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Clinical Cancer Research Vol. 10, 3110-3116, May 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Expression of Matrix Metalloproteinase-9 in Head and Neck Squamous Cell Carcinoma

A Potential Marker for Prognosis

Henni Ruokolainen1, Paavo Pääkkö2 and Taina Turpeenniemi-Hujanen1

Departments of 1 Oncology and Radiotherapy and 2 Pathology, Oulu University Hospital, Oulu, Finland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Previous studies have shown that matrix metalloproteinase-9 (MMP-9) is expressed in malignant head and neck squamous cell carcinoma. The prognostic role of MMP-9 is still unclear. The aim of this study was to investigate the role of MMP-9 immunoreactive protein as a prognostic marker for survival in head and neck squamous cell carcinoma.

Experimental Design: Overexpression of the immunoreactive protein for MMP-9 was evaluated in tissue sections of 74 primary head and neck carcinomas with a monoclonal antibody using a biotin-streptavidin immunohistochemical staining method. The staining results were compared with the clinical data and to the patients’ outcome.

Results: Positive immunostaining for MMP-9 was observed in 82% of the head and neck carcinomas, 39% of the cases being extensively positive. MMP-9 protein expression was independent of the stage or the grade of the tumor. The expression of MMP-9 was prognostic for shortened survival, the 5-year cause-specific survival being 45% in MMP-9 positive cases, and 92% in cases negative for MMP-9 (P = 0.013). MMP-9 positivity also correlated to the relapse-free survival (P = 0.019). At the 5-year follow-up, the cumulative relapse-free survival rate was 79% for patients with MMP-9-negative tumor and 42% for the patients with positive immunostaining for MMP-9. High expression of MMP-9 seemed to be linked with more aggressive relapses, appearing in 33% of the cases in local relapses, in 52% of cases with lymph node relapses, and in 60% of the cases with hematogenic relapses.

Conclusions: This is the first study with a long follow-up showing that the immunoreactive protein of MMP-9 in head and neck carcinoma is associated with shortened relapse-free and cause-specific survival, suggesting that MMP-9 has a role in tumor progression of head and neck carcinomas, as well as in estimation of the prognosis of these diseases.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Head and neck cancer appears to be a heterogeneous group of tumors consisting of multiple histopathologies, Tumor-Node-Metastasis stages, and treatment outcomes. Over 90% of the head and neck tumors are squamous cell carcinomas (HNSCCs; Ref. 1 ). HNSCC is the sixth most common cancer worldwide. In the European Union, 170,000 new cases of HNSCC were diagnosed in 1998. In the same period, ~42,000 new cases of HNSCC were diagnosed in the United States (2 , 3) . The clinical behavior of tumors of the head and neck and their responses to oncological treatment vary and may be partly associated with the biological heterogeneity of these tumors. The major cause of death among these patients is local-regional recurrence. It is known that the clinical stage at the time of the diagnosis is a major effector of the prognosis. Furthermore, other important factors are the location of the tumor and the anatomical factors of the tumor such as localization of the lymph and blood vessels (4, 5, 6) . However, useful markers associated with biological aggressiveness are needed to predict the outcome of the disease. Traditional therapies may cause problems with speech, swallowing, and also cosmetic morbidity. Therefore, it is obvious that prognostic markers could serve also in developing new treatment strategies in this group of cancers.

The matrix metalloproteinases (MMPs) are a large group of secreted proteinases that require zinc for catalytic activity (7) . Type IV collagenases, Mr 72,000 gelatinase A (MMP-2) and Mr 92,000 gelatinase B (MMP-9), are the largest members of this gene family. They are able to degrade connective tissue, among other substrates, the basement membrane collagen, which appears to be very crucial in tumor cell invasion and in the process of metastasis (8, 9, 10, 11, 12, 13) . Gelatinases have also been associated in tumor angiogenesis, especially gelatinase B (14, 15, 16) . They have been shown to have prognostic value in some solid tumors. Elevated expression of MMP-2 immunoreactive protein has been found to correlate with poor prognosis, e.g., in melanoma and breast carcinoma (17, 18, 19) . In non-small cell lung carcinoma and in renal cell carcinoma, increased expression of MMP-9 is of prognostic impact (20 , 21) .

