European Archives of Oto-Rhino-Laryngology

, Volume 270, Issue 3, pp 1137–1142

Expression of matrix metalloproteinase-12 is correlated with extracapsular spread of tumor from nodes with metastasis in head and neck squamous cell carcinoma

Authors

  • Jin Man Kim
    • Department of PathologyChungnam National University, School of Medicine
  • Hak Jun Kim
    • Department of Otolaryngology, Head and Neck SurgeryChungnam National University Hospital, School of Medicine
  • Bon Seok Koo
    • Department of Otolaryngology, Head and Neck SurgeryChungnam National University Hospital, School of Medicine
  • Ki Sang Rha
    • Department of Otolaryngology, Head and Neck SurgeryChungnam National University Hospital, School of Medicine
    • Department of Otolaryngology, Head and Neck SurgeryChungnam National University Hospital, School of Medicine
Head and Neck

DOI: 10.1007/s00405-012-2161-x

Cite this article as:
Kim, J.M., Kim, H.J., Koo, B.S. et al. Eur Arch Otorhinolaryngol (2013) 270: 1137. doi:10.1007/s00405-012-2161-x

Abstract

Matrix metalloproteinases (MMPs) play an important role in tumor invasiveness and metastasis. The aim of this study was to investigate the expression pattern of MMPs in the primary tumor of head and neck squamous cell carcinomas (HNSCC) with cervical node metastasis and to correlate the expression of MMP in the primary tumor with the presence of extracapsular spread (ECS) in nodes with metastasis. A retrospective study was conducted. Paraffin blocks were obtained from 40 HNSCC patients with cervical node metastasis who underwent surgery as an initial treatment between 2004 and 2011. Expressions of MMP-2, MMP-3, MMP-12, and MMP-14 were investigated immunohistochemically. MMP-2, MMP-3, MMP-12, and MMP-14 were expressed in 27, 47.5, 55, and 57.5 % of cases, respectively. MMP-12 expression was found to be significantly associated with ECS and correlated with nodal metastasis (p = 0.024, 0.011). No relation was found between MMP expression and survival. MMP-12 expressed in the primary tumor is a molecular marker that may be useful for predicting ECS in HNSCC patients with metastatic nodal disease.

Keywords

Matrix metalloproteinasesHead and neck squamous cell carcinomas

Introduction

Head and neck squamous cell carcinoma (HNSCC) is frequently associated with cervical lymph node metastasis and the presence of such metastasis has a significant negative impact on prognosis, regardless of the anatomic sub-site of the primary tumor [1]. The presence of extracapsular spread (ECS) in lymph nodes with metastasis was related with increased locoregional recurrence and distant metastasis, resulting in decreased survival and poor prognosis [24].

The adjuvant use of concurrent postoperative chemotherapy and radiotherapy has been recommended to improve the rates of locoregional control and disease-free survival in patients with risk factors such as ECS [5, 6]. The presence of ECS is believed to be the most important factor influencing prognosis as well as in determining the treatment option.

Although it remains to be established, the most likely mechanism by which ECS occurs is via mechanical disruption of the lymph node capsule by an expanding tumor or capsular destruction by type I collagenase produced by the tumor [7].

Matrix metalloproteinases (MMPs) are a diverse group of zinc-dependent endopeptidase that plays a role in tumor invasion and metastasis by degrading extracellular matrix (ECM) components [8, 9]. Several types of MMPs were reported to be expressed in HNSCC and were associated with tumor invasion and metastasis [1013].

MMP-2 has been shown to be overexpressed in oral cavity cancers as well as being associated with tumor invasiveness [10, 13, 14]. MMP-3 has been reported to be related with tumor stages and cervical metastasis of HNSCC [15, 16]. The expression of MMP-12 is associated with tumor progression and invasiveness in several malignant tumors [1720]. MMP-14 can activate other MMPs, especially MMP-2, potentially promoting tumor invasion and progression [21, 22]. However, there have been few reports documenting the relationship between the expression of MMPs and ECS in HNSCC.

We studied the expression of MMP-2, MMP-3, MMP-12, and MMP-14 in the primary tumor of HNSCC patients with cervical lymph node metastasis. The aim of this study was to investigate the relationship between the expression of MMPs and clinical parameters and also to determine whether the expression of MMPs in the primary tumor can predict the presence of ECS in nodes with metastasis.

Patients and methods

Patients

Forty patients who were diagnosed with HNSCC with cervical node metastasis at the time of presentation and underwent simultaneous curative surgery on the primary lesion and the neck as primary treatment between January 2004 and September 2011 were recruited. Patients who underwent salvage surgery after failure of radiation or chemoradiation were excluded from this study.

