Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma

Oral Oncology (2005) 41, 283–293

http://intl.elsevierhealth.com/journals/oron/

Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma Juan Carlos de Vicente a,*, Manuel Florentino Fresno b, Lucas Villalain c, ´ndez Vallejo e ´ Antonio Vega d, Gonzalo Herna Jose a

Department of Oral and Maxillofacial Surgery, University Hospital of Oviedo, Oviedo, Spain Department of Pathology, University Hospital of Oviedo, Oviedo, Spain c ´n, Spain Department of Oral and Maxillofacial Surgery, Hospital of Virgen Blanca, Leo d Department of Anatomy and Embryology, Faculty of Medicine, University of San Pablo-CEU, Madrid, Spain e Department of Oral Medicine and Buccofacial Surgery, School of Dentistry, Complutense University, Madrid, Spain b

Received 2 August 2004; accepted 27 August 2004

KEYWORDS

Summary To successfully establish a metastasis from an invasive carcinoma, the first step involves the degradation of the underlying basement membrane, which is mainly made up of type IV collagen. Matrix metalloproteinases (MMPs)-2 and -9 are thought to play an important role in its degradation because of their ability to destroy this type of collagen. In order to evaluate the prognostic significance of these proteases, we studied the expression of MMP-2 and -9 in series of 68 OSCC by immunohistochemistry. Of the oral carcinomas, 28% (n = 19) expressed MMP-2, and 17.6% (n = 12) expressed MMP-9. MMP-2 immunoreactivity was significantly higher in patients with alcohol consumption (p = 0.028) (OR = 4), and in those younger than 60 years (p = 0.041). MMP-9 immunostaining showed statistically significant association with the tumor grade of differentiation (p = 0.019), the T-stage (p = 0.05), and also with the alcohol intake (p = 0.04) (OR = 7.67). In the present study, although not statistically significant, we observed that immunoexpression of MMP-2 and MMP-9 was stronger in patients with lymph node metastasis (OR = 1.65 and 2.29, respectively).

Immunohistochemistry; MMP-2; MMP-9; Lymph node metastasis; Oral squamous cell carcinoma; N0; Prognosis; Ethanol

*

Corresponding author. Address: Escuela de Estomatologı´a, C/Catedra ´tico Jose ´ Serrano s/n, 33006 Oviedo, Spain. Tel.: +34 985 103638; fax: +34 985 103673. E-mail address: [email protected] (J.C. de Vicente).




1368-8375/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2004.08.013

284

J.C. de Vicente et al. In patients without regional lymph node metastasis, positive MMP-9 immunostaining was related to poor survival rates (p = 0.02; OR = 5.8). MMP-2 and -9 are involved in the invasion process of oral cancer, and MMP-9 is related to poor prognosis in the subset of patients without neck node metastasis. Ethanol could enhance the carcinogenetic process in oral cavity through its influence in the expression of MMP-2 and -9. c 2004 Elsevier Ltd. All rights reserved.




Introduction Oral squamous cell carcinoma (OSCC), the sixth most common cancer, is characterized by a high degree of local invasiveness and a high rate of metastasis to cervical lymph nodes, but a low rate of metastasis to distant organs. Death as a result of cancer is often the result of local recurrence or regional and/or systemic metastasis. Thus, metastasis are a major problem in successful cancer treatment, and it is believed that they begin early in the growth of the primary tumor.1 The process of metastasis consists of series of tumor–host interactions that involve multiple extracellular matrix degrading enzymes, including serine proteinases, cysteine proteinases, and matrix metalloproteinases (MMPs).2 MMPs play an important role in the malignant behavior of the neoplasm. They are a family of Zn2+-dependent proteinases that can degrade all components of extracellular matrix (ECM).3 This family of endopeptidases is associated with ECM degradation in physiological and pathological conditions such as embryonic development, wound healing, angiogenesis, arthritis, inflammation, and tumor metastasis.4 They are classified into four groups according to their substrate specificity: collagenases (MMP1, -8, and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3, -10, and -11), and membrane-type MMPs (MT-MMPs) (MMPs 14–17, 24, and 25). Alignment of the amino acid sequences between these enzymes shows that there is a high degree of homology between the enzymes in each group (about 80%) and between groups (about 50%).5 MMPs are produced by cancer cells or through the induction of surrounding stromal cells. This suggests that tumor cells are capable of utilizing MMPs produced by stromal cells and indicates an active role for stroma in tumor invasion.5 To successfully establish a metastasis, the first step is active migration of cancer cells from their tissue of origin and involves the degradation of the underlying basement membrane, which is made up of matrix macromolecules such as type IV collagen, laminin, and heparan sulfate proteoglicans. Thus, this membrane constitutes the first barrier

