|Year : 2013 | Volume
| Issue : 2 | Page : 272-275
Expression of type IV collagen in different histological grades of oral squamous cell carcinoma: An immunohistochemical study
Pankaj Agarwal, Ram Ballabh
Department of Oral Pathology and Microbiology, KD Dental College and Hospital, Mathura, Uttar Pradesh, India
|Date of Web Publication||13-Jun-2013|
Department of Oral Pathology and Microbiology, KD Dental College and Hospital, Mathura, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study was to evaluate the expression of type IV collagen in three histological grades of oral squamous cell carcinoma (SCC) in comparison with that in normal oral mucosa, to determine whether this protein can be used as a marker in early detection of the biological behavior of phenotypically altered cells.
Material and Methods: The staining intensity, staining pattern, and distribution mode for type IV were compared among the four groups by two pathologists, and the differences between observers and disease groups were statistically analyzed by Chi-square test or Fisher's exact test.
Results: The type IV staining intensity was more enhanced in well-differentiated (w) SCC than poorly-differentiated (p) SCC (P = 0.004). The staining was more linear (P = 0.001) and continuous (P = 0.003) in early invasive SCCs than in highly invasive SCCs. Its distribution was more continuous in wSCC than in less differentiated SCC (P = 0.003). There were statistically significant differences in the staining intensity between wSCC and pSCC (P = 0.005) or between wSCC and moderately differentiated SSC (P = 0.003) and in the staining pattern between wSCC and pSCC (P = 0.001).
Conclusion: The results indicated that there was a direct relationship between the presence of type IV collagen and the differentiation degree of SCC cells and thus that SCC cells loose their capability to form the basement membrane as they become less differentiated.
Keywords: Basement membrane, oral mucosa, squamous cell carcinoma, type IV collagen
|How to cite this article:|
Agarwal P, Ballabh R. Expression of type IV collagen in different histological grades of oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther 2013;9:272-5
|How to cite this URL:|
Agarwal P, Ballabh R. Expression of type IV collagen in different histological grades of oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther [serial online] 2013 [cited 2019 Sep 16];9:272-5. Available from: http://www.cancerjournal.net/text.asp?2013/9/2/272/113382
| > Introduction|| |
Squamous cell carcinoma (SCC) is one of the most common malignant cancers of the oral cavity, en-compassing at least 92.8% of all oral malignancies. Despite improved diagnostic and therapeutic methods over the 20 last years, this tumor is still characterized by a high rate of mortality.  Five percent of all tumors occur in the head and neck, and approximately half of those occur specifically in the oral cavity. As the sixth leading cause of cancer-related mortality, oral cancer accounts for one death every hour in the United States. 
Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells to form metastases at distant sites. These processes require alterations in cell-cell and cell-extracellular matrix interactions.  Loss of basement membrane has been associated with many types of carcinomas, with tumor cells in lymph nodes and organ metastases. 
Basement membranes are complex structures composed of a mixture of collagen, glycoproteins, and proteoglycans.  Four major molecules are present in most basement membranes : t0 ype IV collagen (type IV), laminin, perlecan, and entactin.  Among them, type IV is important as a structural backbone of the basement membrane. Neoplastic invasion and metastases are characterized by the ability of tumor cells to cross tissue compartment boundaries. The subepithelial basement membrane plays an important role in the complex interactions of this process, as it is the first obstacle to be traversed by the neoplastic cells. The ability of malignant cells to destroy the basement membrane has been related to their invasive potential, and utilized as an aid in the early diagnosis and prediction of the biological behavior in various tumors. 
| > Material and Methods|| |
The material for the study included 35 formalin-fixed paraffin-embedded tissue blocks retrieved from the Department of Oral Pathology and Microbiology. Among these, 10 cases (blocks) of well-differentiated (w) SCC, 10 of moderately differentiated (m) SCC, 10 of poorly-differentiated (p) SCC, and 5 of normal oral mucosa as controls were investigated for immunohistochemical staining for type IV collagen.
