Immunohistochemical expression of matrix metalloproteinase-9 and 13 in oral squamous cell carcinoma and their role in predicting lymph node metastasis

Abstract

BACKGROUND

One of the main characteristics of oral squamous cell carcinoma (OSCC) is that it metastasizes to cervical lymph nodes frequently with a high degree of local invasiveness. A primary feature of malignant tumors is their penetration of neighboring tissues, such as lymphatic and blood arteries, due to the tumor cells' capacity to break down the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) constitute a family of proteolytic enzymes that facilitate tissue remodeling and the degradation of the ECM. MMP-9 and MMP-13 belong to the group of extracellular matrix degrading enzymes and their expression has been studied in OSCC because of their specific functions. MMP-13, a collagenase family member, is thought to play an essential role in the MMP activation cascade by breaking down the fibrillar collagens, whereas MMP-9 is thought to accelerate the growth of tumors. Elevated MMP-13 expression has been associated with tumor behavior and patient prognosis in a number of malignant cases.

AIM

To assess the immunohistochemical expression of MMP-9 and MMP-13 in OSCC.

METHODS

A total of 40 cases with histologically confirmed OSCC by incisional biopsy were included in this cross-sectional retrospective study. The protocols for both MMP-9 and MMP-13 immunohistochemical staining were performed according to the manufacturer’s recommendations along with the normal gingival epithelium as a positive control. All the observations were recorded and Pearson’s χ² test with Fisher exact test was used for statistical analysis.

RESULTS

Our study showed no significant correlation between MMP-9 and MMP-13 staining intensity and tumor size. The majority of the patients were in advanced TNM stages (III and IV), and showed intense expression of MMP-9 and MMP-13.

CONCLUSION

The present study suggests that both MMP-9 and MMP-13 play an important and independent role in OSCC progression and invasiveness. Intense expression of MMP-9 and MMP-13, irrespective of histological grade of OSCC, correlates well with TNM stage. Consequently, it is evident that MMP-9 and MMP-13 are important for the invasiveness and progression of tumors. The findings may facilitate the development of new approaches for evaluating lymph node metastases and interventional therapy techniques, hence enhancing the prognosis of patients diagnosed with OSCC.

Key Words: Matrix metalloproteinases; Oral squamous cell carcinoma; Tumor staging; Immunohistochemistry; Invasion; Lymph node metastasis; TNM stage

Core Tip: The most prevalent type head and neck cancer is oral cavity squamous cell carcinoma (OCSCC). The major cause of the poor prognosis is extensive local invasion that spreads to the lymph nodes. Degradation of the matrix and spread of cancer cells are major characteristics of malignant tumors. Hence, in the present study, matrix metalloproteinase (MMP)-9 and 13 expression was investigated to understand and interpret the invasion and metastasis in OSCC. Most of the cases showed various degrees of staining intensity for MMP-9 and MMP-13. MMP-9 and MMP-13 staining intensity had no significant correlation with tumor size, though a significant relationship (P = 0.000) was observed with metastasis.

INTRODUCTION

Oral squamous cell carcinoma (OSCC) is characterized by significant local invasiveness and a strong propensity for cervical lymph node metastasis. Cancer-related mortality often stems from either local recurrence or regional and systemic metastasis[1]. Understanding and predicting metastasis, as well as enhancing prognosis, are major areas of direct and indirect oral cancer research attention. There have been significant efforts in the past few years to investigate the cellular and molecular pathways involved in metastasis[2].

In this regard, matrix metalloproteinases (MMPs) are one notable category of variables that show promise in predicting invasion and metastasis in OSCC. MMPs are a large family of calcium-dependent zinc-containing endopeptidases that belong to the extracellular matrix (ECM) degrading enzyme family[1]. The ECM, which is made up of proteins like proteoglycans, collagens, elastins, and gelatin, is remodeled and broken down by enzymes of this family[1,3]. MMP-9 and MMP-13, as two members of this family, have drawn interest in OSCC because of their unique roles.

A zinc-dependent proteinase called MMP-9 is essential in breaking down type IV collagen, which is a significant part of the basement membrane, and it is believed to significantly contribute to tumor invasion by facilitating tumor cell migration and the release of cytokines and other factors within the tumor environment, thereby promoting tumor growth and development[3-6].

