Transmembrane 4 superfamily (TM4SF) is a recently described gene family, and TM4SF members are known to play roles in the signal transduction pathways and to regulate cell activation, development, proliferation, and motility. MRP-1/CD9, KAI1/CD82, and ME491/CD63, members of the TM4SF, have been reported to suppress tumor progression or metastasis. Previously, we showed that MRP-1/CD9 suppressed cell motility and metastatic potential to lungs. Moreover, reduction of MRP-1/CD9 and KAI1/CD82 gene expression was found to be a factor in a poor prognosis for patients with non-small cell lung cancer. However, among TM4SF, CD151 is identical to an existing gene, PETA-3, which may promote tumor metastasis of malignant cells, and its expression may be involved in the malignant progression of cancer. The function of CD151 is opposite that of the metastasis suppressor genes, MRP-1/CD9 and KAI1/CD82. On the basis of these results, we used reverse transcription-PCR and immunohistochemical techniques for a retrospective study of CD151 gene expression in tumor tissues from 145 lung cancer patients; 72 tumors were stage I, 29 stage II, 27 stage IIIA, and 17 stage IIIB. Whereas 86 patients had tumors positive for the CD151 gene, 59 had tumors that were negative for the CD151 gene. The overall survival rate of patients with CD151-positive tumors was much lower than that of CD151-negative patients (51.9% versus 73.1%; P = 0.013). Our findings suggest that high CD151 gene expression in lung cancer may be associated with a poor prognosis. Assessment of CD151 could be instrumental for improvements in lung cancer diagnosis and therapies.

TM4SF3 is a recently described gene family, and TM4SF members are known to play roles in signal transduction pathways and to regulate cell activation, development, proliferation, and motility (1). Among TM4SF, MRP-1/CD9 (2), KAI1/CD82 (3), and ME491/CD63 (4) have been reported to modulate tumor progression or metastasis. In our previous reports, we showed that tumor cells overexpressing MRP-1/CD9 had reduced cell motility and metastatic potential (2). In addition, reduced MRP-1/CD9 expression was associated with a poor prognosis in breast cancer (5), lung cancer (6), and pancreatic cancer (7). These data suggest that MRP-1/CD9 expression may be associated with metastatic ability and the degree of malignancy. KAI-1/CD82 is also a member of TM4SF, and KAI-1/CD82 expression suppressed experimental metastasis of rat prostate tumor cells and reduced their motility (8). KAI1/CD82 is considered a metastasis-suppressor gene of prostate cancer, and low levels of KAI1/CD82 expression have been reported to be involved in the malignant progression of prostate cancer (3). We also showed that reduced KAI1/CD82 gene expression was an indicator of a poor prognosis in lung cancer (9), breast cancer (10), and pancreatic cancer (7). These findings also indicate that KAI1/CD82 is an important gene in cancer metastasis and progression.

On the other hand, CD151, a TM4SF protein also known as SFA-1 and PETA-3 (11, 12), was recently reported to be the first member of the TM4SF to be linked to a positive effecter of metastasis (13). This is contrary to the function of the metastasis suppressor genes, MRP-1/CD9 and KAI1/CD82. In the present study, we investigated the clinical biological function of CD151 as a member of TM4SF, using RT-PCR and immunohistochemical techniques to evaluate the gene and protein levels of CD151 in lung cancer tissues as a prognostic factor in predicting the clinical behavior of lung cancer.

Clinical Characteristics of Patients.

Tumor specimens were obtained from 145 of the initial 166 patients with NSCLC who underwent surgery at the Department of Thoracic Surgery of the Kitano Hospital, Tazuke Kofukai Medical Research Institute (Osaka, Japan) between March 1991 and February 1996. Because of advanced stage lung cancer (stage IV), which involves several ill-defined factors and includes distant metastases, eight patients were excluded from this study. Six patients with two or more different types of cancers and seven patients who died of causes other than lung cancer were also excluded from the study. As a result, we investigated 145 patients with lung cancer up to stage IIIB, and all cases were classified according to the Tumor-Node-Metastasis classification of malignant tumors: 72 tumors were stage I, 29 were stage II, 27 were stage IIIA, and 17 were stage IIIB (14). The clinical records and histopathological diagnoses of all of the patients were fully documented. The salient clinical characteristics of the patients are presented in Table 1. This report includes follow-up data up to July 31, 2000. The median follow-up for all patients was 48.0 months (range, 15.9–104.0 months).