A monoclonal mouse anti-human type IV collagen antibody was obtained from Sigma Aldrich Chemicals Co. Ltd. (St. Louis, MO, USA), and biotinylated goat anti-mouse IgG and reagents for immunohistochemistry were obtained from Dako (Glostrup, Denmark). Four-micron-thick sections were taken and mounted onto poly-l-lysine adhesive-coated glass slides and incubated for 1 h at 48°C in a slide warmer for proper adhesion of the section to the slide. Following the standard protocol using the avidin-biotin system, immunoperoxidase staining was performed.
Sections were dewaxed through three changes of xylene for 5 min each and hydrated through descending grades of alcohol (100%, 90%, and 70%), 5 min each, and brought to water. The sections were dipped in freshly prepared 3% H 2 O 2 in methanol for 10 min to block endogenous peroxidase activities. They were washed in two changes of phosphate bovine serum (PBS) for 5 min each. The sections were then digested with 2 mg/ml pepsin in 0.1 M HCl for 30 min at room temperature for optimal antigen retrieval. The sections were further treated with 10% goat serum for 30 min at room temperature to block non-specific antigen sites. They were incubated with the mouse monoclonal primary antibody against type IV (1:50 dilution) for 60 min at 37°C in a moisture chamber. For reagent control, the primary antibody was replaced with preimmune mouse IgG. Incubation with biotinylated goat anti-mouse IgG was carried out for 30 min at room temperature. Finally, the sections were incubated with streptavidin-peroxidase conjugate for 30 min at room temperature. In each step, sections were washed in three changes of PBS thoroughly for 5 min each, after incubation. For visualization, sections were incubated with diaminobenzidine tetrahydrochloride for 5 min, and then lightly counterstained with Mayer's hematoxylin for 45 s and then gently washed in water. The sections were dehydrated through ascending grades of alcohol and mounted with resinous media (Kirkpatrick and Lendrum's DPX).
Evaluation of immunohistochemical staining
Finally, the stained slides were evaluated using a light microscope at ×25 and ×40 magnifications. Two observers viewed all the slides to eliminate subjective bias, and the data were analyzed using Kappa statistics to eliminate interobserver bias. Comparisons within the different grades of oral SCCs were subjected to Chi-square test.
For the intragroup comparison, Fisher's exact test was applied. Fisher's exact test is used when we have two nominal variables. The P < 0.05 was significant, P < 0.01 was highly significant, P < 0.001 was very highly significant, and P > 0.05 was considered as not significant.
| > Results|| |
In the present study, the control sections of normal oral mucosa showed type IV expression at the junction of epithelium and connective tissue stroma as a linear continuous brown staining [Figure 1]. Following the evaluation of normal oral mucosa, 30 cases of oral SCC were then subjected to type IV staining. In each case, 3 unit fields were randomly selected. In each field, the staining intensity was graded as bright or weak. The staining pattern was evaluated as linear or granular, and the distribution mode was classified into continuous or discontinuous around the tumor cell nests.
|Figure 1: Normal oral mucosa. Immunoperoxidase stain for type IV collagen, hematoxylin counterstain, ×40. Type IV collagen was immunolocalized along the basement membrane of the mucosal epithelium and vascular channels|
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Immunoreactivity for type IV was observed to be bright around tumor islands in cases of early invasive SCC [Figure 2]a, whereas the staining intensity became weaker in highly invasive SCCs [Figure 2]b. In wSCC cases, there were obviously continuous type IV depositions around tumor cell nests [Figure 3]a; while less-differentiated SCC cell nests had discontinuous stainings [Figure 3]b. In early invasive SCCs, linear type IV staining was obtained around carcinoma cell nests [Figure 4]a. However, the staining became granular as SCC cells became more anaplastic [Figure 4]b.