Another member of the collagenase family, MMP-13, assumes a critical role in the MMP activation cascade by degrading various fibrillar collagens (types I, II, III, IV, X, and XIV), tenascin, fibronectin, aggrecan, versican, and fibrillin-I. MMP-13's involvement in oral cancer lies in its influence on metastasis through the formation of focal adhesions, protein synthesis during the G1 phase, and induction of cell cycle arrest in the G2 phase leading to the accumulation and activation of transcription factors like P53 and cyclin B1/CDK1.

During the breakdown of the ECM, collagen type II is more efficiently cleaved than collagen types I and III[7-9]. MMP-13 expression is elevated in several malignancies and is linked to tumor behavior and patient prognosis. Hence, this study aimed to evaluate the immunohistochemical expression of MMP-9 and MMP-13 in tissue specimens of OSCC and their correlation with clinical and histopathological grades.

MATERIALS AND METHODS

Approvals

Approval from the local Institutional Ethics Committee was obtained under the reference SVIEC/ON/DENT/BN-PG12//D12102, and written informed consent was obtained from each participant. The participants' demographic information, clinical presentation, and radiological findings, including size, location, lymph node status, and clinical stage, were documented in the provided format.

Case selection

This retrospective study involved a total of 40 cases of OSCC which were previously histologically confirmed by incisional biopsy.

The exclusion criteria included having other systemic disorders, refusing to provide informed consent, or having received OSCC treatment in the past.

Sectioning and staining procedures

Paraffin-embedded blocks were used to prepare four sections measuring 4 to 5 microns each. Hematoxylin and eosin (HE) staining was applied to two of these sections, and immunohistochemistry (LSAB) expression of MMP-9 and MMP-13 (DAKO Corporation, Denmark, Clone-DCS-6) was detected on the remaining two sections that were mounted on slides coated with silane.

Tissue sections after deparaffinization in xylene were rehydrated through a descending ethanol series (100%, 95%, 90%, 80%, and 70%) at room temperature for 5 min. Antigen retrieval was done using a microwave, followed by a wash in distilled water after allowing it to cool for 10 min. Endogenous peroxidase activity was blocked with hydrogen peroxide for 5 min. Incubation with the prediluted primary rabbit monoclonal MMP-9 antibody (DAKO Corporation, Denmark, Clone-DCS-6) was then done for 30 min at room temperature. The slides were then rinsed with Tris buffered saline (TBS) three times and incubated with the secondary antibody reagent: A labeled secondary antibody (biotinylated anti-mouse, anti-rabbit, anti-goat immunoglobulin) in phosphate buffered saline (PBS) with carrier protein and sodium azide, streptavidin peroxidase (streptavidin conjugated to horseradish in PBS with carrier protein and an antimicrobial agent), and buffered substrate containing hydrogenperoxide (H₂O₂) and a preservative (pH 7.5) for 15-30 min at room temperature. 3'3-diaminobenzidine (DAB) was then applied for minutes. For counterstaining, the slides were rinsed with deionized water, incubated for 5 min with hematoxylin, and rinsed with PBS for 1 min.

Evaluation of staining

Using the Bryne's grading scheme, the HE stained areas were evaluated and categorized as grade I, grade II, or grade III[10]. Both markers were processed following the manufacturer's instructions. Positive immunoreactivity was indicated at the target antigen site by the presence of a brown-colored end product. Normal gingival epithelial tissue sections were used as positive controls, while the absence of staining was used as a negative control. MMP-9 immunopositivity was detected in the cytoplasm of tumor cells, and Table 1 provides an assessment of its intensity. MMP-13 exhibited predominantly positive staining in the cell nucleus, occasionally in the cytoplasm, and its intensity was assessed as indicated in Table 2[11].

Table 1 Staining intensity and grading of matrix metalloproteinase-9 expression.

Grade	Interpretation
0	No positivity
Mild	Up to 10% of positive tumor cells
Moderate	11%-50% of positive tumor cells
Intense	Above 50% of positive tumor cells

Table 2 Staining and grading of matrix metalloproteinase-13.

Grade	Interpretation
0	No staining of the tumor or stromal cells
1+	Mildly (< 50%) positive staining of the tumor cells and/or weak staining of stromal cells
2+	Moderately (> 50%) positive staining of the tumor cells and/or moderate staining of stromal cells
3+	Intense staining of the tumor cells and/or strong staining of stromal cells. No normal epithelial cells were stained

In order to reduce observer bias, all observations were recorded by three separate observers. The surgically removed lymph node samples were subjected to histological examination to ascertain whether tumor cells were present in the lymph nodes.