Tumor Specimens.

To ascertain the presence of cancer cells, half of the fresh tissue specimen from each tumor was immediately embedded in OCT compound (Miles, Kankakee, IL), and frozen sections were cut on a cryostat to a thickness of 6 μm and immediately stained with H&E. The other half of a given tumor specimen containing only cancer cells was used for RT-PCR.

RT-PCR Analysis.

Total cellular RNA was purified from fresh or frozen tumor tissues by the acid guanidinium thiocyanate procedure (15). The RNA concentration was determined by spectrophotometry and adjusted to a concentration of 500 ng/μl. First strand cDNA synthesis was performed using a cDNA synthesis kit as indicated by the manufacturer (Pharmacia, Piscataway, NJ) from 5 μg of total RNA. For PCR amplification, a 1-μl aliquot of the reaction was used. To ensure reproducible quantitative performance of the RT-PCR assay, we titrated the amount of starting cDNA and the number of amplification cycles for CD151. Subsequent experiments were performed using the parameters that allowed amplification of both CD151 and β-actin DNA (as the internal control) within a linear range. On the basis of the nucleotide sequence of CD151, 5′-ATGGGTGAGTTCAACGAGAAG-3′ was used as the sense primer and 5′-TCAGTAGTGCTCCAGCTTGAG-3′ was used as the antisense primer (12). This CD151 primer pair amplifies a 761-bp fragment (nucleotides 85–846). The reaction mixture was subjected to 20 PCR amplification cycles of 40 s at 94°C, 40 s at 60°C, and 90 s at 72°C. β-Actin amplification was used as internal PCR control (16) with 5′-GATATCGCCGCGCTCGTCGTCGAC-3′ as the sense primer and 5′-CAGGAAGGAAGGCTGGAAGAGTGC-3′ as the antisense primer. The same conditions were used to amplify β-actin DNA. Tubes containing all ingredients except templates were included in all runs as negative reaction controls. Preparations of the CD151 positive human endothelial cell line, ECV304, were used as positive controls (17). Ethidium bromide staining of the 1% agarose gel after migration of the PCR products identified a band of the expected size for the pair of primers designed. To ensure reproducibility, all PCR amplifications were performed in duplicate. Densitometric analysis of the photographic negatives was used for band quantification.

Specimen Classification Based on RT-PCR Results.

The value obtained for CD151 by densitometry in a given sample was divided by that of β-actin and referred to as the CD151 expression ratio. The expression ratio of the primary tumor was then divided by that of the human endothelial cell line, ECV304, which was used as a control to obtain the relative rate of CD151 expression (17).

Immunohistochemical Assays.

The assays were carried out as described previously (5). Endogenous peroxidases were blocked by incubation with 0.3% H2O2 in absolute methanol for 30 min. The sections were then incubated with 5% BSA for 2 h at room temperature, followed by incubation of replicate sections for 2 h with the anti-CD151 monoclonal antibody SFA1.2B4, three washings in PBS, and incubation for 1 h with biotinylated horse antimouse IgG (Vector Laboratories Inc., Burlingame, CA). Antibody binding was visualized with 3,3′-diaminobenzidine tetrahydrochloride, and the sections were lightly counterstained with Mayer’s hematoxylin. As negative reaction controls, sections were incubated with mouse myeloma SP2 supernatant.

All immunostained sections were reviewed by two pathologists who had no knowledge of the patients’ clinical status.

Statistical Analysis.

The overall cancer-specific survival was defined as the period from the date of operation to the date of cancer death. The statistical significance of the difference between the incidence of CD151-positive expression and several clinical and pathological parameters was assessed with the χ2 test or the Mann-Whitney U test. The survival curves were estimated with the Kaplan-Meier method, and the resulting curves were compared using the log-rank test (18, 19). The joint effect of covariables was analyzed with the aid of the stepwise Cox proportional hazards regression model (SAS Institute, Cary, NC; Ref. 20), and two factors (nodal status and CD151 status of the tumor) were studied by assigning scores to each of the variables for the regression analysis. All Ps were based on two-tailed statistical analysis and P < 0.05 was considered to indicate statistical significance.

