion was assessed by classification of the mode of invasion.19 Tumor differentiation was evaluated using G categories of the TNM classification of malignant tumors.18
Mouse monoclonal antibody (MoAb) DF3/MUC1 (Toray-Fuji Bionics, Tokyo, Japan) was used in immunohistochemistry to detect the MUC1 mucin antigen. MoAb DF3 identifies the TRPAPGS epitope in the MUC1 core peptide,20, 21 although DF3 binding to the protein may be enhanced by the presence of carbohydrates.22 Immunohistochemistry was performed by the immunoperoxidase method as follows. Antigen retrieval was performed using CC1 antigen retrieval buffer (Ventana Medical Systems, Tucson, Ariz) for all sections. After incubation with the primary antibody (MoAb DF3/MUC1, 1:10) in phosphate-buffered saline (PBS) (pH 7.4) with 1% bovine serum albumin, sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (Ventana Medical Systems). Reaction products were not present when hybridoma culture medium, normal mouse serum, or PBS was used instead of the primary antibodies. Positive controls for antibody staining were run using pancreatic tissues.
Two blinded investigators (T.H. and M.N.) independently evaluated the results of immunostaining without knowledge of patient outcomes. When their evaluations differed, a consensus decision was reached. Immunohistochemical staining was evaluated by the percentage of positively stained carcinoma cells. According to preliminary statistical analysis using receiver operating characteristic (ROC) analysis, specimens that demonstrated > 5% positively stained carcinoma cells were graded as being positive for DF3/MUC1.
Associations between DF3/MUC1 expression and clinicopathologic characteristics were evaluated using the chi-square test. Overall survival (OS) and disease-free survival (DFS)of the patients was compared between DF3/MUC1-positive and DF3/MUC1-negative groups according to the Kaplan-Meier method. Other potential clinical and histopathologic variables were also tested for prognostic significance according to 3-year and 5-year OS and DFS rates. OS was measured from the date of surgery to the date of the last follow-up examination or death, whereas DFS was measured from the date of surgery to the date on which recurrence and/or subsequent lymph node metastasis was first detected. For the analysis of prognostic factors, 6 tumor locations were classified into 1 of 2 groups: the better-survival group (lip, buccal, tongue, and mandible [n = 157 patients]) and the poor-survival group (maxilla and floor of mouth [n = 49 patients]) according to the preliminary survival analyses described previously.23 Differences in survival rates were evaluated using the log-rank test.
Univariate and multivariate analyses of OS and DFS were performed using the Cox proportional hazards regression model. Variables that demonstrated a P value < .10 on univariate analysis were used to create a multivariate model to determine whether aberrant DF3/MUC1 expression was an independent prognostic factor. On multivariate analysis, tumor stage was excluded for the model because it contains both the T and the N classification, which would negate the statistical significance of each. In addition to survival analysis, univariate and multivariate analyses of relative risk of subsequent lymph node metastasis were performed using the Cox proportional hazards regression model. Statistical analysis was performed using JMP software (version 9.0.2; SAS Institute, Inc, Cary, NC). All statistics were 2-sided, and a probability of P < .05 was considered statistically significant.
Clinicopathologic Characteristics and Outcomes
The clinicopathologic characteristics of 206 patients with OSCC are listed in Table 1. There were 133 men and 73 women, with a median age of 66.3 years (range, 25 years-89 years). The rate of pathological regional lymph node metastasis at the time of surgery was 18.4%. The majority of tumor differentiation and mode of invasion was the well-differentiated type (75.7%) and grade 3 of mode of invasion (61.2%), respectively. At the time of last follow-up, 159 of the 206 patients (77.2%) were alive, 38 (18.4%) had died of OSCC, and 9 (4.4%) had died of other causes. Overall, 64 patients developed recurrent disease, including 26 local recurrences (12.6%), 36 subsequent regional lymph node metastases (17.5%), and 2 distant recurrences (1.0%). The mean observation time was 61 months (range, 1 month-206 months).