Extracapsular spread (ECS) was found in the cervical node specimens of 19 patients (47.5 % of patients). The patients with ECS consisted of 17 men and two women and the mean age was 61.7 years (range 46–79 years). The patients without ECS consisted of 20 men and 1 woman and the mean age was 63.5 years (range 42–81 years). The primary tumor sites among the patients with ECS were the oral cavity in six patients, the larynx in seven patients, the pharynx in five patients, and the submandibular gland in one patient. The primary tumor sites among the patients without ECS were the oral cavity in eight patients, the larynx in nine patients, and the pharynx in four patients. The average follow-up period of patients with or without ECS was 28.4 and 29 months, respectively. All the patients were staged according to the 7th AJCC TNM staging system. This study was approved by the Chungnam National University Hospital’s Institutional Review Board.

Immunohistochemical staining

The expression of MMPs in the primary tumor was examined from the biopsies or tissue samples taken during the panendoscopy or in the outpatient clinic in a diagnostic setting. Immunohistochemical staining was performed on the formalin-fixed paraffin-embedded sections (5 μm). The sections were deparaffinized, rehydrated, and microwave treated for 10 min. Endogenous peroxidase activity was blocked by 3 % hydrogen peroxide for 15 min at room temperature. After rinsing the sections in tris-buffered saline, any non-specific binding was blocked by incubation with goat serum for 30 min. Sections were then incubated with the primary antibodies for 2 h at room temperature. Antibodies used in this study were monoclonal mouse antihuman MMP-2, MMP-3, MMP-12, and MMP-14 (all were purchased from Neomarkers, Fremont, CA, USA). After incubation with secondary antibodies (Zymed Co., South San Francisco, CA, USA), the sections were incubated with avidin-biotinylated peroxidase complex for 20 min at room temperature. Immunohistochemical reactions were developed with diaminobenzidine as the chromogenic peroxidase substrate, and slides were counterstained with Meyer hematoxylin.

Interpretation

The slides were analyzed separately by two experienced pathologists blinded to the clinical data. Microscopic examinations (400×) were performed at three sites per section showing high tumor cell density. A positive reaction was considered when more than 10 % of the tumor cells showed positive staining for the marker of interest (Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs00405-012-2161-x/MediaObjects/405_2012_2161_Fig1_HTML.jpg
Fig. 1

MMP-12 immunostaining in head and neck squamous cell carcinoma. a Tumor with negative staining; b tumor with positive staining (×400)

Statistical analysis

Data were analyzed using SPSS ver.17.0 (SPSS Inc., Chicago, IL, USA). The Chi-square test was used to investigate the relationship between MMP expression and clinical data including sex, age, primary site, tumor stage, nodal stage, and ECS. Survival was estimated using the Kaplan–Meier method and the statistical differences in survival were compared by a log-rank test. Statistical significance was defined as p < 0.05.

Results

Relationship between clinical parameters and ECS

Clinical parameters including the patient’s sex, age, primary site of tumor, T stage, and N stage were not significantly correlated with ECS (Table 1).
Table 1

Relationship between the presence of extracapsular spread and clinical parameters

Characteristics

ECS (+) (n = 19)

ECS (−) (n = 21)

p value

Sex, no (%)

Male

17 (89.5)

20 (95.2)

0.462

Female

2 (10.5)

1 (4.8)

Age

28–65 year, no (%)

10 (52.6)

11 (52.4)

0.775

66–88 year, no (%)

9 (47.4)

10 (47.6)

Tumor stage, no (%)

T1

2 (10.5)

6 (28.6)

0.326

T2

8 (42.1)

4 (19)

T3

3 (15.8)

4 (10)

T4

6 (31.6)

7 (33.3)

Nodal stage, no (%)

N1

2 (10.5)

4 (19)

0.284

N2a

2 (10.5)

0

N2b

13 (68.5)

11 (52.4)

N2c

2 (10.5)

5 (23.8)

N3

0

1 (4.8)

Primary tumor site, no (%)

Oral cavity

6 (31.6)

8 (38.1)

0.521

Larynx

7 (36.8)

9 (42.8)

Pharynx

5 (26.3)

4 (19)

Others

1 (5.2)

0

Values are presented as number (%)

ECS extracapsular spread, MMP matrix metalloproteinase

Relationship between the expression of MMPs and ECS

Expression rates of MMP-2, MMP-3, MMP-12, and MMP-14 in the primary tumors were 27, 47.5, 55, and 57.5 %, respectively. The expression rates of MMP-12 was significantly higher in patients with ECS (73.7 %) than those without ECS (38.1 %) (p = 0.024). There was no significant difference in the expression rates of MMP-2, MMP-3, and MMP-14 according to the presence or absence of ECS (Table 2).
Table 2

