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Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 6  |  Page : 842
Stromal response in different histological grades of oral squamous cell carcinoma: A histochemical study


1 Department of Oral Pathology and Microbiology, Babu Banarasi Das College of Dental Sceinces, Faizabad Road, Lucknow, Uttar Pradesh, India
2 Department of Oral Pathology, Sri Guru Ramdas Institute of Dental Sciences and Research, Amritsar, Punjab, India
3 Department of Oral Pathology, Manipal College of Dental Scienes, Manipal, India

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Date of Submission17-Jun-2011
Date of Decision24-Feb-2012
Date of Acceptance03-Mar-2012
Date of Web Publication3-May-2013
 

   Abstract 

Objectives: The aim of the study is to evaluate the connective tissue changes in different grades of Oral Squamous Cell Carcinoma (OSCC's) and the influence of these changes in predicting the biological behavior of these tumors.
Materials and Methods: A total of 39 cases of OSCC's and 6 sections of controls were examined using seven connective tissue special histochemical stains.
Results: Staining intensity of collagen, reticulin, acid mucins, fibrin, glycoproteins, sulfated mucins, elastic fibers around the tumor islands and within the connective tissue was observed.
Conclusions: Tumor cells which grow progressively in the host stroma have the capacity to pre-empt and subvert the response of host, which is essential for their growth and spread.
Clinical Relevance: The reactive changes in the tumor stroma may alter the biological aggressiveness of oral cancer, and by incorporating this concept into a prognostic system, we may help to reflect the biologic diversity of oral cancer and predict its clinical outcomes.

Keywords: Grades of oral carcinomas, oral squamous cell carcinoma, reactive changes, special stains, stromal response

How to cite this article:
George J, Narang RS, Rao NN. Stromal response in different histological grades of oral squamous cell carcinoma: A histochemical study. Indian J Dent Res 2012;23:842

How to cite this URL:
George J, Narang RS, Rao NN. Stromal response in different histological grades of oral squamous cell carcinoma: A histochemical study. Indian J Dent Res [serial online] 2012 [cited 2019 Nov 13];23:842. Available from: http://www.ijdr.in/text.asp?2012/23/6/842/111291
Oral Squamous Cell Carcinoma (OSCC) is one of the most formidable health problems, in terms of morbidity and mortality, facing mankind today. Recurrences, second primary, field cancerization and skipped nodes are major hindrances in the successful therapy of OSCC's and therefore prognosis has remained poor for the past several decades. In the past 3 decades, highest incidences have been observed in Southeast Asian countries, with almost no significant improvement in mortality from these cancers. Carcinomas are malignant epithelial tumors in which epithelial cells show an atypical arrangement with varying degrees of differentiation. Epithelial tumor cells invade the stroma in groups or alveoli, which are embedded in; or surrounded by an extracellular matrix (ECM); producing reactive changes in the stroma. ECM is a dense lattice network of collagen and elastin, embedded in a viscoelastic ground substance composed of proteoglycans and glycoproteins. [1]

The mature ECM consists of a supramolecular aggregate of connective tissue proteins, including fibrillar and non-fibrillar collagens, elastin, glycoproteins and glycosaminoglycans, which interact with one another through covalent and non-covalent bonds to form highly insoluble materials. [2] Extracellular Matrix molecules influence differentiation, proliferation and migration of epithelial tumor cells; and also have stabilizing and separating functions. [3] The extracellular microenvironment of tumors is determined by matrix synthesized by normal and tumor cells, as well as the host stromal components secreted by surrounding host fibroblasts. Even in a single tumor there may be variations in the stroma, from one area to another; and compostion of the stroma may evolve over time. [4] However the role of fibrous components (collagen, elastin and reticulin fibers) and ground substance (glycoproteins, mucins and fibrin) in different stages of neoplasia still remains to be defined. The reactive changes in the tumor stroma may alter the biological aggressiveness of oral cancer. Incorporation of these concepts into a prognostic system may help to reflect the biologic diversity of oral cancer and predict clinical outcomes. A total of 39 cases of OSCC's were examined using various special stains and 6 sections were taken as controls. An attempt was made to evaluate the connective tissue changes in different grades of OSCC's and the influence of these changes in predicting the biological behavior of these tumors.