CD151 Expression in Lung Tumor Tissues Analyzed by Quantitative RT-PCR.

The relative expression rates for CD151 ranged from 0 to 2.0 with a distribution as shown in Fig. 1. The mean relative expression rate for CD151 was 0.886, and the distribution of the relative expression rate was bimodal. Because most prognostic factors are usually considered as dichotomized, discontinuous variables, a cutoff point was selected to give the optimal separation between low and high risk for overall survival as described previously (21). Values >0.7 for the CD151 relative expression rate in a given specimen were considered as indicating positive gene expression, values ≤0.7 were considered as denoting negative expression. Of the 145 primary lung cancers studied, a total of 86 carcinomas (59.3%) were evaluated as positive for CD151 and 59 (40.7%) as negative for CD151 (Fig. 2). Although 22 tumors showed only reduced CD151 gene expression (0.1 < CD151 relative expression rate ≤ 0.7), the remaining 37 were quite negative for CD151 (CD151 relative expression rate ≤ 0.1).

CD151 Protein Expression Analyzed by Immunohistochemistry.

When ≥50% of the tumor cells in a given specimen were positively stained, the sample was classified as positive, and when <50% of tumor cells were stained, it was classified as reduced. The RT-PCR cutoff point was almost consistent with the cutoff point (50%) for immunohistochemistry (Table 2). Of the 145 lung cancers studied with the immunohistochemical method, 79 (54.5%) were classified as CD151 positive (Fig. 3,A). In these cases, CD151 immunostaining expression was intense and uniform on the surface of fixed, nonpermeabilized HEp-3 cells as seen in our study. Furthermore, with the aid of two-color confocal microscopy, we found similar localization of CD151 and integrin chains within cytoplasmic vesicles and endothelial cell junctions (13). Our study identified 66 cases (45.5%) with negative CD151 expression (Fig. 3,B), and immunostaining of most of these tumors was heterogeneous. CD151 gene expression was readily evident in the primary tumors that were classified as positive in the immunohistochemical assays. In contrast, CD151 gene expression was weak or entirely absent in the lung cancers that immunohistochemically showed negative CD151. Overall, the immunohistochemical results showed good agreement (91.0%) with those of the RT-PCR assays (Table 2). In cases of discrepancy, the RT-PCR results were used for specimen classification.

Relationship between CD151 Gene Expression and Clinical and Pathological Variables.

We analyzed the relationships of CD151 gene expression with several clinical and pathological variables. However, there were no statistically significant relationships by the χ2 test and the Mann-Whitney U test between gene expression and the patients’ gender, age, tumor size, lymph node status, histological subtypes, grade of differentiation, or smoking history (Table 1).

Association of CD151 Gene Expression with 5-Year Survival in Lung Cancer.

When we compared the survival among all 145 patients according to CD151 gene expression, the 5-year survival rate for patients with CD151 gene-positive tumors was much lower than that of those with CD151 gene-negative tumors (Table 3 and Fig. 4). The 5-year survival rate for the 86 patients with CD151-positive tumors was 51.9%, and that of the 59 patients with CD151-negative tumors was 73.1% (P = 0.013). Similarly, the patients with adenocarcinomas showed the same tendency for CD151-positive tumors to be associated with a progressive reduction in overall survival compared with those with negative tumors (47.4% versus 71.9%; P = 0.047). No such difference was found among the patients with squamous cell carcinomas.

Prognostic Value of CD151 Status.

The variables used for the Cox regression analysis are shown in Table 4. The estimated prognostic value of each of the variables in relation to the overall survival is expressed as P. Both variables (nodal status and CD151 gene expression) were found to be significant factors for survival; CD151 was significant for overall survival (P = 0.0252).