Statistical analysis

Pearson’s χ² test was used to establish a relationship between two variables.

RESULTS

Case demographics

This study examined a sample of 40 people, of whom 25 were male and 15 were female (Table 3). The majority of the 40 participants were in their fifth or sixth decade, and there was a noticeable male predominance.

Table 3 Gender frequency distribution of cases.

	Male	Female	Total
Cases	25	15	40

The most frequently implicated areas were the vestibule and the buccal mucosa, followed by the tongue; the least frequently involved site was the hard palate (Table 4).

Table 4 Site distribution of cases.

Site	No of cases	Percentage
Buccal mucosa	20	50
Buccal vestibule	01	2.5
Tongue	16	40
Hard palate	03	7.5
Total	40	100.0

The majority of patients used tobacco, primarily in non-smoking forms. Histopathologically, according to the Bryne’s grading system, the majority of cases, as shown in Table 5, were grade I (62.5%) lesions, followed by grade II (30%) and grade III (7.5%).

Table 5 Bryne’s histological grading of oral squamous cell carcinoma cases.

Grade	No of cases	Percentage
I	12	30.0
II	25	62.5
III	3	7.5
Total	40	100.0

Staining analysis and interpretation

Details of different MMP-9 and MMP-13 staining intensities are displayed in Table 6. All the cases were positive for MMP-9, with the majority showing intense expression. Important details of the associations between tumor size and MMP-9 and MMP-13 expression are given in Table 7. For MMP-13, categorized by expression intensity ('None', 'Mild', 'Moderate', and 'Intense'), a χ² analysis revealed a non-significant association (P = 0.27) with tumor size. Comparably, categorized MMP-9 exhibited a non-significant association (P = 0.80) with tumor size. Table 7 displays MMP expression patterns in relation to tumor size, even though no statistical significance was found. 'Intense' MMP-9 and MMP-13 expression was frequently associated with greater tumor sizes.

Table 6 Comparison of matrix metalloproteinase-9 and matrix metalloproteinase-13 expression.

Metastasis	MMP-9			MMP-13			Intense
	Mild	Moderate	Intense	Nil	Mild	Moderate
Absent	1	5	12	2	2	6	8
	5.6%	27.8%	66.7%	11.1%	11.1%	33.3%	44.4%
Present	0	6	16	1	7	5	9
	0.0%	27.3%	72.7%	4.5%	31.8%	22.7%	40.9%
Total	1	11	28	3	9	11	17
	2.5%	27.5%	70.0%	7.5%	22.5%	27.5%	42.5%

MMP: Matrix metalloproteinase.

Table 7 Association between tumor size with matrix metalloproteinase-9 and matrix metalloproteinase-13 expression.

MMP-13					MMP-9
Tumor size	T1	T2	T3	T4	T1	T2	T3	T4
None	1	3	0	0
Mild	5	6	0	0	5	5	0	0
Moderate	1	11	1	0	2	6	0	0
Intense	1	8	2	1	1	17	3	1
χ2 (P) value				11.02 (0.27)				11.26 (0.80)

MMP: Matrix metalloproteinase.

Table 8 presents the results, which indicate a significant (P = 0.000) correlation between MMP-9 expression (classified as 'Mild', 'Moderate', and 'Intense') and lymph node involvement. Notably, 'Intense' MMP-9 expression predominated in cases with nearby lymph nodes (19 cases). Similarly, MMP-13 exhibited a strong association (P = 0.000) with lymph node involvement, with the highest frequency of "Moderate" expression in cases with nearby nodes (10 cases). The results highlight the clinical importance of MMP-9 and MMP-13 in the advancement of OSCC, irrespective of the histological grade. Table 9 indicates the striking connections between the formation of metastasis and the expression of MMP-9 and MMP-13 in our study. MMP-9 expression, categorized as 'Mild', 'Moderate', and 'Intense', displayed a significant relationship (P = 0.000) with metastasis. Notably, 'Intense' MMP-9 expression was predominantly seen in cases where metastasis was present (21 cases). MMP-13, categorized as 'None', 'Mild', 'Moderate', and 'Intense', also displayed a strong association (P = 0.000) with metastasis. 'Intense' MMP-13 expression was most prevalent in cases with metastasis (12 cases).

