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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 17  |  Issue : 2  |  Page : 185-189
 

Immunohistochemical expression of basement membrane laminin in histological grades of oral squamous cell carcinoma: A semiquantitative analysis


1 Department of Oral and Maxillofacial Pathology, Sharavathi Dental College and Hospital, Shimoga, India
2 Department of Oral and Maxillofacial Pathology, Amrith Educational and Cultural Society Maaruti College of Dental Sciences and Research Centre, Bangalore, Karnataka, India

Date of Web Publication11-Oct-2013

Correspondence Address:
R Shruthy
Sharavathi Dental College and Hospital, Alkola, T H Road, Shimoga - 577 204, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-029X.119755

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   Abstract 

Background and Objectives: To determine the immunohistochemical (IHC) localization of basement membrane component laminin in histological grades of oral squamous cell carcinoma (OSCC). The purpose of this study was to demonstrate the loss of continuity of the basement membrane in OSCC using an antibody directed against laminin using advanced polymer staining system. Materials and Methods: A total of 30 cases of OSCC: 10 cases of well differentiated squamous cell carcinom (WDSCC), 10 cases of moderately differentiated squamous cell carcinoma (MDSCC), and 10 cases of poorly differentiated squamous cell carcinoma (PDSCC) were subjected to heat-induced antigen retrieval method using ethylene-di-amine-tetraacetic acid buffer in a microwave oven. Then the sections were stained with anti-laminin polyclonal antibody and visualized using super sensitive polymer horseradish peroxidase detection system. In each case, the integrity of the basement membrane laminin was assessed by using statistical analysis. Results: Statistical analysis showed a decreased distribution of laminin from WDSCC to MDSCC to PDSCC (P value 0.0573). The intracytoplasmic staining of laminin gradually increased from WDSCC to MDSCC to PDSCC (P value 0.0198). Interpretation and Conclusion: WDSCC cases showed more laminin expression in basement membrane around the tumor islands and less loss of continuity compared to MDSCC and PDSCC cases suggesting a greater enzymatic degradation of basement membrane components in MDSCC and PDSCC than WDSCC. The loss of structural basement membrane laminin and the presence of laminin in the tumor cells of PDSCC cases suggest that laminin helps in tumor invasion. The expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histologic differentiation and aggressiveness.


Keywords: Basement membrane, immunohistochemistry, laminin, oral squamous cell carcinoma, tumor invasion


How to cite this article:
Shruthy R, Sharada P, Swaminathan U, Nagamalini B R. Immunohistochemical expression of basement membrane laminin in histological grades of oral squamous cell carcinoma: A semiquantitative analysis. J Oral Maxillofac Pathol 2013;17:185-9

How to cite this URL:
Shruthy R, Sharada P, Swaminathan U, Nagamalini B R. Immunohistochemical expression of basement membrane laminin in histological grades of oral squamous cell carcinoma: A semiquantitative analysis. J Oral Maxillofac Pathol [serial online] 2013 [cited 2019 Dec 6];17:185-9. Available from: http://www.jomfp.in/text.asp?2013/17/2/185/119755



   Introduction Top


Squamous cell carcinoma (SCC) is defined as "a malignant epithelial neoplasm exhibiting squamous differentiation as characterized by the formation of keratin and/or the presence of intercellular bridges". [1]

The etiology of oral cancer is almost certainly multifactorial. The carcinogenic changes may be influenced by oncogenes, tumor suppressor genes, carcinogens, and mutations caused by chemicals, viruses, irradiation, drugs, tobacco, alcohol, hormones, nutrients, or physical irritants. [2]

Laminin is a large (900 kDa) mosaic protein which is a component of the basement membrane and is composed of many distinct domains with different structures and functions. Laminins are large heterotrimeric extracellular glycoproteins composed of α, β, and γ subunits. [3]

Laminin 5 (α3, β3, γ2) is a typical component of epithelial basement membrane and is considered as a biochemical equivalent of the anchoring filaments fixing basal keratinocytes to the basement membrane. [4]

Basement membrane degradation must occur for most tumors to invade tissues and is essential for metastasis. As a readily detectable basement membrane component, laminin can be used as a marker for presence of intact or degraded basement membrane during tumorigenesis. [5]

The purpose of this study is to demonstrate the break in the continuity of the basement membrane and correlate the same with the histological grading.

