Expression level of valosin-containing protein (VCP) as a prognostic marker for gingival squamous cell carcinoma

S. Yamamoto1, Y. Tomita2,*, Y. Hoshida2, S. Toyosawa3, H. Inohara4, M. Kishino3, M. Kogo5, M. Nakazawa5, S. Murakami6, N. Iizuka2, S. Kidogami2, M. Monden1, T. Kubo4, N. Ijuhin3 and K. Aozasa2

1 Department of Surgery and Clinical Oncology, 2 Department of Pathology, 4 Department of Otolaryngology and Sensory Organ Surgery, Osaka University Graduate School of Medicine, Suita, Osaka; 3 Department of Oral Pathology, 5 Department of Oral and Maxillofacial Surgery, 6 Department of Oral and Maxillofacial Radiology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan

* Correspondence to: Dr Y. Tomita, Department of Pathology (C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81 6 6879 3711; Fax: +81 6 6879 3719; Email: yt{at}molpath.med.osaka-u.ac.jp


    Abstract
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 Abstract
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 Patients and methods
 Results
 Discussion
 References
 
Background: Valosin-containing protein (VCP) is associated with anti-apoptotic function and metastasis via activation of the nuclear factor-{kappa}B signaling pathway. In the present study, association of VCP expression with prognosis of gingival squamous cell carcinoma (GSCC) was examined.

Patients and methods: VCP expression in 74 patients with GSCC (34 males and 40 females) with ages ranging from 42 to 85 (median 66) years was evaluated by immunohistochemistry, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells.

Results: Twenty-four (32.4%) cases showed level 1 and 50 (67.6%) level 2 VCP expression. Patients with level 1 GSCC showed a significantly better 5-year survival rate than those with level 2 GSCC (5-year overall survival: 100% versus 84.9%, P<0.05). Multivariate analysis revealed VCP expression level, lymph node metastasis and pT(TNM) to be independent factors for overall survival. Patients with GSCC at stages I and II showed favorable prognosis regardless of VCP expression status, whereas at stages III and IV, patients with level 1 VCP expression showed better survival rates than those with level 2 expression.

Conclusion: Prognostic significance of VCP expression level in GSCC was demonstrated.

Key words: gingival squamous cell cancer, prognosis, valosin-containing protein


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Head and neck squamous cell carcinomas (HNSCCs) are the sixth most frequent cancer in the world. Annual incidence of HNSCCs is 500 000 cases all over the world and it is increasing gradually [1Go]. Gingival squamous cell carcinoma (GSCC) is a relatively rare tumor comprising less than 10% of all HNSCCs, but shows poor prognosis even after curative surgery [2Go, 3Go]. Several prognostic factors have been proposed for GSCC, among them, tumor size and lymph node metastasis are the main factors affecting prognosis [1Go, 3Go]. Therefore, TNM (tumor–node–metastasis) classification has been widely employed for the staging of GSCC [4Go], but could not always predict clinical outcome accurately. Therefore, identification of additional prognostic factors for GSCC is necessary to establish appropriate treatment modalities for patients with GSCC.

Recently, we have identified the gene encoding valosin-containing protein (VCP, also known as p97) to be associated with metastasis of a murine osteosarcoma cell line by using an mRNA subtraction technique [5Go]. VCP, a member of the ATPases associated with various cellular activities (AAA) superfamily, is known to be involved in the ubiquitin-dependent proteasome degradation pathway of Inhibitor {kappa}B{alpha} (I{kappa}B{alpha}), an inhibitor of nuclear factor-{kappa}B (NF{kappa}B) [6Go]. Cell lines transfected with VCP showed the constant activation of NF{kappa}B, rapid degradation of phosphorylated I{kappa}B{alpha}, decreased apoptosis rates after tumor necrosis factor-{alpha} stimulation and increased metastatic potential [5Go]. These findings suggest that expression level of VCP could be used for prediction of metastasis and prognosis in patients with various cancers. Indeed our previous study showed that VCP expression level correlated with the recurrence rate and prognosis in patients with hepatocellular carcinoma [7Go], gastric carcinoma [8Go] and prostate carcinoma [9Go]. In the present study, VCP expression in GSCC was immunohistochemically examined, and its correlation with clinicopathologic features and patients' prognosis was investigated.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and tissue samples
Tissue samples were obtained from 74 patients who underwent curative resections for GSCC at the Division of Oral and Maxillofacial Diseases, Osaka University Dental Hospital and the Division of Otorhinolaryngology, Osaka University Hospital, Suita, Japan, during the period from February 1988 to March 2002. There were 34 males and 40 females with ages ranging from 42 to 85 (median 66) years. Tumors were located in the upper gingiva in 22 patients and lower gingiva in 52. Samples obtained from the gingival lesions and dissected lymph nodes were fixed in 10% formalin and routinely processed for paraffin-embedding. Histologic sections cut at 4 µm were stained with hematoxylin and eosin (H and E) and immunoperoxidase procedures [avidin–biotin complex (ABC) method]. Histologic sections were reviewed by one experienced pathologist (Y.H.) to determine histologic subtype and lymph node metastasis. Tumor stages were defined based on the pTNM classification [4Go].