Table 8 Association between node involvement and matrix metalloproteinase-9 and matrix metalloproteinase-13 expression.

		MMP-9			MMP-13
		Mild	Moderate	Intense	Mild	Moderate	Intense
Node involvement	No involvement	9	6	1	10	2	0
	Nodes nearby	1	2	19	1	10	11
	Distant nodes	0	0	2	0	1	1
χ2 (P) value		26.17 (0.000)			29.18 (0.000)

MMP: Matrix metalloproteinase.

Table 9 Association of presence of metastasis with matrix metalloproteinase-9 and matrix metalloproteinase-13 expression.

		MMP9			MMP13
		Mild	Moderate	Intense	None	Mild	Moderate	Intense
Presence of metastasis	Presence	1	2	21	0	1	11	12
	Absence	9	6	1	4	10	2	0
χ2 (P) value		26.03 (0.000)			29.16 (0.000)

MMP: Matrix proteinase.

Tumor size and grade of node involvement

Table 10 sheds light on the relationships between tumor size, grade of mode involvement, and the presence of metastasis. Tumor size T1 was associated with 'Good' involvement (2 cases) and 'Moderate' involvement (6 cases), while no cases were observed in the 'Poor' involvement category (χ² = 4.31, P = 0.63). For tumor size T2, 10 cases exhibited 'Good' involvement, 15 cases exhibited 'Moderate' involvement, and 3 cases exhibited 'Poor' involvement. Tumor sizes T3 and T4 did not exhibit any cases of 'Poor' involvement.

Table 10 Association between tumor size, grade of node involvement, and presence of metastasis.

Tumor size	Good	Moderate	Poor	χ2 (P) value
T1	2	6	0	4.31 (0.63)
T2	10	15	3
T3	0	3	0
T4	0	1	0
Node involvement
No involvement	4	12	0	3.55 (0.47)
Nodes nearby	7	12	3
Distant node	1	1	0
Presence of metastasis
Presence	8	13	3	2.88 (0.23)
Absence	4	12	0

Node involvement and grade

Among cases with 'No Involvement' of nearby nodes, 4 cases were categorized as 'Good' involvement and 12 cases as 'Moderate' involvement, with no cases in the 'Poor' category (χ² = 3.55, P = 0.47). In cases with 'Nodes Nearby', 7 cases had 'Good' involvement, 12 cases had 'Moderate' involvement, and 3 cases had 'Poor' involvement. In the 'Distant Node' category, 1 case each exhibited 'Good' and 'Moderate' involvement, with no cases of 'Poor' involvement observed.

Presence of metastasis and grade

'Presence' of metastasis was associated with 8 cases of 'Good' involvement, 13 cases of 'Moderate' involvement, and 3 cases of 'Poor' involvement (χ² = 2.88, P = 0.23). 'Absence' of metastasis was associated with 4 cases of 'Good' involvement and 12 cases of 'Moderate' involvement, with no cases of 'Poor' involvement.

DISCUSSION

OSCC stand as the most common type of cancer affecting the head and neck region, and it frequently involves lymph nodes and has a predisposition toward substantial local infiltration, which generally translate into a bad prognosis. One of its most distinguishing characteristics is the ability to penetrate surrounding tissues, such as blood and lymphatic vessels, though the ability depends on the tumor's ability to degrade the ECM. Several studies have identified a connection between a high level of enzymatic degradation of the ECM, tumor invasion, metastasis, and elevated expression of MMPs[2,5-7,12].

Malignant cells go through a number of complex stages during their invasion and metastasis, including adhesion to the ECM, disintegration of matrix elements, cell separation, and migration through the deteriorated matrix[13]. Numerous proteases are necessary for this intricate process, and it is vital to keep a local equilibrium between these proteases and protease inhibitors[14].

Among the family of degrading proteases, MMPs are an important group involved in ECM degradation and tissue remodeling[15,16]. More than 20 membrane-type MMPs, collagenases, gelatinases, stromelysins, matrilysins, and other structurally related but genetically distinct MMPs have been found and classified into several families[13,17]. MMP-13, classified as a collagenase, has been linked to the formation and invasion of OSCC and is involved in the regulation of cell proliferation and survival. Its expression can be triggered by various cytokines and growth factors such as tumour necrosis factor-alpha and transforming growth factor-beta[9]. Notably, tumour necrosis factor-alpha can also stimulate the metastatic pathway by attracting factors conducive to metastasis to the cell surface[1-4,18].