Several studies have shown that gelatinases are expressed in head and neck carcinoma cells, and gelatinases may take part in the progression and invasion of these tumors (22, 23, 24) . Only few studies have tried to explore the role of MMP-9 as a prognostic marker for survival. Some studies have suggested an association between gelatinase mRNA, immunoreactive protein, or enzyme activity to invasion or to lymph node metastasis in some tumor types according to their malignancy category (25, 26, 27) .

Here, we evaluated the expression of MMP-9 immunoreactive protein in paraffin-embedded samples of the primary tumors in a prospective series of patients with HNSCC. The role of MMP-9 protein in the progression of these tumors was studied by correlating the MMP-9 expression to clinical behavior of the disease after at least 5 years of follow-up.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
The patients referred to Oulu University Hospital for treatment of a primary head and neck carcinoma between the years 1994 and 1996 were recruited to the study, and they were followed for a minimum of 5 years. Paraffin-embedded tissue samples from 74 cases were available for this study. The study protocol was approved by the Ethical Committee of Oulu University in March 1994 and renewed by the Ethical Committee of Oulu University Hospital in July 2002.

The mean age of the patients was 64 years (minimum 28 and maximum 88 years). The stage of the disease, tumor size, and lymph node involvement were determined according to the International Union against Cancer Tumor-Node-Metastasis classification (28) . The histological grade of the tumors was reviewed and classified according to the WHO Classification of Head and Neck Tumors. All 74 cases represented a squamous cell carcinoma.

The treatment strategies for patients in this series were carried out according to the local protocol for treatment, and the treatment line depended on the stage of the tumor. Radical surgical operation with postoperative radiotherapy (50–60 Gy) was the treatment for 25 of the 74 patients, 20 patients were treated by a radical surgical operation without other treatments, and 11 patients were inoperable and received only radiotherapy (50–64 Gy). Sixteen patients received preoperative radiotherapy (50 Gy) treatment before surgical operation. In our study, there were no patients treated with adjuvant chemotherapy. Two of the patients had an advanced carcinoma, and they received only palliative treatment. In the clinical follow-up, the patients were seen every 3–6 months to assess locoregional tumor control and survival.

Immunohistochemical Staining.
Paraffin-embedded sections (4 µm) from the primary tumors of head and neck carcinomas were stained using the avidin-biotin-immunoperoxidase technique. Paraffin sections were incubated at 37°C for at least 4 h, dewaxed (Histo-Clear, National Diagnostic, Atlanta, GA) and hydrated. The specimens were treated with 0.4% pepsin (Sigma, St. Louis, MO) at 37°C for 20 min. Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxidase/methanol for 15 min, and nonspecific binding was blocked with 10% goat serum for 15 min.

A mouse monoclonal antibody (CA-4001; Diabor Ltd., Oulu, Finland) for MMP-9 was used as a primary antibody (8 µg/ml) mixed with 1% BSA. The antibody recognizes the NH2-terminal end of the latent MMP-9 both as a free enzyme and when it is in a complex with the tissue inhibitor of metalloproteinase.

The specimens were incubated for 60 min at room temperature in a humidity chamber. The immunohistochemical staining was continued using Histostain-bulk kit (Zymed, San Francisco, CA) according to the manufacturer’s protocol. Biotinylated antimouse IgG was used as a second antibody. After that, the peroxidase was introduced with a streptavidin conjugate. The slides were washed thoroughly with PBS between all stages of the procedure. The antibody reaction was visualized by using a fresh substrate solution containing aminoethyl carbazol substrate kit (AEC kit; Zymed). The sections were counterstained with hematoxylin, dehydrated, and mounted in glycerol-vinyl-alcohol (GVA Mount; Zymed). For the negative controls, the primary antibody for MMP-9 was replaced with mouse nonimmuno IgG, and each set of staining always included a separate known positive control.

Evaluation of MMP-9 Immunostaining.
The slides were analyzed separately by two independent observers blinded to clinical data. The immunoreactivity in the malignant cells in each section was graded according to the extent of the positive staining from 0 to 3. The case was considered positive when >1% of the tumor cells showed a positive staining. Cases showing a positive staining in >50% of the tumor cells were considered extensively positive for MMP-9.

Statistical Analysis.
All statistical analyses were performed using the SPSS software system (SPSS for Windows, version 10.0, Chicago, IL).