The representative pattern of immunohistochemical staining of DF3/MUC1-positive cases is illustrated in Figure 1. DF3/MUC1 expression was not detected in normal squamous epithelium of the oral cavity, which is consistent with previous reports.24 Focal aberrant expression of DF3/MUC1 appeared in the cancer-adjacent dysplastic epithelium with increasing grade of epithelial dysplasia (mild, moderate, and severe dysplasia) (Fig. 1 and Figs. 2A and 2B). Weak expression of DF3/MUC1 was observed focally in the basal and/or parabasal layers of the epithelium with moderate dysplasia, whereas strong de novo expression of DF3/MUC1 was often noted almost throughout the entire thickness of the epithelium with severe dysplasia (Fig. 2A). In DF3/MUC1-positive cases, DF3/MUC1 demonstrated a tendency to be expressed in the invasive front and surface of the tumor nests, rather than any other component, such as the central part in tumor nests (Figs. 2C and 2D). DF3/MUC1 was expressed predominantly in the cell membrane of tumor cells, with a high frequency of positivity was noted in the cell cytoplasm (Fig. 2D).
Positive staining (> 5% of cells stained) of DF3/MUC1 was observed in the carcinoma cells in 80 OSCC cases (38.8%), although DF3/MUC1 was not expressed in the normal squamous epithelium in these cases. The mean amount of DF3/MUC1 expression among the 206 patients was 7.7%. The range of staining was 0% to 80%.
Association Between DF3/MUC1 Expression and Clinicopathologic Characteristics
Associations between expression of DF3/MUC1 and clinicopathologic characteristics are summarized in Table 2. The percentages of patients with positive lymph node metastasis (P = .002), advanced tumor stage (P = .02), diffuse invasion of cancer cells (P = .03), and vascular invasion (P = .01) were significantly higher in the DF3/MUC1-positive group compared with the DF3/MUC1-negative group. Overall, aberrant expression of DF3/MUC1 was found to be strongly associated with tumor aggressiveness in patients with OSCC. When comparing clinicopathological factors and survival rates between the group with high expression of DF3/MUC1 (> 50% cells stained) and the group with low expression (5%-49% cells stained), there was no significant difference noted between the 2 groups.
Table 2. Association Between DF3/MUC1 Expression and Clinicopathologic Factors
No. of Patients (%)
DF3/MUC1 Negative (n = 126)
DF3/MUC1 Positive (n = 80)
P value was statistically significant by the chi-square test.
The 3-year and 5-year OS rates of the entire patient population were 84.6% and 78.8%, respectively. Univariate analysis of the OS rate among each variable as determined by the log-rank test is listed in Table 3. The 3-year OS of patients with DF3/MUC1 expression was significantly worse than for other covariates (P = .001) (Table 3). The following variables were also found to be associated with poor prognosis: tumor location in the maxilla and floor of the mouth (P = .05), lymph node metastasis (P = .001), advanced stage of disease (P = .003), poor differentiation (P = .009), diffuse invasion (P = .001), and vascular invasion (P = .0007). The same factors also demonstrated statistical significance in the analysis of the 5-year OS rate (tumor location: P = .01; lymph node metastasis: P = .006; advanced stage of disease: P = .009; poor differentiation: P = .046; diffuse invasion: P = .005, vascular invasion: P = .002; and DF3/MUC1 expression: P = .009) (Table 3). The correlation between DF3/MUC1 expression and the OS rate over the entire observation period as determined by the Kaplan-Meier method is illustrated in Figure 3.
Table 3. Overall Survival Percentage According to Clinicopathologic Variables
Abbreviations: OS, overall survival.
P value was statistically significant by the log-rank test.
The univariate and multivariate analysis of OS using the Cox proportional hazards regression model is summarized in Table 4. On the univariate analysis, lymph metastasis (P = .004), advanced stage of disease (P = .005), poor differentiation (P = .03), diffuse invasion (P = .003), vascular invasion (P = .006), and DF3/MUC1 expression (P = .002) were found to be statistically significant prognostic factors affecting OS. Multivariate analysis of the 7 prognostic factors revealed that lymph node metastasis (P = .03), poor differentiation (poor vs well and moderate; P = .024), vascular invasion (P = .049), and DF3/MUC1 expression (P = .04) were independent prognostic factors for OS (Table 4). Taken together, these data indicate that DF3/MUC1 was a statistically significant independent prognostic factor for OS in patients with OSCC.