CD151 is a member of the TM4SF of cell-surface proteins and is identical to cDNA clones from the megakaryoblastic leukemia cell line designated 14A2.H1 (11), which was independently cloned from the adult T-cell leukemia line SF-HT and termed SFA-1 (SF-HT-activated gene 1; Ref. 12). The CD151 cDNA clone shows an open reading frame that encodes a 253-amino acid protein with a molecular mass if 28 kDa (11). The human CD151 gene is a single gene located on chromosome 11p15.5 (22) and has been reported to regulate platelet aggregation and mediator release (11, 23, 24). CD151 is expressed by a variety of cell types, including the basal cells of the epidermis; epithelial cells; skeletal, smooth, and cardiac muscle; Schwann cells; platelets; and endothelial cells (25), and has been shown to be associated with α3β1 integrin in several cell types (26) as well as with the α5β1 and α6β1 integrins (27, 28). Anti-CD151 monoclonal antibody inhibits endothelial cell migration and modulates in vitro angiogenesis (29). In addition, surprisingly, HeLa cells transfected with and overexpressing CD151 were found to be more migratory than control transfectants expressing little CD151 (13). CD151 belongs to a structurally distinct family of cell membrane glycoproteins, TM4SF, but it was reported to be identified to a positive effecter of metastasis for the human epidermoid carcinoma cell line HEp-3, the first member of TM4SF to be linked to the metastatic effecter (13).

As part of our evaluation of members of the TM4SF as possible prognostic predictors, we further extended our study to the expression of CD151 and performed a retrospective study of the expression of the CD151 gene by tumors of the lung. Subsequently, we demonstrated that CD151 gene expression was associated with a poor prognosis for patients with NSCLC, especially in the case of adenocarcinoma. The patterns of recurrence indicate that distant metastases remain the primary cause of death in patients treated with surgery alone or surgery with chemotherapy and that these patterns are different for various histological types (30, 31, 32). In fact, distant metastases are by far the most common type of recurrence in patients treated for adenocarcinoma of the lung (33). In contrast, the incidence of local and distant recurrence was found to be equal in patients with squamous cell carcinoma, which in view of our earlier data is a very interesting phenomenon. Previously we were able to show that reductions in MRP-1/CD9 and KAI1/CD82 were likely to represent poor prognoses for patients with lung cancer and that the overall survival rate of MRP-1/CD9- and KAI1/CD82-positive patients with adenocarcinomas was much better than that for patients with reduced MRP-1/CD9 or KAI1/CD82(34). These results represent a correlation that is the reverse of that established for CD151. Nevertheless, the gene status of the TM4SF members CD151, MRP-1/CD9, and KAI1/CD82 appears to have prognostic relevance for lung cancer patients with adenocarcinoma.

Our data showed that CD151 was lost to the same degree at all four stages of lung cancer. Metastatic advances by tumor cells have been shown to depend on initial arrest in the secondary organ (35), as well as events that occur after extravasation, such as migration through the stroma to sites of preferred secondary tumor growth (36). Testa et al.(13) reported that CD151 is involved in an early step in the formation of metastatic foci, such as arrest, extravasation, and/or migration into the connective tissue stroma of the secondary organ. It is well known that an accumulation of genetic alterations causes the progression of tumors (37), but there have been very few reports on the relationship between the mechanism of metastasis and an accumulation of gene alterations in NSCLC. Many studies have reported that the complex composed of integrin α3, α5, α6, or β1 and TM4SF members might play an important role in cell attachment. There is the notion that the complex might have a significant role in maintaining the integrity of normal cells. Hence, it might be necessary to investigate such an association in normal tissues. Considering the progression of malignant tumors, aberrant complex composed of integrin α3, α5, α6, or β1 and CD151 might also occur even in the early stage of these tumors. On the other hand, as tumor malignancy proceeds, the association between integrins and KAI1/CD82 may diminish rapidly. During the last stage, the levels of MRP-1/CD9 might be reduced as a result of a aberrant methylation of the promoter, or aberrant glycosylation might occur in the first hydrophilic region, leading to a possible loss of the normal functions of MRP-1/CD9 (35). Thus, the malignant cells could eventually acquire metastatic potential. The decline in MRP-1/CD9 expression might occur later than that of aberrant CD151 gene expression. These and other questions about the functions of CD151, MRP-1/CD9, and KAI1/CD82 in various kinds of cancers may well be answered in the near future. Although the precise biological roles of CD151, MRP-1/CD9, and KAI1/CD82 have not been determined, our previous studies together with our present results suggest that aberrant expression of these genes appears to be associated with a poor prognosis in lung cancer and that these genes may therefore be potentially useful prognostic markers of metastasis. However, large-scale prospective and retrospective studies on various types of cancers are needed to establish whether CD151 is indeed of practical utility as a prognostic predictor. We need to establish the cell line and demonstrate the function of the TM4SF in the process of tumor metastasis. Thus, to reveal the process of metastasis related to the TM4SF, we are trying to clarify how the TM4SF affects cancer cell motility. We will report the result of this study in the near future.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to M. M. (Grant 12470280) and a grant from the Vehicle Racing Commemorative Foundation to M. M.