The correlations of tumor stage, Tumor-Node-Metastasis classification, histological grade, and the primary anatomical site were analyzed separately according to the expression of MMP-9 in carcinoma cells. The statistical significance of these correlations was determined with the {chi}2 test. The cause-specific survival and relapse-free survival rates were analyzed using the Kaplan-Meier method, and the statistical differences in survival among subgroups were compared by a log-rank test (29) . The cause-specific survival was defined as the time from the date of the diagnosis to the date of death caused by head and neck carcinoma or the date of the last control. Cases dying from another disease were censored at the time of the death. Similarly, the relapse-free survival was calculated from the date of the diagnosis to the date of the relapse or the last follow-up visit.

To assess the independent prognostic value of MMP-9 expression, we used Cox proportional hazard regression analyses that included MMP-9 and stage or grade that were associated with survival rate.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MMP-9 Expression in Squamocellular Carcinoma of Head and Neck.
We noticed that in positive cases, the immunoreactive protein was prominent in cancer cells and seemed to localize in the cell cytoplasm as a diffuse staining (Fig. 1A)Citation . No granular staining was seen. Overexpression of MMP-9 immunoreactive protein was detected in 82% (61 of 74) of the cases (Table 1)Citation . Extensive positive staining for MMP-9 (>50% of the cells appearing as positive) was marked as +++ and was found in 39% (29 of 74) of the cases; moderate positivity (++; 25% < cells with a positive reaction < 50%) was found in 15% (11 of 74) of the cases; and weak positivity (+, 1% < cells with a positive reaction < 25%) in 28% (21 of 74) of the cases. The staining was negative in 18% (13 of 74) of the cases.



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  		Fig. 1. Cytoplasmic immunostaining of matrix metalloproteinase-9 (MMP-9) in head and neck squamous cell carcinoma. The immunostaining procedure was performed as described using anti-MMP-9 monoclonal antibody. A, tumor with extensive (+++) immunostaining for MMP-9. B, tumor with negative immunostaining for MMP-9.

 

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  		Table 1 Matrix metalloproteinase (MMP)-9 positivity and number of extensive positive cases (+++) in different subgroups of head and neck carcinoma

 
Association of MMP-9 with Clinical Parameters.
No correlation was found between the MMP-9 positivity and the stage of the disease (P = 0.832, {chi}2 test). We could not find any association between the overexpression of MMP-9 and tumor size or cervical lymph node involvement (P = 0.525, {chi}2 test), either. Additionally, there was no correlation between MMP-9 overexpression and the grade of the tumor (P = 0.432, {chi}2 test). Neither was there any correlation between patients’ age or sex and the positive immunoreaction for MMP-9 (P = 0.744, P = 0.583, {chi}2 test; Table 1Citation )

Effect of Tumor Stage, Grade, or Patient’s Sex in Prognosis.
In this material, the stage of disease was significantly associated with the cause-specific survival. The 5-year cumulative survivals for stages I, II, III, and IV patients were 90, 52, 69, and 21%, respectively, in the Kaplan-Meier analysis. The differences were significant (P = 0.0002, log-rank analysis). In addition, stage was here also significantly correlated to the relapse-free survival time (P = 0.0032, log-rank analysis). No correlation was, however, found here between tumor grade and the cause-specific survival (P = 0.548) or relapse-free survival (P = 0.541). In this material, the 5-year cumulative cause specific survival for females was 44%, compared with 57% among men (P = 0.114, log-rank analysis).

MMP-9 Overexpression and Survival.
A statistically significant correlation was found between the cause-specific survival and the overexpression of the MMP-9 protein in immunohistochemical staining. The Kaplan-Meier analysis showed that the cumulative survival of patients with MMP-9 negative tumors was 92% after 5 years of follow-up, whereas the cumulative survival was only 45% with all MMP-9 positive tumors (P = 0.013, log-rank analysis; Fig. 2ACitation ). In MMP-9-positive cases, the cumulative survival was 36% in cases presenting with an extensively (+++) MMP-9-positive tumor when it was 50% in those showing a moderate (++) positivity and 57% in those presenting with a weak (+) positivity for MMP-9 (data not shown).