   Materials and Methods Top


Material for the study was retrieved from the Archives of Department of Oral Pathology, College of Dental Surgery, Manipal. The study included 6 cases of normal oral mucosa (used as controls) and 39 cases of OSCC's (13-well differentiated, 13-moderately differentiated and 13-poorly differentiated). The special stains used for the study were PAS, Alcian Blue-PAS (AB-PAS), Aldehyde Fuschin-Alcian Blue (AF-AB), Martius Scarlet Blue (MSB), Verhoff's and Gomori's silver reticulin stain. Salivary glands in the connective tissue served as positive control for PAS, AB- PAS and AF-AB stains. Blood vessels were taken as control for MSB stain; and collagen fibers in the lamina propria as control for Verhoff's stain. Paraffin embedded tissue blocks were sectioned at 5 μ thickness and incubated at 48°C on slide warmer for 1 hour to ensure adhesion of the sections to the slide. Sections were then deparaffinized in xylene and hydrated through decreasing grades of alcohol and taken to water. Consequently the sections were stained with connective tissue specific stains, using specific protocols for each stain as shown in [Table 1]. Staining Intensity was scored as 0 = absent, 1 = weak, 2 = bright.
Table 1: Techniques used for staining of specific components as in the study

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All the three grades of OSCC's were evaluated for staining intensity of each component around tumor islands. A total of 52 areas in each slide were evaluated. To eliminate the subjective bias, two observers independently evaluated all the slides. To minimize the inter observer bias, Bonferonni 't' test was applied. Results were then statistically analyzed using Chi square test and Mann Whitney 'U' test. Chi square test was applied to evaluate the staining intensity of the various components in connective tissue and around tumor islands. Mann-Whitney U test was applied for the inter comparison of the 3 grades of OSCC's and the staining intensity of all the 9 components.


   Results Top


Staining intensity around tumor islands

On application of Chi Square test, very highly significant 'P' value (P < 0.001) was noted in the staining of fibrin [Figure 1], [Figure 2] and [Figure 3] and collagen fibers [Figure 4], [Figure 5], [Figure 6], [Figure 7] and [Figure 8] [Bar Diagram 1] [Additional file 1].
Figure 1: Histopathological section revealing acid mucins around the tumor islands; Alcian Blue-Periodic acid-Schiff Stain, 20x

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Figure 2: Histopathological section revealing fibrin strands around tumor islands; Martius scarlet blue stain, 10x

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Figure 3: Histopathological section revealing fibrillar connective tissue stained positive for fibrin; Martius scarlet blue stain, 10x

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Figure 4: Histopathological section revealing glycoproteins in the connective tissue and around tumor islands; Periodic acid-Schiff Stain, 10x

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Figure 5: Histopathological section revealing glycoproteins adjacent to the tumor islands stained positive with Periodic acid-Schiff stain, 20x

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Figure 6: Histopathological section revealing collagen fibers lining the tumor islands; Verhoff's Stain, 10x

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Figure 7: Histopathological section revealing collagen fibers around the tumor islands; Verhoffs method, 20x

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Figure 8: Histopathological section revealing elastic fibers in the connective tissue; Verhoffs method, 40x

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Staining intensity in the connective tissue