                
3

The abbreviations used are: TM4SF, transmembrane 4 superfamily; CD, cluster of differentiation antigen; RT-PCR, reverse transcription-PCR; NSCLC, non-small cell lung cancer.

Fig. 1.

Distribution of relative CD151 expression (increasing rate). The values were obtained from the expression ratio of a given sample divided by that of a human endothelial cell line, ECV304, used as a positive control.

Fig. 1.

Distribution of relative CD151 expression (increasing rate). The values were obtained from the expression ratio of a given sample divided by that of a human endothelial cell line, ECV304, used as a positive control.

Close modal
Fig. 2.

A, agarose gel electrophoresis of RT-PCR-amplified 1031-bp CD151 cDNA. Lane 1, size marker; Lane 2, human endothelial cell line ECV304 (positive control); Lanes 3 and 5, primary adenocarcinoma of lung with CD151 gene expression evaluated as positive; Lanes 4 and 6, primary adenocarcinoma of lung with CD151 gene expression evaluated as negative; Lanes 7 and 9, primary squamous cell carcinoma of lung evaluated as CD151 gene positive; Lanes 8 and 10, primary squamous cell carcinoma of lung evaluated as CD151 gene negative. B, agarose gel electrophoresis of amplified β-actin DNA (internal PCR control) of each of the specimens.

Fig. 2.

A, agarose gel electrophoresis of RT-PCR-amplified 1031-bp CD151 cDNA. Lane 1, size marker; Lane 2, human endothelial cell line ECV304 (positive control); Lanes 3 and 5, primary adenocarcinoma of lung with CD151 gene expression evaluated as positive; Lanes 4 and 6, primary adenocarcinoma of lung with CD151 gene expression evaluated as negative; Lanes 7 and 9, primary squamous cell carcinoma of lung evaluated as CD151 gene positive; Lanes 8 and 10, primary squamous cell carcinoma of lung evaluated as CD151 gene negative. B, agarose gel electrophoresis of amplified β-actin DNA (internal PCR control) of each of the specimens.

Close modal
Fig. 3.

A, CD151 expression in adenocarcinoma of the lung is intense and is seen uniformly at the cell surface and cytoplasm (positive staining). B, negative immunostaining of adenocarcinoma.

Fig. 3.

A, CD151 expression in adenocarcinoma of the lung is intense and is seen uniformly at the cell surface and cytoplasm (positive staining). B, negative immunostaining of adenocarcinoma.

Close modal
Fig. 4.

Overall survival of 145 patients with lung cancer in relation to tumor CD151 gene status. Ps were determined with the log rank test.

Fig. 4.

Overall survival of 145 patients with lung cancer in relation to tumor CD151 gene status. Ps were determined with the log rank test.

Close modal
Table 1

Relationship between CD151 gene expression and various clinical and pathological variables

VariablesTotal (n)CD151P
Positive (n)Negative (n)
Gender     
 F 35 21 14 NSa 
 M 110 65 45  
Age (yr)     
 >60 101 60 41 NS 
 ≤60 44 26 18  
Tumor status     
 T1 36 20 16 NS 
 T2 73 42 31  
 T3 27 19  
 T4  
Nodal status     
 N0 91 51 40 NS 
 N1 22 14  
 N2 23 15  
 N3  
Pathological status     
 I 72 43 29 NS 
 II 29 15 14  
 IIIA 27 19  
 IIIB 17  
Histology     
 AD 88 53 35 NS 
 SQ 49 28 21  
 LA  
Differentiation     
 Well 24 11 13 NS 
 Moderately 74 44 30  
 Poorly 47 31 16  
Smoking     
 Smoker 102 63 39 NS 
 Nonsmoker 43 23 20  
Total 145 86 59  
VariablesTotal (n)CD151P
Positive (n)Negative (n)
Gender     
 F 35 21 14 NSa 
 M 110 65 45  
Age (yr)     
 >60 101 60 41 NS 
 ≤60 44 26 18  
Tumor status     
 T1 36 20 16 NS 
 T2 73 42 31  
 T3 27 19  
 T4  
Nodal status     
 N0 91 51 40 NS 
 N1 22 14  
 N2 23 15  
 N3  
Pathological status     
 I 72 43 29 NS 
 II 29 15 14  
 IIIA 27 19  
 IIIB 17  
Histology     
 AD 88 53 35 NS 
 SQ 49 28 21  
 LA  
Differentiation     
 Well 24 11 13 NS 
 Moderately 74 44 30  
 Poorly 47 31 16  
Smoking     
 Smoker 102 63 39 NS 
 Nonsmoker 43 23 20  
Total 145 86 59  
a