Table 4. Univariate and Multivariate Analysis of Overall Survival in OSCC
The 3-year and 5-year DFS rates of the entire patient population were 70.0% and 62.4%, respectively. Univariate analysis of DFS among each variable as determined by the log-rank test is listed in Table 5. The 3-year DFS of patients with lymph node metastasis (P = .002), an advanced stage of disease (P = .03), diffuse invasion (P = .01), vascular invasion (P < .0001), and DF3/MUC1 expression (P = .0003) was significantly worse than that observed with another covariates (Table 5). These factors (lymph node metastasis [P = .02], advanced stage of disease [P = .02], diffuse invasion [P = .005], vascular invasion [P = .0009], and DF3/MUC1 expression [P = .002]), in addition to tumor location (P = .04) and perineural infiltration (P = .008), also demonstrated statistical significance in the analysis of the 5-year DFS rate. The correlation between DF3/MUC1 expression and the DFS rate over the entire observation time as determined by the Kaplan-Meier method is illustrated in Figure 4.
Table 5. Disease-Free Survival Percentage According to Clinicopathologic Variables
Abbreviation: DFS, disease-free survival.
P value was statistically significant using the log-rank test.
The univariate and multivariate analyses of DFS by the Cox proportional hazards regression model are summarized in Table 6. On the univariate analysis, pathological lymph node metastasis (P = .005), advanced stage of disease (P = .03), diffuse invasion (P = .02), vascular invasion (P = .0001), and DF3/MUC1 expression (P = .0006) were found to be statistically significant prognostic factors for DFS. Multivariate analysis of the 4 prognostic factors revealed that lymph node metastasis (P = .02), vascular invasion (P = .004), and DF3/MUC1 expression (P = .04) were independent prognostic factors for DFS (Table 6). Along with analysis of OS, DF3/MUC1 was found to be a statistically significant independent prognostic factor for DFS in patients with OSCC.
Table 6. Univariate and Multivariate Analysis of Disease-Free Survival in Patients With OSCC
Analysis of Risk Factors Correlated With Subsequent Regional Lymph Node Metastasis
Subsequent lymph node metastasis occurred in 21 of 80 patients (26.3%) with DF3/MUC1 expression and in 15 of 126 patients (11.9%) without DF3/MUC1 expression (P = .008, chi-square test). Univariate and multivariate analyses of variables correlated with subsequent lymph node metastasis are summarized in Table 7. On the univariate analysis, diffuse invasion (P = .045), vascular invasion (P = .046), and DF3/MUC1 expression (P = .009) were found to be associated with subsequent lymph node metastasis. On multivariate analysis, only DF3/MUC1 expression was found to be an independent risk factor for subsequent lymph node metastasis in patients with OSCC (P = .03) (Table 7).
Table 7. Univariate and Multivariate Analysis of Variables Correlated With Subsequent Lymph Node Metastasis
The results of the current study demonstrate that aberrant DF3/MUC1 expression in cancer cells is correlated with tumor aggressiveness such as lymph node metastasis, advanced tumor stage, diffuse invasion, and vascular invasion of OSCC and that DF3/MUC1 is an independent prognostic factor for OS and DFS in patients with OSCC. In addition, aberrant DF3/MUC1 expression was found to be a significant risk factor for subsequent regional lymph node metastasis. Taken together, these observations indicate that aberrant DF3/MUC1 expression in patients with OSCC is associated with unfavorable events and poor survival after surgery. To the best of our knowledge, the current study is the first to provide evidence of the clinical prognostic value of DF3/MUC1 expression in patients with OSCC. The determination of DF3/MUC1 expression status in biopsy specimens has the potential to influence decisions regarding treatment, including the need for elective neck dissection. Moreover, OSCC patients with positive DF3/MUC1 expression should be followed carefully.