After surgery, all patients received laboratory examination such as routine peripheral blood cell counts at 1–6-month intervals, and chest X-ray at 6–12-month intervals. Adjuvant therapy was performed in 61 patients; chemotherapy alone in 25 (pre-operatively in six, post-operatively in three and combined pre- and post-operatively in 16), radiotherapy alone in 13 (post-operatively in three, and combined pre- and post-operatively in 10) and combined chemo- and radiotherapy in 23 (post-operatively in two, and combined pre- and post-operatively in 21). Cisplatinum (CDDP) and fluorouracil (5-FU) were the main chemotherapeutic agents in the present study. Follow-up period for survivors ranged from 3.5 to 176.9 (median 68.3) months.

Immunohistochemistry
For the detection of VCP, immunoperoxidase procedure (ABC method) was carried out on paraffin-embedded sections as previously described [7Go, 8Go]. Briefly, antigen retrieval was carried out by heating the sections in 10 mM citrate buffer for 5 min. Mouse monoclonal anti-VCP (p97) antibody (PROGEN Biotechnik, Heidelberg, Germany) was used as the primary antibody at a dilution of 1:3000. Sections were lightly counterstained by methyl green. Positive staining in endothelial cells was used as internal positive control. For negative controls, non-immunized mouse IgG serum (Vector Laboratories, Burlingame, CA) was used as the primary antibody, and uniformly gave negative results. Stained sections were examined in a blind manner without any prior information of the clinical features of the patients. Staining intensity in the cytoplasm of the tumor cells was evaluated in comparison to that of endothelial cells, and categorized as follows: weaker (VCP expression level 1) or equal to or stronger (level 2) than that in endothelial cells. Cases showing combined level 1 and 2 intensity of staining in different areas of the tumors were classified as level 2 expression.

Strong correlation between VCP expression at mRNA level, as determined by reverse transcription polymerase chain reaction (RT–PCR) or in situ hybridization (ISH), and at protein level, as determined by immunohistochemistry, has been described previously [7Go, 9Go, 10Go].

Statistical analysis
Overall survival was measured from the date of surgery. Disease-free survival was measured from the date of surgery until local recurrence of the disease, the occurrence of distant metastases or death due to any cause. The data were statistically analyzed using JMP software (SAS Institute Inc, Cary, NC). The chi-square test and Fisher's exact test were used to analyze the association between VCP expression measured by immunohistochemistry and clinicopathologic features of GSCC. Kaplan–Meier methods with the log-rank test were used to calculate survival rates and estimate differences in survival curves [11Go]. The Cox proportional hazard regression model with a stepwise procedure was used to analyze the independent prognostic factors [12Go]. P values of <0.05 were considered as statistically significant.


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 Patients and methods
 Results
 Discussion
 References
 
Histologic findings
Histologically, 40 tumors were classified as well-differentiated, 18 moderately-differentiated and 16 poorly differentiated SCC. Tumor size was less than 2 cm (pT1) in five patients, 2–4 cm (pT2) in 28 and more than 4 cm (pT3) in 21. Tumors in the remaining 20 patients invaded into adjacent tissue (pT4). Dissected lymph nodes were histologically analyzed for the presence or absence of lymph node metastasis; 27 patients had metastasis-positive nodes.

VCP expression in GSCC
Cancer cells in 24 cases (32.4%) and 46 cases (62.2%) showed constant level 1 and level 2 VCP expression, respectively, in cytoplasm in every area of the specimens. Four GSCC (5.4%) cases showed combined level 1 and 2 staining. In total, 50 cases (67.6%) were judged as having level 2 VCP expression (Figure 1).



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Figure 1. (A, B) VCP level 1 GSCC with well-differentiated subtype. Tumor cells exhibited weak VCP staining in the cytoplasm. (C, D) VCP level 2 GSCC with well-differentiated subtype. Tumor cells exhibited strong cytoplasmic VCP staining. Magnification x50. Endothelial cells show strong cytoplasmic VCP staining (B). H and E, hematoxylin and eosin; VCP, VCP immunohistochemistry.

 
Uni- and multivariate analysis of prognostic factors in GSCC patients
Five-year disease-free and overall survival rates were 73.0% and 89.8%, respectively. Tumor recurrence was found in 23 patients; local recurrence in 17, metastasis to cervical node in six, and skin and liver in one. Of these, nine patients died due to the tumor. Association of VCP expression with clinical factors is listed in Table 1. In comparison to GSCC with level 1 VCP expression, level 2 GSCC showed significantly advanced rates of pT factor of pTNM classification (P<0.01) and lymphatic invasion (P<0.05).