|Figure 2: Oral squamous cell carcinoma. (a) Separated tumor cell nests, (b) packed tumor cell nests. Immunoperoxidase stain for type IV collagen, hematoxylin counterstain, ×25. In the invading front of squamous cell carcinomas, tumor cell nests were surrounded by strong type IV immuno-positive lines (a), while the type IV immunostaining intensity for type IV collagen was weaker in densely packed tumor cell nests in the central part of tumor tissues|
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|Figure 3: Oral squamous cell carcinoma. (a) Well localized tumor islands, (b) tumor cells invading the surrounding stroma. Immunoperoxidase stain for type IV collagen, hematoxylin counterstain, (a) ×25; (b) ×40. In early carcinomas, tumor islands were localized by continuous deposition of type IV collagen (a), while type IV collagen was disrupted as discontinuous deposition of antibody in later stages of carcinomas (b)|
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|Figure 4: Oral squamous cell carcinoma. (a) demarcated invading tumor front, (b) diffuse metastasis of tumor cells. Immunoperoxidase stain for type IV collagen, hematoxylin counterstain, (a) ×25; (b) ×40. The invading front of carcinoma was well demarcated from the adjacent stroma by linear deposition of type IV collagen in early grades of carcinoma (a), and higher grades of carcinomas showed granular deposition of immunoreactive type IV collagen (b)|
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Two observers independently evaluated the microslides for the staining intensity, pattern, and mode for type IV.
The Kappa test was applied for each parameter to minimize the inter-observer errors. Thus, the observation made by one observer was then subjected to further statistical analysis. When chi square test was applied to the data obtained after observing type IV-stained slides, statistically significant differences were confirmed for the staining intensity (P =0.004), staining pattern (P =0.001), and distribution mode (P =0.003) [Table 1]. Finally, when Fisher’s exact test was applied to the inter-group relation, statistically significant differences were obtained for the staining intensity between wSCC and pSCC (P =0.005) [Table 2] and between wSCC and mSCC (P =0.003) [Table 3]. In terms of the staining pattern, the difference was statistically significant between wSCC and pSCC (P= 0.001) [Table 4].
|Table 1: Comparison of staining intensity, pattern, and distribution of type IV collagen in oral squamous cell|
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|Table 2: Intragroup comparison of staining intensity in different grades of oral squamous cell carcinoma|
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|Table 3: Intragroup comparison of distribution of type IV collagen in different grades of oral ssquamous cell carcinoma|
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|Table 4: Intragroup comparison of staining pattern in different grades of oral squamous cell carcinoma|
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| > Discussion|| |
In the present study, the bright staining of the antibody was confined to the aggregates of organized epithelial cells, which was similar to the finding from a study done by Sakr et al.,  who hypothesized that basement membrane material forms a strong indicator of focal tumor differentiation and lacks invasion in to adjacent tissues. Kannan et al.,  supported these findings in their study on basement membrane proteins and noted a relationship between differentiation status of the lesions and expression of Type IV collagen. Differentiated lesions showed a more conspicuous basement membrane than less- differentiated lesion. Hence, the bright staining of Collagen IV in the present study secreted by tumor cells appears to reflect one aspect of the differentiated phenotype of tumor cells.
In the present study, discontinuity of basement membrane showed a gradual increase, parallel to the increasing tumor progression from well-to-poorly-differentiated stage. Similar observations were demonstrated by Kawata et al.,  who hypothesized that Matrix metalloproteianase (MMP)-2 was mainly expressed in cancer cells. Because MMP-2 degrades type IV, the level of MMP-2 in carcinomas may be a useful indicator of the degree of invasion and metastasis.
van Cauwenberge et al.,  reported that remnants of basement membrane material may represent areas of partial regression of the neoplasm. It has been well established that these non-cohesive irregular small cords and single cells are more likely to attain access to vasculature and develop metastasis. The invasive front should always be the field of study for alterations in basement membrane in ORAL SCC.
Further, moderately differentiated and well-differentiated carcinoma showed a linear and continuous deposition of type IV indicating that neoplastic cells still maintain their ability to produce basement membrane components. In pSCC, all microslides showed a patchy and granular distribution of collagen IV antibody. These can be considered as areas of reduplication, or the origin of this deposition may also be from compressed stromal elements. Re-duplication of the basal lamina has been observed by Kwon et al.,  in clear cell carcinoma of ovary and viewed as a criterion of differentiation. Thin disordered pattern of deposition in the present study can also be related to the secretory product by dedifferentiated malignant cells. Altered cells are able to migrate through the basement membrane in to adjacent connective tissue and may secrete new basement membrane component for a while at the periphery before local infiltration.
The results of the present study shows a definite alteration in distribution of collagen type IV with a significant loss in all cases of pSCC, which is a sign of appearance of phenotypically altered malignant cells at invasive front reflecting one of the inherent biological parameters of tumor aggressiveness.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]