The presence of regional (neck) lymph node metastases at the time of diagnosis, which is associated with a 5-year survival rate of < 50%, is one of the most important prognostic factors in patients with OSCC.3, 4 Although lymph node metastasis often occurs during the early stage of OSCC, it is hard to detect by clinical examination or conventional imaging techniques.4 Generally, neck dissection is the most reliable method with which to address lymph node metastasis within the neck, but this procedure can also result in lymphatic leak, facial nerve injury, and dysfunction of the trapezius muscle. Therefore, the indications for elective neck dissection in patients with clinically N0 OSCC remain controversial. In this study, DF3/MUC1 expression in biopsy specimens was found to be a predictor of subsequent lymph node metastasis, and thus DF3/MUC1 expression may be an indication for elective neck dissection.
The results of the current study demonstrated that normal oral mucosa (squamous epithelium) did not express DF3/MUC1, whereas the majority of dysplastic epithelium in the area adjacent to DF3/MUC1-positive carcinoma showed focal DF3/MUC1 expression. This finding is consistent with observations in a previous study of patients with OSCC24 and another study of patients with esophageal squamous cell carcinomas.17 These data suggest that aberrant DF3/MUC1 expression is involved at an early stage of carcinogenesis and that it is activated during the process of squamous dysplastic transformation in patients with SCC. Thus, DF3/MUC1 could be a marker of the carcinogenic risk of OSCC or could be used for the early detection of OSCC. Furthermore studies are required to determine the level and significance of DF3/MUC1 expression in precancerous lesions/conditions, such as oral leukoplakia and lichen planus. Moreover, if intraoral staining of DF3/MUC1 is possible, it has potential to help guide the positioning of the incisional line during surgery for DF3/MUC1-positive tumors.
In the current study, aberrant DF3/MUC1 expression was found to be significantly correlated with several parameters related to tumor aggressiveness of OSCC, such as lymph node metastasis, advanced tumor stage, diffuse invasion, and vascular invasion of tumor cells. These clinical results are consistent with those reported in numerous in vitro studies and support a role for MUC1 in tumor progression. MUC1 can stimulate cell proliferation through its interaction with growth factor receptor, β-catenin, and estrogen receptor-α.7 For example, the interaction with MUC1 stabilizes β-catenin and promotes β-catenin–mediated activation of Wnt target genes,25 which have been linked to growth and tumorigenesis.6 MUC1 also contributes to the malignant phenotype by blocking stress-induced apoptosis and necrosis and by attenuating activation of death receptor pathways.26, 27 For example, MUC1 directly binds to the p53 regulatory domain and selectively promotes the transcription of growth arrest genes and decreases the transcription of apoptotic genes as a survival response to stress and thereby decreases cell death.28 Conversely, human tumors use mucins to detach from the tumor mass and surrounding stroma (antiadhesive effect), to attach to endothelia and invade (adhesive effect), and to escape immune surveillance.5 Recently, MUC1 has been shown to serve as a natural ligand of galectin-3 in human cancer cells. The interaction between circulating galectin-3 and MUC1 enhances cancer cell-endothelial adhesion and hence promotes metastasis.29 Targeted inactivation of murine Muc1 was found to significantly reduce the growth rate of oncogene-induced mammary tumors and mildly affected the propensity of these tumors to metastasize.30 Taken together, these studies support data from the current study indicating that DF3/MUC1 expression is correlated with tumor aggressiveness and malignant potential in patients with OSCC.