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  		Fig. 2. Effect of matrix metalloproteinase (MMP)-9 immunoreactive protein on the cause-specific survival (A and B) or relapse-free survival (C and D) in head and neck carcinomas. Negative cases (–) n = 13, weak (+) positivity n = 21, moderate (++) positivity n = 11, and extensively strong (+++) positivity n = 29.

 
MMP-9 immunoreaction also correlated significantly with the relapse-free survival. Eleven of the 13 patients with a primary tumor negative for MMP-9 were free of relapse after the 5 years of the follow-up. The cumulative relapse-free survival rate was 79%. On the other hand, 24 of 61 patients with an MMP-9-positive primary tumor were relapse-free at the 5 years of the follow-up. The cumulative relapse-free survival rate for these patients was 42% (Fig. 2C)Citation . This difference was significant (P = 0.019) in the log-rank analysis. For patients with either weakly or moderately MMP-9-positive tumors, the cumulative disease-free survival rate was 52%, whereas it was only 33% for the patients with a tumor showing an extensive positive staining for MMP-9 (data not shown).

During the follow-up time, 39 patients had relapses. In 37 of 39 relapsed cases, the MMP-9 immunostaining was positive. It is notable that only 2 of all 39 relapsed cases were negative for MMP-9. This difference was statistically significant (P = 0.003, {chi}2 test). In patients with a local relapse during the follow-up, the MMP-9 immunostaining was extensively positive in 33% of the cases, in lymph node relapses in 52% of the cases and in cases with a hematogenic relapse in 60% of the cases (Table 2)Citation . This difference was not statistically significant (P = 0.161, {chi}2 test).


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  		Table 2 First site of the relapse and occurrence of expression and extensively strong expression of matrix metalloproteinase (MMP)-9 immunoreactive protein in the head and neck carcinomas

 
The Cox regression multivariate analysis was performed to evaluate whether the correlation with MMP-9 immunohistochemistry and survival depended on the two other major factors associated with prognosis: the stage or grade of the tumor. The most significant prognostic factor for survival was in this material the stage of the disease. The patients with a stage IV disease had an 8.7-fold relative risk for death compared with the patients with a stage I disease (P = 0.005, 95% confidence interval 1.95–38.96). In the same analysis, MMP-9 still retained its prognostic effect. Patients with an extensively positive staining for MMP-9 had a 7.3-fold relative risk for death when compared with patients with a MMP-9-negative immunostaining (P = 0.057, 95% confidence interval 0.94–55.80; Table 3Citation ). We also similarly tested the dependence between MMP-9 and the grade of tumor. MMP-9 retained its role as an independent marker predicting shortened survival, but the grade of the tumor did not have a prognostic role in this analysis. Patients with an extensively MMP-9-positive tumor had an 11.4-fold relative risk for death compared with those with an MMP-9-negative tumor (P = 0.018, 95% confidence interval 1.51–86.30; Table 4Citation ). Because of the limited number of patients, it was not possible to include MMP-9, grade, and stage as variables in same analysis.


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  		Table 3 Cox regression analysis including the matrix metalloproteinase-9 status and the stage of the disease

 

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  		Table 4 Cox regression analysis including the matrix metalloproteinase-9 status and the grade of the tumor

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we show that MMP-9 immunoreactive protein is a prognostic factor for unfavorable survival in head and neck carcinoma. This is the first time that such a correlation is shown when using patient material with a long follow-up time. Here, the 5-year cause-specific survival was found to be strongly dependent on MMP-9 overexpression. Only less than half (45%) of the patients with an MMP-9-positive primary tumor survived 5 years after diagnosing the malignancy, whereas only 1 person in the MMP-9-negative patient group died during that time, resulting in a 92% cause-specific 5-year survival rate. This major difference was also significant in the log-rank analysis (P = 0.013). A remarkable difference was also found in 5-year relapse-free survival between patient groups presenting with MMP-9 positive or negative tumors (42 and 79%, respectively; P = 0.019). It is interesting that in this study the stronger the intensity of MMP-9 immunostaining, the more remarkable the effect in Kaplan-Meier analyses. These results suggest that MMP-9 may be a strong indicator for prognosis. These results were in line with those of Riedel et al. (30) who reported that MMP-9 immunohistochemical staining was associated with poorer survival in 52 cases of HNSCC tumors. However, the follow-up time in that study was only <2.5 years (30) . This is also supported by Hong et al. (31) showing an association between MMP-9 and metastasis in oral squamous cell carcinoma. In 2002, Yorioka et al. (32) reported that zymographic measurements of the MMP-2 and MMP-9 activities in oral squamous cell carcinoma specimens correlated to the disease-free survival period. The number of tumor samples in that study was 44, and the follow-up time was <30 months (32) . On the contrary, Ondruschka et al. (33) failed to see any correlation between MMP-9 tumor cell positivity and survival. Nathan et al. (34) have recently reported an immunohistochemical study where they found that 92% of the tumors representing HNSCC were positive for MMP-9 immunohistochemical staining, but they failed to show a significant difference in disease-free interval.