However, MMP-9, also referred to as gelatinase B, is involved in the breakdown of collagen in the basement membrane and connective tissue. This role is essential for promoting the invasion of tumor cells and the metastatic process[17,19,20].

The present study aimed to assess the expression of MMP-9 and MMP-13 and their association with different tumor characteristics. The results revealed that the expression increased with tumor size. Also, MMP-13 expression intensified with respect to the presence of metastasis and grade. Nonetheless, it was observed that the expression had a significant association with the existence of metastases. A similar observation was made regarding nodal involvement, demonstrating a strong association between the intensity of MMP-9 and MMP-13 expression and nodal involvement[21-23]. A recent study witnessed that MMP-13 expression was associated with poor tumour differentiation and malignancy in patients at younger age, indicating tumour aggressiveness[24]. Elahi et al[25] in their study, observed that MMP-9 expression remained independent of the stage/grade of the tumor. Nevertheless, when taking into account different histological grades, the clinical significance of MMP-9 overexpression was noteworthy, indicating its potential function in promoting tumor cell infiltration. Champatyray et al[23] reported a contrasting finding, indicating that MMP-9 expression displayed a significant correlation with different histological grades of oral cancer. In this study, MMP-9 expression levels increased from well-differentiated to poorly differentiated OSCC[23]. A similar study observed that higher expression of MMP-9 was correlated with greater possibilities of lymph node metastases and tumor stage[26]. Comparable results in concurrence to these studies were noted in the present study. Thus, the expression of MMP-9 may help clinicians with better management of this malignancy. In a separate investigation conducted by Xia et al[5], they identified MMP-9 as a promoter of metastasis in OSCC by regulating the mRNA stability of the parental gene. This finding implies that targeting circular MMP-9 could be a potential treatment strategy for metastatic OSCC[5].

It was found that MMP-9 and MMP-13 were both overexpressed during the early phases of OSCC, suggesting that these two MMPs might function together. It is hypothesized that MMP-9 and MMP-13, via different mechanisms, each contribute significantly, albeit independently, to the invasiveness and advancement of OSCC.

Remarkably, high levels of MMP-9 and MMP-13 expression showed a strong link with TNM stage but no association with the histological grade of OSCC. This result draws attention to possible subjectivity in the grading scheme, which may have been brought about by an unequal distribution of cases across grades and a small sample size. Furthermore, differences in their expression patterns may be attributed to the intricate and multifactorial nature of metastasis in squamous cell carcinoma, which involves the activity of numerous MMPs.

This observation further emphasizes the need for a thorough understanding of the pathogenesis of OSCC by highlighting the possibility that some well-differentiated OSCCs may display clinical aggressiveness, which is defined by regional invasion and metastasis.

The subjectivity in this study will be greatly reduced by a larger sample size. Additionally, a sample that is evenly distributed based on histological grading will facilitate improved comparison and correlation between various variables. Although the current study provides evidence in support of the role of tumor tissue MMP-9 and MMP-13 in lymph node metastasis, other MMPs have also been proposed.

CONCLUSION

OSCC is the most prevalent type of cancer in the world. MMPs change the way that cells behave and accelerate the progression of the disease, leading to invasion and metastasis. MMP-9 has prognostic significance in OSCC due to its overexpression, which facilitates metastasis and advances tumor growth.

The current study provides insight into the roles played by MMP-9 and MMP-13 in tumor invasiveness and progression. It may also aid in the creation of novel methods for evaluating lymph node metastasis, which could enhance patient outcomes and treatment options for those with OSCC. Further research and investigation into MMP-9 and MMP-13 in OSCC can use it as a template. Further research is needed on this to establish the correlation and interdependence of MMP-9 and MMP-13 with other MMPs in early stages of OSCC. This could make it easier to create new interventional therapy strategies that could enhance the prognosis and available treatment modalities for OSCC.

ACKNOWLEDGEMENTS

The authors wish to thank Jadhav KB for his valuable opinion during the preparation of the manuscript.