The current series of immunohistochemical studies of mucin expression in various human tumors has shown that DF3/MUC1 expression is correlated with invasive proliferation of tumors and poor patient outcomes.8-15, 17 The results of the current study also demonstrated the prognostic value of DF3/MUC1 expression in OSCC, reinforcing the broad utility of DF3/MUC1 as a prognostic factor related to poor outcome. MUC1 was the first mucin to be cloned and is the most intensively studied member of the mucin family, with many antibodies available for MUC1 detection.31, 32 In fact, there are > 50 MoAbs that can be used to detect several glycoforms of MUC1, including underglycosylated (eg, MoAbs NCL-MUC-1-CORE and DF3), sialylated (eg, MoAb MY.1E12), and fully glycosylated (eg, MoAb HMFG-1) forms.16, 32 Among these, the DF3 MoAb identifies TRPAPGS in the MUC1 core peptide,20 although DF3 binding to the protein may be enhanced by the presence of carbohydrates.22 It has been reported that the human DF3/MUC1 protein functions as an oncogene through interaction with beta-catenin.16 The current study used the DF3 MoAb for immunohistochemistry, and previous studies have described a correlation between expression of the DF3/MUC1 antigen and poor outcomes.8-15, 17 This specific association between poor outcomes and overexpression of the DF3/MUC1 epitope in various human cancers suggests that the DF3 MoAb could have therapeutic potential within an immunological approach.6 In addition, studies examining the interaction of DF3/MUC1 with other molecules, such as β-catenin, may improve our understanding of the role of DF3/MUC1 in the progression of OSCC.
To our knowledge, before the current study, there have only been limited published data regarding the relation between MUC1 expression and clinicopathologic factors in patients with OSCC33-36; there have been no published data regarding MUC1 expression and outcomes in patients with OSCC. Results among these previous studies have varied widely, possibly because of differences in the MoAb being used. According to a recently published review article of MUC1 expression in patients with SCC of the head and neck,33 7 MoAbs have been tested in patients with OSCC to date, and only 3 were found to be associated with clinicopathologic parameters, although none of these demonstrated an association with patient survival. Furthermore, there have been no reports of an association between the C595 MoAb, which recognizes a MUC1 core protein epitope, and clinicopathologic factors. Croce et al36 described an association between the CT33 MoAb, which defines the carboxy-terminal 17 amino acids of the cytoplasmic tail of MUC1, and tumor differentiation. This finding was not observed in an earlier work by Rabassa et al,35 and there was no relation noted between the CT33 MoAb and tumor stage in either study.33 Conversely, the HMFG-1 MoAb was found to be negatively correlated with advanced tumor stage in a study of 56 oral SCCs and 64 SCCs of the pharynx, larynx, and tonsils,36 whereas DF3 staining increased during disease progression from dysplasia to invasive and/or metastatic carcinoma in a study of 77 oral SCC cases.24, 33 A possible mechanism by which DF3/MUC1 promotes the invasive phenotype was described earlier in this text. To our knowledge, the current study is the first to demonstrate an association between DF3/MUC1 expression and prognosis in patients with OSCC.
MUC1 expression varies a great deal depending on tissues and organs,37 and this diversity may be caused by epigenetic regulations such an DNA methylation and histone modification.38 Yamada et al have demonstrated that MUC1 gene expression is regulated by DNA methylation and by histone H3 lysine 9 modification at the MUC1 promoter in pancreatic, breast, and colon cancer cell lines.39 In addition, Jin et al demonstrated that MUC1 expression is suppressed by miR-1226 and that miR-1226–induced downregulation of MUC1 was correlated with cell death in MUC1-positive breast cancer cells.40 These findings suggest the possible significance of the epigenetic control of MUC1 under carcinogenesis of human malignancies. However, epigenetic regulation of MUC1 expression in SCC is still poorly understood. We recently established a noninvasive method that can assess comprehensive epigenetic status, including DNA methylation and histone modification, using oral rinse samples.41 The detection of dysfunctional epigenetic regulation of MUC1 expression from clinical samples obtained from patients with OSCC is an important area for future research.
The current study is the first to demonstrate that aberrant expression of DF3/MUC1 is an independent risk factor for poor prognosis in patients with OSCC. DF3/MUC1 is a useful marker for predicting subsequent lymph node metastasis in patients with OSCC, and aberrant expression of DF3/MUC1 may be an indication for elective neck dissection. Patients with OSCC demonstrating positive DF3/MUC1 expression should be followed carefully.
Supported in part by Grants-in-Aid 23390466 (to K. Sugihara), 21792020 (to T. Hamada), and 23390085 (to S. Yonezawa) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.