to tumor invasion and because one of its major constituents is type IV collagen, the gelatinases (MMP-2 and MMP-9) are thought to play an important role in its degradation because of their ability to cleave this type of collagen. Most of the MMPs except MT-MMPs and MMP-11 (stromelysin 3) are secreted as inactive zymogens and are activated extracellularly or pericellularly by serine proteinases, such as trypsin, plasmin, and neutrophil elastase6 that cleave their amino-terminal domains. Similarly to other MMPs, MMP-2 is secreted in an inactive zymogen form (pro-MMP2), but its conversion to an active form (active-MMP2) is due to membrane type 1-metalloproteinase (MT1-MMP or MMP-14).7 In fact, pro-MMP-2 binds to TIMP-2 in combination with MT1-MMP on the cell surface, forming a ternary complex. Then, pro-MMP-2 in the complex is activated by adjacent MT1-MMP that is free from TIMP-2.8 Together with the degradation of the basement membrane, cancer expansion requires the induction of angiogenesis in the malignant tissue to provide nourishment for proliferating cancer cells.1 Schnapper et al.9 have demonstrated the importance of type IV collagenases in new blood vessel formation. Neck lymph node metastasis are found in approximately 50% of patients with OSCC treated by neck dissection,10 and is considered to be the most important clinical prognostic factor in these patients. Although treatment guidelines are based on tumor site, node and distant metastasis (TNM system), they do not always predict clinical outcome accurately, because patients of the same TNM groups have different survival rates. As a consequence, from a prognostic point of view, it is of paramount importance for the clinician to know the invasiveness of the cancer and the likelihood of metastasis. The expressions of MMP family members in OSCC have been reported. Specifically, previous studies have shown that MMP-2 (72-kDa type IV collagenase or gelatinase A)11–13 and MMP-9 (92-kDa type IV collagenase or gelatinase B)13 are expressed in OSCC. However, the correlation of their expressions with clinical and histopathological features is still controversial.

Expression and clinical significance of MMp-2 & MMP-9 Studies that elucidate the mechanism of metastasis of cancers may result in the development of treatments which will decrease, or even inhibit this process, improving the survival rate of patients. The purpose of this study was to investigate the expression of MMP-2 and MMP-9 in a series of 68 OSCC, and to evaluate the implications of their expression on clinicopathologic factors, principally on prognosis, using immunohistochemistry.

Materials and methods Patients This study is based on a retrospective cohort of 68 patients suffering from a primary OSCC who were diagnosed at the Department of Oral and Maxillofacial Surgery, Asturias University Hospital, Oviedo, Spain, between January of 1990 and December of 1992. Inclusion criteria were surgical treatment performed according to standard procedures and consisting of the resection of the primary tumor and a radical or selective ipsi- or bilateral neck dissection, complete clinicopathologic data and availability of sufficient paraffin-embedded tumor material. Clinicopathologic information on each case, including age, gender, smoking and alcohol intake history, tumor size, nodal status, location, histologic grade, treatment, and presence or absence of tumor recurrence was obtained from patient files. There were 15 (22%) women and 53 (78%) men, ranging in age from 27 to 87 years (mean, 59.8 years). The main clinical characteristics of the 68 patients selected for this study are detailed in Table 1. All patients were staged according to the 1997 UICC TNM Classification of Malignant Tumors.14 The follow-up period ranged from 4 to 128 months (average: 56.4 months). All patients had been treated surgically with curative intention, and 29 (42.6%) underwent postoperative radiotherapy, receiving 40–70 Gy, according to the following indications: T4, poorly differentiated carcinomas, tumor-positive resection margins, and metastases in neck nodes. Surgical margins of resection were free of tumor infiltration in 63 of 68 cases (92.6%). Extracapsular extension was observed in 6 of 23 cases (26%) of neck node metastases. The recurrence-free survival and the survival of the patients were quantified. Clinical outcome was measured by two end points: death caused by disease recurrence and non-treatable disease presence at the end of the follow-up time. At the end of this period 27 patients (39.7%) had died of tumoral recurrence, and 41 cases (60.3%) were alive and free of recurrence.

285 Table 1

Patient characteristics

Variable

Number (68)

Age (years) Mean (range)

60 (27–87)

%

Gender Male Female

53 15

77.9 22.1

Tumor site Lip Tongue Floor of the mouth Gingiva Buccal mucosa Palate

4 29 17 8 6 4

5.9 42.6 25.0 11.8 8.8 5.9

T-category T1 T2 T3 T4

20 29 3 16

29.4 42.6 4.4 23.5

N-category N0 N1 N2 N3

45 13 10 0

66.2 19.1 14.7 0.0

Stage I II III IV

14 19 12 23

20.6 27.9 17.6 33.8

Histologic grade Poorly Moderately Well

2 15 51

2.9 22.1 75.0

Immunohistochemistry Immunohistochemistry was performed on paraffin sections of the 68 OSCC mounted on glass slides. Surgical specimens were fixed in 10% neutralbuffered formalin (pH 7.4) at 4 °C for 72 h and then embedded in paraffin. Four-lm-thick tissue sections were mounted on poly-L -lysine-coated slides. Briefly, the sections were dewaxed with xylene, and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by immersion of slides in methanol with 0.03% hydrogen peroxide for 30 min. The sections were then heated in 10 mM citrate buffer (pH 6.0) three times for 10 min each in a microwave oven at 500 W to retrieve antigenicity. The sections were rinsed in distilled water, and then in phosphate-buffered saline (PBS). Non-specific conjugation was blocked with a solution of