On evaluation for staining intensity in the connective tissue, very highly significant 'P' value (P < 0.001) was noted in the staining of glycoproteins [Figure 4], [Figure 9], acid mucins, neutral mucins, collagen [Figure 10], [Figure 11] and reticulin fibers [Figure 2], [Figure 12] [Bar Diagram 2] [Additional file 2]. When well-differentiated carcinomas were compared to moderately differentiated, for staining around the tumor islands, 'P' value of acid mucins was highly significant. Comparing well-differentiated carcinomas with poorly differentiated for fibrin staining revealed a very highly significant 'P' value. Staining of glycoproteins and fibrin revealed very highly significant 'P' value, on comparing moderately differentiated OSCC's with poorly differentiated OSCC's. When cases of well-differentiated OSCCs were compared to those of moderately differentiated for staining intensity of different components in the connective tissue, acid mucins and fibrin revealed very highly significant 'P' values. Comparing well-differentiated OSCCs to poorly differentiated, for staining intensity in the connective tissue, glycoproteins, acid mucins and collagen fibers revealed very highly significant 'P' values. Glycoproteins, neutral mucins, fibrin, collagen and reticulin fibers revealed very highly significant 'P' value on comparing moderately differentiated OSCCs to poorly differentiated OSCCs for staining intensity in the connective tissue. Comparison of staining intensity of each component in the connective tissue with their corresponding staining intensity around tumor islands in all the three grades of OSCC's revealed significantly high 'P' value for fibrin and reticulin fibers, very highly significant 'P' value for glycoproteins, neutral mucins and collagen fibers in well differentiated OSCC's as against the other two grades.
Figure 9: Histopathological section revealing sulfated mucins in the edematous connective tissue; Aldehyde fuchsin-alcian blue stain, 10x

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Figure 10: Histopathological section revealing fibrillar connective tissue positive for aldehyda fuchsin; Aldehyde fuchsin-alcian blue stain, 40x

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Figure 11: Histopathological section revealing collagen fibers in the connective tissue; Verhoff's Stain, 10x

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Figure 12: Histopathological section revealing reticulin fibers in the adjacent connective tissue; Gomori's silver reticulin staining method, 20x

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   Discussion Top


Solid tumors are composed of two discrete interdependent components, the malignant cells themselves and the stroma in which they are dispersed. Initially it was thought that stroma acts as an active antagonist to tumor cell invasion. Recent evidences however suggest that tumor stroma is not just a passive structure but is associated with tumor progression. Endothelial cells, pericytes, inflammatory cells, fibroblasts and extracellular matrix constitute the tumor milieu or tumor microenvironment which helps in tumor growth, invasion and metastasis. In the present study, the relationship between connective tissue stroma of the host and the invading tumor cells, showed some observable changes in the host response in all the grades of squamous cell carcinoma (SCC) when compared to the control stroma. Among the 52 fields observed in all the grades of SCC, most of the fields showed a weak staining of glycoproteins particularly around the tumor islands/nests. This may be a regressive change such as the lysis of stromal components, creating pathway for cell migration. There may be a change in stromal protein composition which is induced by mediators derived from tumor cells, supporting the in vitro studies of Iozzo. [5] Bernacki et al.[6] in their review described the presence of glycosidases in the interstitial fluid of various carcinomas, which support the observations made in this study.

Many studies have proposed that malignant epithelial cells continue to synthesize, secrete and assemble basement membrane (BM) materials, such as laminin, entactin and heparan sulphate. In the present study, few fields in all grades of SCC's showed a bright staining of PAS around the tumor islands / nests, which were probably the secretion of basement membrane components by the tumor cells. Combinations of special stains were employed to differentiate the different types of mucins, components of the ground substance. Observations revealed weak staining for acidic mucins with AB- PAS and AF-AB stains around the tumor cell nests in all grades of SCC; thus strongly sulphated mucin may be the product of stromal cells. Majority of the fields in different grades of OSCC showed an increased amount of neutral mucins and maximum staining of neutral mucins was seen in poorly differentiated OSCC (Bar Diagram 1). These neutral mucins could either be epithelial in origin as evident by their presence adjacent to the tumor cells with an increased amount of hexose components or accumulation of uronic acid containing substances like hyaluronic acid, chondroitin sulphate and heparan sulphate, or could be products of stromal cells produced in response to the invading heterogenous tumor cell population, which act probably as a scaffold around the tumor cells. The weak staining of these neutral mucins in a few areas may also be attributed to an increased breakdown caused by tumor cells or by the host-derived proteases, supporting the observation of Liotta [1] on tumor metastasis.