Oral SCC is one of the most invasive human tumors. Unravelling how laminin influences tumor development and invasion is fundamental in the development of new prognostic indicators and treatment strategies for OSCC. [5]


   Materials and Methods Top


Thirty cases of OSCC were retrieved from the archives of our college which included 10 cases of WDSCC, 10 cases of MDSCC and 10 cases of PDSCC oral squamous cell carcinoma. Normal salivary gland tissue was used as control. All the blocks were sectioned using a semiautomatic microtome (Microm HM340E) and subjected to immunohistochemical (IHC) study using polyclonal antihuman laminin primary antibody.

Immunohistochemical procedure followed for laminin staining

Tissue sections 3 μm thick mounted on poly-lysine coated slides were incubated at 37°C overnight and then for an hour at 60°C before staining. The slides were deparaffinized in xylene and rehydrated through graded alcohol into water and subjected to antigen retrieval using microwave oven, two cycles at 96°C for 6 min. The tissues were cooled to room temperature and incubated with peroxide block for 12 min to block endogenous peroxidase activity and subsequently for 10 min with protein block to eliminate background staining. The sections so treated were then incubated with primary antibody for 2 h followed by post primary for 30 min. Subsequently, they were incubated with Novolink polymer for 30 min and finally with fresh 3,3'-diaminobenzidine (DAB) chromogen for 1-2 min (prepared in a ratio of 1:20). The slides were then washed in water to remove the excess DAB and counterstained with Mayer's hematoxylin, dehydrated, cleared, and mounted with DPX and assessed for staining characteristics. Tris buffer was used as wash buffer as and when required.

Interpretation of staining

Normal salivay gland tissue showed positive expression around the basement membrane. [Figure 1] Presence of brown colored end product was indicative of positive immunoreactivity. Basement membranes of the epithelium, blood vessels, nerves, and muscles were used as internal positive control. The distribution of the stain in each case of OSCC was observed in the following areas: around the basement membrane of malignant epithelial islands and its continuity and within the cytoplasm of the cells.
Figure 1: Photomicrograph of control tissue: Positive expression of laminin around the basement membrane of salivary gland (IHC stain, ×200)

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   Results Top


The present study included a total of 30 confirmed cases of OSCC of which 10 cases were WDSCC, 10 cases MDSCC, and 10 cases PDSCC.

In each case, the integrity of the basement membrane laminin and intracytoplasmic laminin was assessed and subjected to appropriate statistical analysis.

The number of positive and negative cases in each parameter (basement membrane, continuity around the basement membrane and intracytoplasmic staining) under each grade (well, moderate, and poor) were tested for significance using binomial test and compared using the Fisher exact test.

[Table 1] shows the results of parameters in WDSCC, where 90% cases were positive for laminin around the basement membrane out of which 80% showed continuous positivity and was statistically significant [Figure 2] and [Figure 3]. Thirty percent of the cases showed intracytoplasmic positive staining [Figure 4].
Figure 2: Positive expression of laminin around the basement membrane in WDSCC (IHC stain,×200)

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Figure 3: Loss of continuity of expression of laminin around the basement membrane in WDSCC (IHC stain, ×200)

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Figure 4: Positive intracytoplasmic staining for laminin in WDSCC (IHC stain, ×200)

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Table 1: Results of parameters in well-differentiated oral squamous cell carcinoma