286 20% rabbit serum (DAKO, Glostrup, Denmark) applied to the sections for 10 min. The sections were incubated with primary antibody against MMP-2 (mouse monoclonal anti-MMP-2, clone 36006.211, RD systems, diluted 1:20) for 24 h, and against MMP-9 (mouse monoclonal anti-MMP-9, clone 15W2, Novocastra, diluted 1:20) for 30 min at room temperature. They were then rinsed in PBS, and bound antibody was detected using EnVision polymer technology (K4001, DAKO, Carpinteria, CA) for 30 min according to the manufacturer’s instructions. After washing in PBS, staining was developed with 3, 30 -diaminobenzidine-tetrahydrochloride in 50 mM Tris–HCL (pH 7.5) containing 0.001% hydrogen peroxide for 5 min, and then lightly counterstained with Mayer’s hematoxylin. One positive and one negative control were included in each batch of immunostained sections. Formalin-fixed, paraffin-embedded samples of colon carcinoma were used as positive controls for MMP-2, and liver for MMP-9. For negative control, the primary antibody was replaced with non-immune mouse serum. All slides were scored by two investigators without knowledge of the clinical outcome. Occasional disagreements were discussed to reach a consensus. In cases of persistent differences between them, the sections were studied by a third independent observer and the majority decision was thus considered. Staining for MMP-2 and MMP-9 was measured as the percentage of positively stained nuclei, and assigned to four categories, whereby the intense immunoreaction observed in colon carcinoma (MMP-2) and liver (MMP-9) served as strongly positive control. The degree of staining was scored as follows: 0, negative; 1, less than 10%; 2, more than 10% and less than 50%; 3, more than 50% positive staining. Group 0 was defined as negative MMP-2 or MMP-9 expression, and groups

J.C. de Vicente et al. 1, 2, and 3 as positive MMP-2 or MMP-9 immunoexpression. Areas with pronounced inflammation or necrosis were avoided.

Statistical analysis An SPSS for Windows computer program (Release 11.5, SPSS Inc., Chicago, IL) was used for statistical analyses. The association between clinical parameters (tumor size, regional lymph node status, tumor location, and histopathologic grade) and immunohistochemical results was analyzed with the chisquare or Fisher’s exact test (if N\ 5). Survival analysis was performed with the Kaplan–Meier product limit method and the log rank test, used to compare survival among groups of patients. A significance level of 0.05 was used for all statistical tests.

Results Immunohistologic expression of MMP-2 in primary OSCC and regional lymph node metastasis Immunoreactivity for MMP-2 was detected in 19 of 68 cases (28%) and primarily in cancer nests of the advancing front of neoplastic tissue (Fig. 1). Of these 19 cases, 3 cases (4.4%) strongly expressed MMP-2; 5 (7.4%) showed immunostaining between 10% and 50% of cancer cells, and finally, 11 (16.2%) expressed less than 10% of positive cell staining. The remaining 49 cases (72%) were negative for MMP-2. Of the 23 cases with lymph node metastasis, 8 cases (34.8%) expressed MMP-2, with 2 (8.7%) belonging to group 3, 1 (4.3%) to group 2, and 5 (21.7%) to group 1. Of the 45 cases with-

Figure 1 Immunolocalization of MMP-2 in OSCC. (a) MMP-2 is localized predominantly in cancer nests of the advancing front of the tumor (original magnification 400). (b) The MMP-2 is diffusely localized in the carcinoma cells (original magnification 1000).

Expression and clinical significance of MMp-2 & MMP-9

287

Table 2 Association between MMP-2 immunoexpression and lymph node metastasis

Table 3 Association between MMP-9 immunoexpression and lymph node metastasis

Node statusa

Negative

Positive

Total

Node statusa

Negative

Positive

Total

N0 N+ Total

34 (75.6%) 15 (65.2%) 49 (72.1%)

11 (24.4%) 8 (34.8%) 19 (27.9%)

45 23 68

N0 N+ Total

39 (86.7%) 17 (73.9%) 56 (82.4%)

6 (13.3%) 6 (26.1%) 12 (17.6%)

45 23 68

p value was not significant. a N0: absence, and N+: presence of neck node metastasis.

out lymph node metastasis 11 (24.4%) expressed MMP-2 (Table 2). The odds ratio (OR) for nodal metastasis in the cases that expressed MMP-2 was 1.65.