Relationship between extracapsular spread and the expression of MMPs

Characteristics

ECS (+) (n = 19)

ECS (−) (n = 21)

p value

MMP expression, no (%)

 MMP-2

  (−)

13 (68.4)

16 (76.2)

0.583

  (+)

6 (31.6)

5 (33.8)

 

 MMP-3

  (−)

10 (52.6)

11 (52.4)

0.987

  (+)

9 (47.4)

10 (47.6)

 

 MMP-12

  (−)

5 (26.3)

13 (61.9)

0.024*

  (+)

14 (73.7)

8 (38.1)

 

 MMP-14

  (−)

8 (42.1)

9 (42.9)

0.342

  (+)

11 (57.9)

12 (57.1)

 

Values are presented as number (%)

ECS extracapsular spread, MMP matrix metalloproteinase

* p < 0.05

Relationship between clinical parameters and expression of MMP

Significant association was not observed between the expression of MMP-2, MMP-3, and MMP-14 and the patient’s sex, age, primary site of tumor, T stage, and N stage. MMP-12 expression was not related to the patient’s sex, age, primary site of tumors, and T stage, but was significantly correlated with advanced N stage (p = 0.011) (Table 3).
Table 3

Comparative analysis of the correlation between MMP expression profiles and clinical parameters

Variables

MMP-2

p value

MMP-3

p value

MMP-12

p value

MMP-14

p value

(−)

(+)

(−)

(+)

(−)

(+)

(−)

(+)

Sex

Male

26 (65.0)

11 (27.5)

0.267

18 (45.0)

0

0.233

15 (37.5)

22 (55)

0.083

16 (40.0)

21 (52.5)

0.738

Female

3 (7.5)

0

 

3 (7.5)

19 (47.5)

 

3 (7.5)

0

 

1 (2.5)

2 (5.0)

 

Age

28–65 year

19 (47.5)

3 (7.5)

0.130

12 (30.0)

10 (25.0)

0.775

11 (27.5)

11 (27.5)

0.482

11 (27.5)

11 (27.5)

0.289

66–88 year

10 (25.0)

8 (20.0)

 

9 (22.5)

9 (22.5)

 

7 (17.5)

11 (27.5)

 

6 (15.0)

12 (30)

 

T stage

T1

8 (20.0)

  

7 (17.5)

1 (2.5)

 

5 (12.5)

3 (7.5)

 

6 (15.0)

2 (5.0)

 

T2

10 (25.0)

2 (5.0)

0.055

8 (20.0)

4 (10.0)

0.330

5 (12.5)

7 (17.5)

0.612

4 (10.0)

8 (12.0)

0.203

T3

3 (7.5)

4 (10.0)

 

2 (5.0)

5 (12.5)

 

2 (5)

5 (12.5)

 

3 (7.5)

4 (10.0)

 

T4

8 (20.0)

5 (12.5)

 

4 (10.0)

9 (22.5)

 

6 (15)

7 (17.5)

 

4 (10.0)

9 (22.5)

 

N stage

N1

4 (10.0)

2 (5.0)

 

2 (5.0)

4 (10.0)

 

6 (15.0)

0

 

3 (7.5)

3 (7.5)

 

N2a

1 (2.5)

1 (2.5)

 

1 (2.5)

1 (2.5)

 

0

2 (5)

 

0

2 (5.0)

 

N2b

17 (42.5)

7 (17.5)

0.802

15 (37.5)

9 (22.5)

0.359

10 (25)

14 (35)

0.011*

10 (25.0)

14 (35.0)

0.562

N2c

6 (15.0)

1 (2.5)

 

2 (5.0)

5 (12.5)

 

1 (2.5)

6 (10)

 

4 (10.0)

3 (7.5)

 

N3

1 (2.5)

0

 

1 (2.5)

0

 

1 (2.5)

0

  

1 (2.5)

 

Values are presented as number (%)

MMP matrix metalloproteinase

* p < 0.05

Relationship between MMP expression and ECS and survival

MMP expression was not significantly associated with survival in this study. There was no significant difference in the 5-year overall survival rate between patients with ECS and those without ECS.

Discussion

It has been established that the presence or absence of cervical node metastases in patients with head and neck squamous cell carcinoma (HNSCC) is a powerful prognostic indicator. Metastases to regional lymph nodes reduce the 5-year survival rates to 50 % of patients with disease still confined to the primary site. Therefore, the appropriate management of cervical metastasis is a key element in the management of patients with HNSCC.