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Table 1. Relationship between VCP expression and clinicopathologic factors of 74 patients with GSCC

 
Patients with level 1 GSCC showed a significantly better 5-year overall survival rate than those with level 2 GSCC (5-year disease-free survival: 86.3% versus 63.9%, P=0.07; 5-year overall survival: 100% versus 84.9%, P<0.05) (Table 2 and Figure 2). Univariate analysis revealed that lymph node metastasis and pT(TNM) were significant factors for overall survival together with VCP expression (Table 2). Multivariate analysis with factors proven to be significant in the univariate analysis revealed VCP expression level, lymph node metastasis and pT(TNM) to be independent factors for overall survival (Table 3).


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Table 2. Univariate analysis of clinicopathologic factors for disease-free and overall survival of patients with GSCC

 


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Figure 2. Disease-free (A) and overall (B) survival rate of patients with VCP expression level 1 and 2 GSCC. A significant difference was observed for overall survival.

 

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Table 3. Multivariate analysis of clinicopathologic factors for overall survival of patients with GSCC

 
Prognostic significance of VCP expression in pTNM stage
Prognostic significance of VCP expression was further analyzed according to the pTNM classification [4Go]. Patients with GSCC at stages I and II showed favorable prognosis regardless of VCP expression status (Figure 3A and B). However, at stages III and IV, patients with level 1 VCP expression showed a better disease-free survival rate compared to level 2 patients (Figure 3C and D; 5-year disease-free survival: 82.5% versus 50.9%, P<0.05; 5-year overall survival: 100% versus 81.6%, P=0.06).



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Figure 3. Disease-free and overall survival rate of patients with VCP expression level 1 and 2 GSCC in stages I–II (A, B) and III–IV (C, D) groups. Patients with level 1 VCP expression showed better disease-free survival rate compared to level 2 patients at stages III–IV.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Precise estimation of the clinical course of GSCC is difficult, in part due to the complexity of its anatomical location. TNM staging is used as a standard method for prediction of prognosis of GSCC, however, prognoses of patients are inconsistent even among patients in the same stage [2Go]. Therefore, several biological indices, including aberrant expression or mutation of tumor-suppressor genes such as p53, cell-cycle related molecules such as p27 and chromosomal aberrations have been proposed as additional prognosticators reinforcing the TNM staging system [reviewed in 13Go]. However, utility of these factors for prediction of prognosis in GSCC has been controversial in previous reports [14Go].

VCP is involved in the regulation of activation of NF{kappa}B [5Go, 6Go], which is a transcription factor correlated with various cellular activities such as anti-apoptosis, cell proliferation and invasion [15Go]. NF{kappa}B activates matrix metalloprotainase 9 and its activator urokinase-like plasminogen activator; both play important roles in intravasation and extravasation of cancer cells during metastasis [16Go]. Moreover, high expression levels of NF{kappa}B and NF{kappa}B-regulated gene products have been reported to associate with oral cancer metastasis [17Go, 18Go]. Therefore, there is an indication that VCP could play a critical role in tumor invasion and metastasis in GSCC through activation of NF{kappa}B. To clarify whether VCP expression level could be used as a new prognostic factor for GSCC, the present study was employed.

VCP expression level in GSCC was evaluated by immunohistochemistry. Clear correlation in VCP expression between mRNA and protein level has been shown by us in cases with hepatocellular carcinoma and prostate carcinoma using RT–PCR and immunohistochemistry [7Go, 9Go], and by Muller et al. in murine non-tumoral tissue using ISH and immunohistochemistry [10Go]. These results support the reliability of immunohistochemistry for the evaluation of VCP expression.

Among the clinicopathologic factors examined, high VCP expression was associated with increased pT classification and stage, and lymphatic invasion. These results indicate utility of VCP expression for prediction of aggressive character of growth and invasiveness of GSCC, and are consistent with our previous reports showing the relationship of VCP over-expression with increased metastatic potential of tumor cells in the experimental metastasis model [5Go], and increased recurrence rate and poor prognosis of hepatocellular carcinoma, gastric carcinoma and prostate carcinoma in clinical analysis [7Go–9Go]. In the present study, high VCP expression correlated with lymphatic invasion of the primary tumor, but not with lymph node metastasis. Since the anatomical structure of gingiva is complex and diverse, other factors such as location of the primary tumor might influence the occurrence of lymph node metastasis.

The present uni- and multivariate analyses have revealed VCP expression level to be an independent prognosticator for overall survival of GSCC, together with pT classification and lymph node metastasis. These findings indicate that VCP expression could be used as a useful prognosticator reinforcing pTNM classification. Favorable outcome can be expected for patients at early stage (I and II) irrespective of VCP expression. However, in patients at advanced stage (III and IV), those with level 1 VCP expression showed better prognosis compared to level 2 patients; in this group, 5-year disease-free survival rate was 82.5% and 50.9% for level 1 and 2 patients, respectively.

In conclusion, VCP expression as determined by immunohistochemistry could be used as a new prognosticator for GSCC. Stratification of GSCC patients based on the stage of disease and VCP expression level in the tumor cells would be a useful tool for predicting tumor recurrence and patient prognosis. This system might provide a novel way to explore effective treatment modalities for GSCC.

Received for publication January 6, 2004. Revision received March 22, 2004. Accepted for publication May 10, 2004.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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