Immunohistologic expression of MMP-9 in primary OSCC and regional lymph node metastasis Immunoreactivity for MMP-9 was detected in 12 of 68 cases (17.6%) and both in cancer cells and in the surrounding stroma (Fig. 2). Of these 12 cases, 2 cases (2.9%) strongly expressed MMP-9; 3 (4.4%) showed immunostaining in 10–50% of cancer cells, and finally, 7 (10.3%) expressed less than 10% of positive cell staining. The remaining 56 cases (82.4%) were negative for MMP-9. MMP-9 labelling was mainly localised in the stroma rather than within the tumoral areas. Of the 23 cases with lymph node metastasis, 6 cases (26.1%) expressed MMP-9, with 0 belonging to group 3, 3 (13%) to group 2, and 3 (13%) to group 1. Of the 45 cases without lymph node metastasis 6 (13.3) expressed MMP-9 (Table 3). The OR for nodal metastasis in the cases that expressed MMP-9 was 2.29.

p value was not significant. a N0: absence, and N+: presence of neck node metastasis.

Immunohistologic expression of MMP-2 in OSCC with regard to clinicopathological data The MMP-2 immunoexpression according to the localization of the tumor showed no difference of statistical significance (p = 0.456). Moreover, we could not find a correlation between the MMP-2 expression level and the gender (p = 0.335), the T-stage (p = 0.308), the N-stage (p = 0.263), the TNM stage (p = 0.140), the histological grading (p = 0.380), the tumor recurrence (p = 0.232) or the smoking habit (p = 0.360). However, MMP-2 immunoreactivity was significantly higher in patients with alcohol consumption (Fisher exact test, p = 0.028) (Table 4), and in those younger than 60 years (p = 0.041) (Table 5) on which MMP-2 expression was positive in 40% compared with patients older than 60 (17%). The OR for MMP-2 expression in patients who drink alcohol was 4. Performing Kaplan–Meier analysis for MMP-2 expression, statistical significance was not reached (p = 0.20). Therefore, MMP-2 immunoexpression was not related to survival of the patients.

Figure 2 Immunolocalization of MMP-9 in OSCC. (a) The immunoreactivity to MMP-9 was expressed in cancer cells (original magnification 400) as well as in the stromal cells. (b) Representative example of MMP-9 immunostaining in OSCC (original magnification 1000).

288

J.C. de Vicente et al.

Table 4 Association between MMP-2 immunoexpression and alcohol consumption MMP-2

Total

Negative

Positive

Alcohol intake No 21 (87.5%) Yes 28 (63.6%) Total 49 (72.1%)

3 (12.5%) 16 (36.4%) 19 (27.9%)

24 44 68

Table 7

Association between MMP-9 and T-stage Negative

Positive

Total

T1 T2 T3 T4

19 24 1 12

1 5 2 4

20 29 3 16

Total

56

12

68

Fisher exact test, p = 0.05.

Fisher exact test, p = 0.028.

Table 5 Association between age and MMP-2 immunoexpression Negative

Positive

Total

Age \60 P60

20 (60.6%) 29 (82.9%)

13 (39.4%) 6 (17.1%)

33 35

Total

49 (72.1%)

19(27.9%)

68

v2, p = 0.041.

Table 8 Association between MMP-9 immunoexpression and alcohol consumption Alcohol intake

MMP-9

Total

Negative

Positive

No Yes

23 (95.8%) 33 (75.0%)

1 (4.2%) 11 (25.0%)

24 44

Total

56 (82.4%)

12 (17.6%)

68

Fisher exact test, p = 0.04.

Immunostaining of MMP-9 did also not correlated with clinical variables, such as the age (p = 0.344), the gender (p = 0.276), the tumor location (p = 0.261), the N-stage (p = 0.359), the TNM stage (p = 0.287), the tumor recurrence (p = 0.183), or the smoking habit (p = 0.738). However, MMP-9 immunostaining showed statistically significant association with the tumor grade of differentiation (Fisher exact test, p = 0.019) (Table 6), the T(p = 0.05) stage (Table 7), and also with alcohol intake (Fisher exact test, p = 0.04) (Table 8). The OR

Table 6 Association between MMP-9 immunoexpression and histological grade of differentiation MMP-9 Negative

Total

for MMP-9 expression in patients who drink ethanol was 7.67. Neither MMP-2 nor MMP-9 was significantly associated with lymph node status or patients survival when all patients were considered together. There was also no difference between MMP-2 or MMP-9

1.1 1.0 .9

Cumulated survival

Immunohistologic expression of MMP-9 in OSCC with regard to clinicopathological data

.8 .7

MMP-9

.6

Positive .5 Censored Negative

Positive .4

Undifferentiated Moderately Well

0 14 42

2 1 9

2 15 51

Total

56

12

68

Fisher exact test, p = 0.019.

Censored

.3 0

20

40

60

80

100

120

140

Time

Figure 3 Survival curve for pN0 45 patients with OSCC according to the immunostaining of MMP-9 (p = 0.02).