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[Table 2] shows the results of parameters in MDSCC, where 80% of the cases were positive for laminin both around the basement membrane and within the cytoplasm [Figure 5] and [Figure 6]. Eighty percent showed continuous positivity around the basement membrane. These results were statistically significant.
Figure 5: Loss of continuity of expression of laminin around the basement membrane in MDSCC (IHC stain, ×200)

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Figure 6: Positive intracytoplasmic staining for laminin in MDSCC (IHC stain, ×200)

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Table 2: Results of parameters in moderately differentiated oral squamous cell carcinoma

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[Table 3] details the results of parameters in PDSCC. In PDSCC cases, 40% were positive for laminin around the basement membrane of which 30% showed continuous positivity. Ninety percent of the cases showed intracytoplasmic positive staining which was statistically significant [Figure 7].
Figure 7: Positive intracytoplasmic staining for laminin in PDSCC (IHC stain, ×200)

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[Table 4] compares the expression of laminin around the basement membrane between the three grades. The expression was statistically significant between WDSCC and PDSCC cases.
Table 3: Results of parameters in poorly differentiated oral squamous cell carcinoma

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Table 4: Comparison of parameters (around the basement membrane) between the three grades


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[Table 5] compares the continuous expression of laminin around the basement membrane between the three grades which was not statistically significant.

[Table 6] compares the intracytoplasmic staining between the three grades. Comparison between WDSCC and PDSCC cases showed statistical significance.
Table 5: Comparison of parameters (continuity around the basement membrane) between the three grades


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Table 6: Comparison of parameters (intracytoplasmic staining) between the three grades


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Comparison of all the three parameters in different grades of OSCC is shown in [Figure 8].
Figure 8: Graph showing comparison of all the three parameters in different grades of oral squamous cell carcinoma

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   Discussion Top


OSCC is the most prevalent malignant neoplasm. Despite the ready accessibility of the oral cavity to direct examination, these malignancies are often still not detected until it is very late and as a result, the survival rate for oral cancer has remained essentially unchanged over the past decades. In recent years, numerous prognostic factors associated with OSCC have been identified. [6]

Basement membrane is a dynamic structure that undergoes quantitative and qualitative changes during the progression of SCC, which is essentially important in tumoral invasion and metastasis. [7]

Laminin is a glycoprotein that is present in the basal membrane and has specific actions including an adhesion function. Tumor cells bind to laminin receptors on the basement membrane and are subsequently stimulated to produce metalloproteinases, which begin fragmentation and degradation of the membrane and help in tumor invasion. [7]

The aim of the present study was to demonstrate the expression of laminin in different grades of OSCC. All the 30 cases of OSCCs, 10 in each category were stained using the polyclonal laminin antibody and assessed for positive and continuous expression around the basement membrane and within the cytoplasm.

Positive laminin expression around the basement membrane was seen in 90% cases of WDSCC, 80% cases of MDSCC, and 40% cases of PDSCC. The expression was continuous around the basement membrane in 80% of WDSCCs and MDSCCs, which was statistically significant. Only 30% of PDSCCs showed continuous positive expression around the basement membrane which was not statistically significant.

These results are in accordance with previous studies by Tosios et al., [8] Souza et al., [7] and Mostafa et al., [9] where positive continuous staining of laminin was observed around the basement membrane. This indicates that the cells adjacent to basement membrane and the tumor cells secrete laminin.

Intracytoplasmic staining was observed in all the grades of OSCC. 30% of WDSCCs, 80% of MDSCCs, and 90% of PDSCCs showed positive staining indicating a statistical significance in case of MDSCC and PDSCC. This may be related to increased production and reduced secretion of laminin by the cells as they become progressively anaplastic. [10]

This is in accordance with the study by Mostafa et al., [9] where varied intracytoplasmic staining was observed in all the grades.