In the present study, PAS positivity was high in the connective tissue indicating an increased amount of neutral mucin secretion by the stromal cells while the acidic mucin secretion was scanty in all grades of OSCC.

Numerous studies on clinical and experimental cancers elicited by carcinogens have described microscopic changes indicating that stroma undergoes radical changes like collagen disintegration, fibroblastic proliferation and increased secretion of glycosaminoglycans antecedent to malignant growth. Observations made on tumor stroma revealed inflammatory cells derived from circulating blood along with fibrin deposits, possibly from clotting of extravasated plasma fibrinogen. Application of the histochemical stain MSB clearly demonstrated the presence of fibrin in all the grades of OSCC, seen as bright staining in poorly differentiated cases [Bar Diagram 1]; particularly around the tumor cell nests, forming an interface between tumor cells and the surrounding host tissue. This is in agreement with previous immunohistochemical studies by Dvorak et al.[7] and Brown et al.[8]

The accumulation of fibrin closely corresponds to leaky blood vessels or hyper permeability of tumor vessels, attributed to the release of permeability mediators by tumor cells. However, studies have stated that fibrin deposits form a three dimensional gel enveloping the tumor cells, which later on matures to a vascularized connective tissue, thereby inhibiting the migration of tumor cells drastically. [4] Though the tumor cells in poorly differentiated OSCC in the present study were in the form of short strands or small nests, abundant mature-fibrin staining red with MSB was evident around the tumor cells. Few workers have observed clotting abnormalities in patients with malignant neoplasms, [9] although no such observations were revealed in the history of our patient group. Ghosh et al. observed increased blood levels of fibrin degradation products in oral cancer patients. [10] Though all these findings suggest the role of fibrin; very little is known about the relationship of fibrin to tumor growth and metastasis. In the present study, however the amount of fibrin distribution correlated well with the amount and distribution of mature stroma that subsequently has replaced it, more so in the supporting connective tissue as well as in the stroma adjacent to the tumor islands. The results of the present study were concurrent with the observations made by Dvorak at the site of accumulation of fibrin, wherein there would be proliferation of stromal cells like fibroblasts and endothelial cells, resulting in neovascularization, [7] and fibroblasts synthesizing the components of matrix, together transform the fibrin- fibronectin stroma. Subsequently this highly vascularized tissue acquires more collagen and loses its cellularity and vascularity as observed immunohistochemically. [7] However Bano et al.[11] and Sakakibura et al.[12] in their culture study on gastric carcinoma cell lines, observed the synthesis of type IV collagen, the component of basement membrane.

In the present study among the fibrous proteins, collagen was more abundant than elastic and reticulin in the stroma as these are probably the products of benign fibroblasts or of the phenotypically altered tumor cells having a capacity to express stromal components, thereby localizing the tumor cells and preventing metastasis. [10] Cancer associated fibroblasts have been shown to alter the phenotype of malignant epithelial tumor cells and enhance tumor progression.

The results of the present study thus indicate that tumor cells which growing progressively in the host stroma have the capacity to pre-empt and subvert the response of host, essential for their growth and spread. Observable changes were seen in the stroma, in all the three grades of OSCC's. There was an increased stromal response in poorly differentiated carcinomas, when compared to the other grades. Abundant fibrin staining was visualized in the stroma particularly adjacent to the tumor islands/ nests in all the grades of OSCC; but it was comparatively more in poorly differentiated OSCC's. Acid mucins were scanty in the connective tissue and weakly stained around the tumor cells. However, neutral mucins showed strong PAS positivity around the tumor cells. Collagen was abundant in the stroma, particularly around the tumor cell nests like a scaffold, preventing the tumor cell from migration.