Expression and clinical significance of MMp-2 & MMP-9 positive and negative tumors regarding the disease specific survival. However, when considering only patients without regional lymph node metastasis, positive MMP-9 immunostaining was related with poor survival rates (Fig. 3). Thus, patients with MMP-9 negative tumors (mean survival: 101 months––95% confidence interval from 86 to 115 months) versus MMP-9 positive ones had a longer survival (mean survival: 47 months; 95% confidence interval from 18 to 76 months) (p = 0.02 by log-rank test; OR = 5.8).

Discussion Matrix metalloproteinases (MMPs) are a family of proteases commonly expressed in invasive tumors and adjacent stroma, and it is thought that they play an important role in tumor invasion and metastasis. Initially, a carcinoma develops within the epithelium, confined by the basement membrane. This structure has several components (laminin, type IV collagen, and heparan-sulphate proteoglycan) and its degradation is an essential step for progression from carcinoma in situ to invasive carcinoma. Tumor cells initially penetrate the basement membrane and lately migrate through the stroma. Thus, proteolysis of extracellular matrix macromolecules is a crucial step in cancer invasion and metastasis. The cancer cells produce different extracellular matrix degrading enzymes, such as MMPs, cathepsins, and plasminogen activators, and it is considered that all of them play a role in the malignant behavior of the neoplasms.11 MMPs are overexpressed in many tumor types, and several studies have been carried out to explore their prognostic significance, but without conclusive results. These reports have used mainly immunohistochemistry, in situ hybridation and gelatin zymography to evaluate expression of MMPs and their tissue inhibitors (TIMPs). Previous attempts to correlate MMPs expression with clinical outcome for patients with oral or head and neck cancer have been inconclusive. For a summary overview see Table 9.8,11,13,15–22 Immunohistochemistry allows the study of the presence of MMPs in tumoral and surrounding tissues matching the histologic and immunohistochemical findings. However, with this method it is not possible to assess the enzymatic activity of MMPs, or to discriminate between latent and active forms. Because of the enzymatic activity is more informative than antigenicity, many authors used in their studies gelatin zymography instead of immunohistochemistry. However, Ikebe et al.17

289 have demonstrated that the zymography-detected gelatinolytic activity of MMP-2 and MMP-9 significantly correlate with the degree of immunohistochemical staining detected in frozen sections of the same biopsy specimens. As a consequence, immunohistochemistry is a valuable method to study the expression of MMPs in OSCC because it allows a direct and visual localization of these proteases and a correlation with morphology, especially with the cellular events on the tumor– stromal interface. Furthermore, immunohistochemistry can be performed on paraffin embedded specimens. In the present study, immunohistochemical examination of 68 OSCC showed that the production of MMP-2 and MMP-9 by carcinoma cells was of 28% and 17.6%, respectively. These findings suggest that OSCC is heterogeneous for the potential to produce MMP-2 and MMP-9, and these figures are lower than the reported by others, that could be explained by the antibodies and the tissue processing used, as well as the heterogeneity of OSCC that could be attributed to epidemiological or biological differences between countries and populations. Because the production of MMPs by normal cells is regulated by growth factors and/or cytokines, the patient heterogeneity also could be due to the expression of specific receptors for these enzymes.11 MMP-2 and MMP-9 degrade type IV collagen in the basement membrane. However, tumor cells must also penetrate interstitial stroma, composed of collagens I and III. Degradation of interstitial collagen is accomplished by MMP-1, -8, -13, and MMP-2 and MT1-MMP.23 Thus, MMP-2 and/or MMP-9 are not exclusively responsible for the invasive phenotype of a carcinoma. The combined action of several MMPs is essential for the efficient degradation of the basement membrane and the interstitial stroma, and this suggests that carcinoma invasion and metastasis are somewhat independent processes. In relation to the cell localization of these proteases, there is no consensus in the literature. Tumor cells can produce MMPs themselves, facilitating their own invasive behavior. However, some studies reported MMP-2 mRNA signals in neighboring stromal fibroblasts surrounding carcinoma cells but not in the carcinoma cells themselves24,25 which means that there are interactions or synergism between these two types of cells, enhancing invasion of surrounding tissues by the neoplastic cells.23 Yorioka et al.13 suggest that tumor cells stimulate the expression of MMPs in the surrounding environment in order to favor the proteolytic degradation of the extracellular matrix or activate latent MMPs synthesized by cancer cells.