This work showed no correlation between MMP-9 immunohistochemical staining and the traditional histopathological or clinical prognostic factors such as the stage or the grade of the tumor. This is in line with the data of Ranuncolo et al. (35) , who did not see correlation between plasma MMP-9 and the traditional clinicopathological prognostic factors. In the Cox regression analysis, the most significant prognostic factor for survival in the present study was the stage of the disease. It was observed that MMP-9 immunohistochemical staining had also prognostic value independent of the stage of the tumors. The Cox regression analysis could be performed including only two factors at a time because of the relatively limited number of the cases. Consequently, the second analysis included MMP-9 and grade. MMP-9 was more powerful in predicting the patient’s relative risk of dying, the relative risk being 11-fold for cases with MMP-9 extensive positive staining (P = 0.018). The lack of correlation between the MMP-9 and the stage or the grade of the tumor and our results in the Cox regression analysis suggested that MMP-9 could be independent of the stage and the grade. It is notably that in the Cox regression analysis, the relative risk of dying was dose dependent of MMP-9 immunohistochemical staining. Accordingly, the relative risk of dying was linear, decreasing with less immunoreaction. The relative risk of dying being highest among the cases with extensive positivity.

We demonstrate here that the patients with MMP-9-positive tumors had more relapses than patients with MMP-9-negative tumors (P = 0.003). Lymph node metastases were the most frequent relapses, and hematogenous metastases appeared in only 5 cases, all positive for MMP-9. The extensive positive immunostaining (+++) for MMP-9 was also more frequent in cases with hematogenous metastasis than with local relapses. The association of the expression of MMP-9 and MMP-2 with mode of tumor invasion and nodal involvement has previously been found in oral squamous cell carcinoma (27) . The correlation between high expression of MMP-9 protein and relapses might indicate that MMP-9 could have a role in some mechanisms important for tumor progression such as angiogenesis. Association between MMP-9 and vascular endothelial growth factor expression or microvessel density has been found in head and neck carcinoma (36) . On the other hand, in other studies of HNSCC, the MMP-9 mRNA or protein has failed to correlate with invasion and metastasis (27 , 37) .

The role of gelatinases in progression of neoplasias is far from clear. MMP-2 is more often identified as a prognostic factor than MMP-9. In fact, the effect of MMP-9 to survival is in some way contradictory. Scorilas et al. (38) have reported that MMP-9 overexpression in breast cancer was associated with a favorable prognosis in node-negative patients. Our study suggests that MMP-9 could be important in progression of at least squamocellular carcinomas such as head and neck cancers.

The discovery of new prognostic markers could promote entirely new treatment possibilities, especially in adjuvant treatment. For instance, synthetic MMP inhibitors have been introduced as an option in cancer treatment. Moreover, our study promotes additional clinical investigations on MMPs.

In conclusion, we show here that MMP-9 immunoreactive protein is a prognostic indicator for relapse-free and cause-specific survival in patients with head and neck squamous cell carcinoma. Additional studies are still needed to find the subgroups of patients who might potentially benefit from the clinical use of the marker.


   ACKNOWLEDGMENTS
 
We thank Kaisu Järvenpää and Anne Bisi for their skillful assistance during this work.


   FOOTNOTES
 
Grant support: The Finnish Cancer Society and the Cancer Society of North Finland.

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.

Requests for reprints: Taina Turpeenniemi-Hujanen, Department of Oncology and Radiotherapy, Oulu University Hospital, P. O. Box 22, FIN-90211 Oulu, Finland. Fax: 358-8-3156449; E-mail: turpeenniemi.hujanen{at}ppshp.fi

Received 11/ 7/03; revised 1/ 7/04; accepted 1/20/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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