   Conclusion Top


The findings of the study infer that tumor cells act as obligatory parasites in the host stroma, and have the capacity to produce certain proteins which can attack the surrounding stroma or induce the proliferation of stromal cells to produce some of the stromal components with the resultant alteration of the normal stroma, possibly an auxiliary factor in tumorigenesis. Subsequently, tumor becomes more malignant with disruption in the local regulatory mechanisms between the epithelial and stromal cells. By considering the comprehensive view of interdependence of cancer cells and stroma, it can be concluded that carcinogenesis is a multistep process, each step reflecting the activities of another cellular gene necessary for the cellular transformation. Altered staining reactions of the stromal components reveal an underlying change in the biochemical level which needs to be studied further. Role of the stroma is therefore like a double edged sword, at times helping in tumor invasion and otherwise warding off the tumor cells. Therapies have to be developed to modulate the stromal behavior antagonizing metastasis.

 
   References Top

1.Liotta LA. Tumor invasion and metastases-Role of ECM: Rhoads Memorial Awards Lecture. Cancer Res 1986;46:1-7.  Back to cited text no. 1
    
2.Zucker S, Cao J, Malloy CJ. Role of matrix metalloproteinases and plasminogen activators in cancer invasion and metastasis: Therapeutic strategies. In: Baguley BC, Kerr DJ, editors. Anticancer drug development. 1st Ed. San Diego: Acedemic Press; 2002.p.92.  Back to cited text no. 2
    
3.Kosmehl H, Berndt A, Katenkamp D. Molecular variants of fibronectin and laminin: Structure, physiological occurrence and histopathological aspects. Virchows Arch 1996;429:311-22.  Back to cited text no. 3
    
4.Nagy JA, Brown LF, Senger DR, Lanir N, Van de Water L, Dvorak AM, et al. Pathogenesis of tumor Stroma generation: A critical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta 1988;948:305-26.  Back to cited text no. 4
    
5.Iozzo RV. Tumor stroma as regulator of neoplastic behaviour. Lab Invest 1995;73:157-60.  Back to cited text no. 5
    
6.Bernacki RJ, Niedbala MJ, Korythyk W. Glycosidases in cancer and invasion. Cancer Metastasis Rev 1985;4:81-104.  Back to cited text no. 6
    
7.Dvorak HF. Tumors: Wounds that do not heal. N Engl J Med 1986;315:1650-9.  Back to cited text no. 7
    
8.Brown PD, Whittaker M. Matrix metalloproteinase inhibitors. In: Teicher BA, Editor. Antiangiogenic Agents in Cancer Therapy. Totowa, N.J: Humana Press; 1999. p. 205-23.  Back to cited text no. 8
    
9.Sack GH Jr, Levin J, Bell WR. Trousseau's syndrome and other manifestations of chronic disseminated coagulopathy in patients with neoplasms: Clinical, pathophysiologic, and therapeutic features. Medicine (Baltimore) 1977;56:1-37.  Back to cited text no. 9
    
10.Ghosh M, Aroor AR, Raghavan MR. Clinical utility of serum fibrinogen degradation products in the diagnostic and prognostic evaluation of oral cancer. Ann Dent 1990;49:11-45.  Back to cited text no. 10
    
11.Bano M, Lewko WM, Kidwell WR. Characterisation of rat mammary tumor cell populations. Cancer Res 1984;44:3055-62.  Back to cited text no. 11
    
12.Sakakibara K, Suzuki T, Motoyama T, Watanabe H, Nagai Y. biosynthesis of an interstitial type of collagen by cloned human gastric carcinoma cells. Cancer Res 1982;2:2019-27.  Back to cited text no. 12
    

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Correspondence Address:
Jiji George
Department of Oral Pathology and Microbiology, Babu Banarasi Das College of Dental Sceinces, Faizabad Road, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.111291

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    Figures

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