290

Table 9

Reported findings of MMP-2 and/or MMP-9 in head and neck carcinoma

Author/year (Reference)

Sample size/Country Method

MMP-2 positivity (%) MMP-9 positivity (%) Association with clinicopathological parameters

Kusukawa et al. (1993)11

46 Japan

Immunohistochemistry

67.4

–

Charous et al. (1997)15

29\ USA

In situ hibridation

67

24

Kurahara et al. (1999)16 Ikebe et al. (1999)17

96 Japan 57 Japan

80.21 70.8

83.33 –

Hong et al. (2000)18

44 South Korea

34.1

52.3

Tokumaru et al. (2000)19

12\ Japan

Not reported

–

Lymph node metastasis

Imanishi et al. (2000)20

70\\ Japan

70.8

–

Riedel et al. (2000)21

52\\\ Germany

Immunohistochemistry Immunohistochemistry, gelatin zymography Immunohistochemistry, gelatin zymography Gelatin zymography, Northern blot Immunohistochemistry, in situ hibridation Immunohistochemistry

Lymph node metastasis and mode o invasion Not differences between metastatic and no metastatic cases were found Lymph node metastasis Not differences between metastatic and no metastatic cases were found Metastasis (MMP-9 but no MMP-2)

–

52

Not differences between metastatic and no metastatic cases were found Not differences between metastatic and no metastatic cases were found. Worse survival Not differences between metastatic and no metastatic cases were found Not differences between metastatic and no metastatic cases were found. Age (MMP-2 but not MMP-9) Metastasis and worse survival (only MMP-9)

O-charoenrat et al. (2001)22 54\\\ Thailand, England Yorioka et al. (2002)13 44 Brazil

Katayama et al. (2004)8

53 Japan

RT-PCR Immunohistochemistry, Not reported gelatin zymography

Not reported

Immunohistochemistry

58.5

86.8

\\

tongue, oral cavity, oropharynx, hypopharynx, larynx, maxilla, and

J.C. de Vicente et al.

All reports concern to OSCC, except \ supraglotis, oropharynx, hypopharynx, larynx, oral cavity, maxillary sinus; nasal cavity, \\\ oral cavity, oropharynx, hypopharynx, larynx.

Expression and clinical significance of MMp-2 & MMP-9 In the present study MMP-2 was expressed in epithelial cells, but MMP-9 was expressed in neoplastic cells and also within fibroblasts adjacent to the tumor. Tsai et al.26 have observed that normal oral keratinocytes and oral cancer cell lines produce MMP-2 and -9 and they showed an immunolocalization of both proteases in cancer nests. Sutinen et al.12 have detected immunostaining for MMP-2 in the peripheral cell layer of the neoplastic islands and in some fibroblast-like cells of tumor stroma. Imanishi et al.20 have noted that the immunopositivity for MMP-2 was localized predominantly in carcinoma cells, but also in stromal cells. Katayama et al.8 have observed that MMP-2 and -9 were mainly expressed in the tumor cells and also in some endothelial cells and in the stromal fibroblasts surrounding the tumor cells. Several studies have shown that MMP-9 is expressed by malignant keratinocytes located at the tumor/stroma interface.27–29 However, Charous et al.15 found no differences in the amount of MMP-2 or MMP-9 secreted between tumor centers and margins. They concluded that expression of MMPs appears to be a property of the whole tumor, and not just a reaction of the advancing tumor to the hostÕs tissues.30 Cervical lymph node metastasis are found in approximately 50% of patients with OSCC, and they strongly correlate with prognosis.31 Relationship between MMP-2 and/or -9 expression and lymph node extension of the tumor remains unclear. Several studies demonstrated statistically significant correlations between lymph node metastasis and MMP-2 and/or MMP-9 expression. Kusugawa et al.,11 Hong et al.,18 Tokumaru et al.,19 Miyajima et al.,32 Kawamata et al.,33 have reported that MMPs, especially MMP-2 and/or MMP-9 were closely related to lymph node metastasis. In the present study, we have observed that immunoexpression of MMP-2 and MMP-9 is stronger in patients with lymph node metastasis. Thus, MMP-2 was expressed in 34.8% of patients with neck node metastasis, compared with 24.4% of patients without nodal involvement. Of the 23 cases of OSCC which metastasized to neck nodes, 26.1% revealed positive immunostaining for MMP-9, while of the 45 which did not metastasize, 13.3% showed positive immunoreactivity. Furthermore, the odds ratios for nodal metastasis in the cases that expressed MMP-2 or -9 were 1.65 and 2.29, respectively. However, we did not reach statistically significance although our sample size was bigger than the majority of the studies reviewed (Table 9). This discrepancy between previous studies and the present one suggests that the complexity of the metastatic process on which and for the OSCC many types of MMPs need to be produced,16 and also