The presence of extracapsular spread (ECS) from lymph nodes with metastasis was related with increased locoregional recurrence, distant metastasis, decreased survival, and poor prognosis [24]. Cooper et al. [5] reported that postoperative concurrent chemoradiation therapy significantly improved the rates of local and regional control and disease-free survival in high-risk patients with extracapsular extension of nodal disease. Eventually, selection of therapeutic option and the patient’s prognosis are greatly influenced by the presence of ECS.

ECS was seen more frequently in large nodes measuring more than 3 cm in diameter (60–100 %), but can also occur in smaller nodes less than 3 cm in diameter (39–59 %) [23, 24].

Radiologic imaging such as CT or MRI is useful to detect ECS preoperatively. Extracapsular spread is suspected when irregular nodal boundaries, infiltration of adjacent fat planes, thickening of adjacent fascia, and apparent invasion of adjacent structures are present [25, 26]. However, radiologic studies alone may be inadequate for detecting all the instances of ECS from cervical lymph nodes with metastasis, in that ECS was later found in 19 % of the clinically node-negative necks [23]. Therefore, other tools, such as molecular markers, are needed to predict accurately the presence of ECS preoperatively.

MMPs have been known to be important in the process of tumor invasion and metastasis. The expression of MMPs, such as MMP-1, MMP-2, MMP-9, MMP-14 (MT1-MMP) and so on, was likely to correlate with the increased rates of cervical lymph node and distant metastasis and decreased survival rates in HNSCC [813].

In this study, we evaluated the expression patterns of MMP-2, MMP-3, MMP-12, and MMP-14 in advanced HNSCC patients with cervical lymph node metastasis and tried to investigate whether the expression of MMPs could predict the presence of ECS in nodes with metastasis.

MMP-2, also known as gelatinase A, has been shown to be overexpressed in oral cavity cancers as well as being associated with tumor invasiveness [10, 13, 14]. MMP-3, also known as stromelysin-1, has been reported to be related to tumor stages and cervical metastasis of HNSCC [15, 16]. MMP-14, also known as membrane type 1 MMP (MT1-MMP), can activate other MMPs, especially MMP-2, potentially promoting tumor invasion and progression [21, 22]. In the present study, MMP-2, MMP-3, and MMP-14 were expressed in 27, 47.5, and 57.5 % of patients, respectively, but its expression was not associated with the presence or absence of ECS, tumor stages, and survival rates.

MMP-12, also known as human macrophage metalloelastase (HME), has shown divergent properties in tumor progression. The expression of MMP-12 is associated with tumor progression and invasiveness in skin, endometrial, esophageal and non-small cell lung cancer [1720]. On the other hand, gastric and colon cancer patients with MMP-12 mRNA expression have better prognosis than those without expression [27, 28]. To date, there is no report on the role of MMP-12 in HNSCC. In the present study, the expression rates of MMP-12 was significantly higher in patients with ECS (73.7 %) than those without ECS (38.1 %) (p = 0.024) and correlated with advanced N stage (p = 0.011). MMP-12 was related to tumor invasiveness in HNSCC, although its expression was not associated with survival in this study.

Modified radical neck dissection (MRND) is considered as the classical treatment for neck metastases of HNSCC, but selective neck dissection (SND) has been studied as adequate treatment for N1 necks without capsule rupture [29]. This study suggests that preoperative evaluation of MMPs status in the primary tumor may be useful to decide whether to perform MRND or SND for N1 necks.

The limitations of this study include its retrospective nature, small number of patients, and the heterogeneity of patient population with tumors originating from different subsites. Studies with larger population and longer follow-ups will be needed to determine the exact role of MMP-12 in the progression of HNSCC.

Conclusion

The presence of extracapsular spread (ECS) from lymph nodes with metastasis was related to increased locoregional recurrence and distant metastasis, resulting in decreased survival and poor prognosis. The adjuvant use of concurrent postoperative chemotherapy and radiotherapy has been recommended to improve the rates of locoregional control and disease-free survival in patients with ECS. Eventually, selection of therapeutic option and the patient’s prognosis are greatly influenced by the presence of ECS. In the present study, the expression rate of MMP-12 in the primary tumor was significantly higher in patients with ECS than those without ECS and correlated with advanced N stage. In conclusion, MMP-12 expressed in the primary tumor is a potential molecular marker that may be useful for predicting ECS in HNSCC patient with metastatic nodal disease.

Acknowledgments

This study was supported by a grant from the National R&D Program for Cancer Control Ministry of Health and Welfare, Republic of Korea (No 0720560).

Conflict of interest

None.

Copyright information

© Springer-Verlag 2012