NS, not significant; AD, adenocarcinoma; SQ, squamous cell carcinoma; LA, large cell carcinoma.

Table 2

Relationship between RT-PCR results and immunohistochemical results of CD151

ImmunohistochemistryRT-PCRTotal (n)
CD151 positive (n)CD151 negative (n)
CD151 positive 76 10 86 
CD151 negative 56 59 
Total 79 66 145 
ImmunohistochemistryRT-PCRTotal (n)
CD151 positive (n)CD151 negative (n)
CD151 positive 76 10 86 
CD151 negative 56 59 
Total 79 66 145 
Table 3

Five-year survival rate of 145 patients with NSCLC in relation to clinical and pathological variables

Variables5-year survival rate (%)P
Gender   
 F 65.1 0.227 
 M 58.7  
Age, years   
 >60 63.2 0.309 
 ≤60 53.3  
Tumor status   
 T1 69.8 0.071 
 T2 61.2  
 T3 51.6  
 T4 37.5  
Nodal status   
 N0 70.3 0.007 
 N1 53.1  
 N2 34.2  
 N3 41.7  
Pathological status   
 I 75.4 <0.0001 
 II 57.2  
 IIIA 36.6  
 IIIB 35.2  
Histology   
 ADa 56.4 0.433 
 SQ 62.5  
 LA 66.9  
Differentiation   
 Well 70.4 0.608 
 Moderately 58.3  
 Poorly 57.7  
Smoking   
 Smoker 60.6 0.575 
 Nonsmoker 60.1  
CD151   
 Positive 51.9 0.013 
 Negative 73.1  
Variables5-year survival rate (%)P
Gender   
 F 65.1 0.227 
 M 58.7  
Age, years   
 >60 63.2 0.309 
 ≤60 53.3  
Tumor status   
 T1 69.8 0.071 
 T2 61.2  
 T3 51.6  
 T4 37.5  
Nodal status   
 N0 70.3 0.007 
 N1 53.1  
 N2 34.2  
 N3 41.7  
Pathological status   
 I 75.4 <0.0001 
 II 57.2  
 IIIA 36.6  
 IIIB 35.2  
Histology   
 ADa 56.4 0.433 
 SQ 62.5  
 LA 66.9  
Differentiation   
 Well 70.4 0.608 
 Moderately 58.3  
 Poorly 57.7  
Smoking   
 Smoker 60.6 0.575 
 Nonsmoker 60.1  
CD151   
 Positive 51.9 0.013 
 Negative 73.1  
a

AD, adenocarcinoma; SQ, squamous cell carcinoma; LA, large cell carcinoma.

Table 4

Multivariate regression analysis of variables for predicting overall survival of 145 patients with NSCLC

VariablesAssigned scoreβSEHazards ratio95% CIaP
CD151       
 Negative 0.660 0.295 0.517 0.290–0.921 0.0252 
 Positive      
Nodal status       
 N1 0.401 0.125 1.493 1.1683–1.907 0.0014 
 N2      
 N3      
 N4      
VariablesAssigned scoreβSEHazards ratio95% CIaP
CD151       
 Negative 0.660 0.295 0.517 0.290–0.921 0.0252 
 Positive      
Nodal status       
 N1 0.401 0.125 1.493 1.1683–1.907 0.0014 
 N2      
 N3      
 N4      
a

CI, confidence interval.

We thank Yuka Yoshida for technical assistance, Mituko Shirata for assistance in preparation of the manuscript, and Drs. Shinji Sawada and Tadashi Obayashi for pathological examinations.

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