291 may be because our study does not discriminate between the pro-form and the active form of both proteases. On the other hand, our results are in agreement with previous reports about MMP-2 and/ or -9 expression in OSCC. Thus, Yorioka et al.,13 Charous et al.,15 Imanishi et al.,20 reported that no difference in MMP-2 and MMP-9 expression was found between primary and metastatic OSCC cases (Table 9). In spite of the statistical significance, our results suggest that the expressions of MMP-2 and MMP-9 measured by immunostaining is related to the invasive behavior of the OSCC. Juarez et al.34 studied MMP-9 using zymography of conditioned medium from two highly invasive OSCC cell lines. They found that the aggressive tumor cells expressed high levels of MMP-9. However, it remains unclear if the overexpression of an enzyme reflects its functional role in the malignant process or whether such overexpression is a sign of the host response against the tumor.30 In the current study, the data indicated that the expression of MMP-9 but not MMP-2 was significantly associated with the histologic grade of differentiation. MMP-9 tended to be expressed more in undifferentiated cases (p = 0.019). Moreover, we did not find an association between MMP-2 or MMP-9 immunoexpression and neck node status or clinical outcome on survival when considered all patients together. However, when looking only at patients without neck node metastasis, MMP-9 but not MMP-2, was found to correlate with a poor clinical outcome. Thus, patients without neck node disease, and with MMP-9 negative tumors versus MMP-9 positive ones had a longer survival (mean survival: 101 months––95% confidence interval from 86 to 115 months––versus 47 months––95% confidence interval from 18 to 76 months, respectively; p = 0.02 by log-rank test; OR = 5.8). This result partially agrees with those of Riedel et al.21 who demonstrated that MMP-9 expression did not correlate with tumor and lymph node stages, but correlated with worse survival in 9 patients with OSCC and other 43 cases located on oropharynx, hypopharynx and larynx. Despite our failure to determine significant prognostic relevance of MMP-9 immunoexpression in cases of OSCC with neck node metastasis, MMP-9 expression may be a useful marker for predicting prognosis in OSCC, especially in early stages located in the primary site, and without regional lymph node metastasis. Tobacco and ethyl alcohol are classically implicated in the genesis of OSCC. Despite epidemiological studies showing a close association between ethanol consumption and oral cancer, ethanol has never been clearly demonstrated to be carcinogenic per se, and the biological basis of this

292 influence remains unclear at present.35 As a consequence, ethanol may be seen as a facilitator in the carcinogenetic process. As far as we known, the present study contains the first description of the association between the overexpression of MMP-2 and MMP-9 and alcohol consumption. We hypothesized that ethanol could increase the aggressiveness of the carcinoma leading to the overexpression of extracellular degrading enzymes, such as MMP-2 and MMP-9 and thus enhancing the invasive potential of the tumor. The results of the present study showed that there is a significant relationship between the expression of MMP-2 on the one hand, and alcohol consumption and age under 60 years on the other. Additionally, a relationship was also observed among the expression of MMP-9, tumor grade of differentiation, tumor size, alcohol intake, and a poor prognosis in patients without invasion of neck nodes by the tumor. Moreover, the expression of both, MMP-2 and MMP-9 was higher in tumors with neck node metastasis than in N0 tumors. In summary, based on our results, we conclude that the expression of MMP-2 and -9 measured by immunohistochemistry is related to the invasion of OSCC, and MMP-9 is related to poor prognosis in a subset of patients without neck node metastasis. Moreover, the role of the ethanol in the behavior of the OSCC could be explained, at least partially, through its influence in the expression of MMP-2 and -9.

Acknowledgments The auhors thank Ms. Aurora Ferna ´ndez Garcı´a for technical assistance. We are grateful to Dr. Jonas Hannestad (Department of Psychiatry, Duke University) for help in reviewing the manuscript for English syntax. This work was supported by a grant for scientific research from the Ministry of Health, Spain (Instituto de Salud Carlosiii, PI020137).

References 1. Aznavoorian S, Murphy AN, Stetler-Stevenson WG, Liotta LA. Molecular aspects of tumor cell invasion and metastasis. Cancer 1993;71:1368–83. 2. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993;73:161–95. 3. Stettler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993;9:541– 73.