Similar studies have been carried out previously. Firth and Reade [11] showed that laminin distribution was continuous in epithelial hyperplasia while dysplastic lesions showed small focal breaks whose number increased in severe dysplasia. Kannan et al., [12] reported a gradual increase in the frequency of laminin discontinuity from normal epithelium to hyperplastic, dysplastic and SCCs, with significant differences among groups. Harada et al., found that the staining pattern of laminin in primary OSCCs was similar to that of metastatic nodules. Santos-Garcνa et al., [13] found discontinuity in dysplastic lesions that progressively increased for in situ and microinvasive carcinomas, being higher in OSCCs and total in the group of metastatic nodules. Our findings confirm and support these studies.


   Conclusion Top


We conclude from our study that, WDSCC cases showed more laminin expression in tumor island basement membrane and less loss of continuity compared to PDSCC cases suggesting a greater enzymatic degradation of basement membrane components than PDSCC.

Therefore, the expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histologic differentiation and aggressiveness. The absence of staining is associated with poor prognosis.

 
   References Top

1.R. Rajendran. Benign and Malignant Tumors of the Oral Cavity. In: R Rajendran and B Sivapathasundharam, editors. Shafer's Textbook of Oral Pathology. 6 th ed. New Delhi-India; Reed, Elsevier; 2009. pp 101.  Back to cited text no. 1
    
2.Rhodus NL. Oral cancer: Leukoplakia and squamous cell carcinoma. Dent Clin North Am 2005;49:143-65.  Back to cited text no. 2
    
3.Beck K, Hunter I, Engel J. Structure and function of laminin: Anatomy of a multidomain glycoprotein. FASEB J 1990;4:148-60.  Back to cited text no. 3
    
4.Kosmehl H, Berndt A, Strassburger S, Borsi L, Rousselle P, Mandel U, et al. Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma. B J Cancer 1999;81:1071-9.  Back to cited text no. 4
    
5.Campbell JH, Terranova VP. Laminin: Molecular organization and biological function. J Oral Pathol 1988;17:309-23.  Back to cited text no. 5
    
6.Pereira MC, Oliveira DT, Landman G, Kowalski LP. Histologic subtypes of oral squamous cell carcinoma: Prognostic relevance. J Can Dent Assoc 2007;73:339-44.  Back to cited text no. 6
    
7.Souza LF, Souza VF, Silva LD, Santos JN, Reis SR. Expression of basement membrane laminin in oral squamous cell carcinomas. Braz J Otolaryngol 2007;73:768-74.  Back to cited text no. 7
    
8.Tosios KI, Kapranos N, Papanicolaou SI. Loss of basement membrane components lamin in and type IV collagen parallels the progression of oral epithelial neoplasia. Histopathology 1998;33:261-8.  Back to cited text no. 8
    
9.Mostafa WZ, Mahfouz SM, Bosseila M, Sobhi RM, Zaki NS. An Immunohistochemical study of laminin in cutaneous and mucosal squamous cell carcinomas. J Egypt Women Dermatol Soc 2007;4:24-33.  Back to cited text no. 9
    
10.d'Ardenne AJ, Kirkpatrick P, Wells CA, Davies JD. Laminin and fibronectin in adenoid cystic carcinoma. J Clin Pathol 1986;39:138-44.  Back to cited text no. 10
    
11.Firth NA, Reade PC. The prognosis of oral mucosal squamous cell carcinomas: A comparison of clinical and histopathological grading and of laminin and type IV collagen staining. Aust Dent J 1996;41:83-6.  Back to cited text no. 11
    
12.Kannan S, Balaram P, Chandran GJ, Pillai MR, Mathew B, Nalinakumari KR, et al. Alterations in expression of basement membrane proteins during tumour progression in oral mucosae. Histopathol 1994;24:531-7.  Back to cited text no. 12
    
13.Santos-García A, Abad-Hernández MM, Fonseca-Sánchez E, Julián-González R, Galindo-Villardón P, Cruz-Hernández JJ, et al. E-cadherin, laminin and collagen IV expression in the evolution from dysplasia to oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 2006;11:E100-5.  Back to cited text no. 13
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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