J.C. de Vicente et al. 4. Matrisian L. The matrix degrading metalloproteinases. Bioessays 1992;14:455–9. 5. Thomas GH, Lewis MP, Speight PM. Matrix metalloproteinases and oral cancer. Oral Oncol 1999;35:227–33. 6. Nagase H. Activation mechanisms of matrix metalloproteinases. Biol Chem 1997;378:151–60. 7. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 1994;370:61–5. 8. Katayama A, Bandoh N, Kishibe K, et al. Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastasis and prognosis. Clin Cancer Res 2004;10:634–40. 9. Schnapper HW, Grant DS, Stetler-Stevenson WG. Type IV collagenases and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol 1993;156:235–46. 10. Yamashita T, Fujii M, Tomita T, et al. The inhibitory effect of matrix metalloproteinase inhibitor ONO-4817 on lymph node metastasis in tongue carcinoma. Anticancer Res 2003;23:2297–302. 11. Kusukawa J, Sasaguri Y, Shima I, et al. Expression of matrix metalloproteinase-2 related to lymph node metastasis of oral squamous cell carcinoma. A clinicopathologic study. Am J Clin Pathol 1993;99:18–23. 12. Sutinen M, Kainulainen T, Hurskainen T, et al. Expression of matrix metalloproteinases (MMP-1 and -2) and their inhibitors (TIMP-1, -2 and -3) in oral lichen planus, dysplasia, squamous cell carcinoma and lymph node metastasis. Br J Cancer 1998;77:2239–45. 13. Yorioka CW, Coletta RD, Alves F, Nishimoto IN, Kowalski LP, Graner E. Matrix metalloproteinase-2 and -9 activities correlate with the disease-free survival of oral squamous cell carcinoma patients. Int J Oncol 2002;20:189–94. 14. Sobin LH, Witterkind Ch, editors. TNM classification of malignant tumors. New York: Wiley-Liss; 1997. p. 17–50. 15. Charous SJ, Stricklin GP, Nanney LB, Netterville JL, Burkey BB. Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in head and neck squamous cell carcinoma. Ann Otol Rhinol Laryngol 1997;106:271–8. 16. Kurahara S, Shinohara M, Ikebe T, et al. Expression of MMPs, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: correlations with tumor invasion and metastasis. Head Neck 1999;21:627–38. 17. Ikebe T, Shinohara M, Takeuchi H, et al. Gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer. Clin Exp Metastasis 1999;17:315–23. 18. Hong S-D, Hong S-P, Lee J-I, Lim C-Y. Expression of matrix metalloproteinase-2 and -9 in oral squamous cell carcinomas with regard to the metastatic potential. Oral Oncology 2000;36:207–13. 19. Tokumaru Y, Fujii M, Otani Y, et al. Activation of matrix metalloproteinase-2 in head and neck squamous cell carcinoma: studies of clinical samples and in vitro cell lines cocultured with fibroblasts. Cancer Lett 2000;150:15–21. 20. Imanishi Y, Fujii M, Tokumaru Y, et al. Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma. Hum Pathol 2000;31:895– 904. 21. Riedel F, Go ¨tte K, Schwalb J, Bergler W, Ho ¨rmann K. Expression of 92-kDa type IV collagenase correlates with angiogenic markers and poor survival in head and neck squamous cell carcinoma. Int J Oncol 2000;17:1099–105. 22. O-charoenrat P, Rhys-Evans PH, Eccles SA. Expression of matrix metalloproteinases and their inhibitors correlates with invasion and metastasis in squamous cell carcinoma of

Expression and clinical significance of MMp-2 & MMP-9

23.

24.

25.

26.

27.

28.

the head and neck. Arch Otol Head Neck Surg 2001;127: 813–20. Ziober BL, Turner MA, Palefsky JM, Banda MJ, Kramer RH. Type I collagen degradation by invasive oral squamous cell carcinoma. Oral Oncol 2000;36:365–72. Poulsom R, Pignatelli M, Stetler-Stevenson WG, et al. Stromal expression of 72 kDa type IV collagenase (MMP-2) and TIMP-2 mRNAs in colorectal neoplasia. Am J Pathol 1992;141:389–96. Soini Y, Paakko P, Autio-Harmainen H. Genes of laminin B1 chain, alpha 1 (IV) chain of type IV collagen, and 72-kd type IV collagenase are mainly expressed by the stromal cells of lung carcinomas. Am J Pathol 1993;142:1622– 30. Tsai C, Hsieh Y, Yang S, Chou M, Chang Y. Matrix metalloproteinase 2 and matrix metalloproteinase 9 expression in human oral squamous cell carcinoma and the effect of protein kinase C inhibitors: preliminary observations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95: 710–6. Pyke C, Ralfkioer E, Huhtala P, Hurskainen T, Dano K, Tryggvason K. Localisation of mRNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridisation. Cancer Res 1992;52:1336–41. Yoshizaki Y, Sato H, Maruyama Y, et al. Increased expression of membrane type-I matrix metalloproteinase in head and neck carcinoma. Cancer 1997;79:139–44.

293 29. Stahle-Backdahl Parks WC. 92-kD gelatinase is actively expressed by eosinophils and stored by neutrophils in squamous cell carcinoma. Am J Pathol 1993;142:995– 1000. 30. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997;89:1260–70. 31. Woolgar JA, Rogers S, West CR, Errington RD, Brown JS, Vaughan ED. Survival and patterns of recurrence in 200 oral cancer patients treated by radical surgery and neck dissection. Oral Oncol 1999;35:257–65. 32. Miyajima Y, Nakano R, Morimatsu M. Analysis of expression of matrix metalloproteinases-2 and-9 in hypopharyngeal squamous cell carcinoma by in situ hybridation. Ann Otol Rhinol Laryngol 1995;104:678–84. 33. Kawamata H, Uchida D, Hamano H, et al. Active-MMP2 in cancer cell nests of oral cancer patients: correlation with lymph node metastasis. Int J Oncol 1998;13:699– 704. 34. Juarez J, Clayman G, Nakajima M, et al. Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous cell carcinoma cell lines of the oral cavity. Int J Cancer 1993;55:10–8. 35. MacIntosh RB. Oral malignant disease: management and investigational directions. In: Fonseca RJ, editor. Oral and Maxillofacial Surgery Vol 5 Surgical Pathology. Philadelphia: WB Saunders Co.; 2000. p. 152–235.