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ORIGINAL ARTICLE
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Immunohistochemical evaluation of tumor angiogenesis and the role of mast cells in oral squamous cell carcinoma


1 Department of Oral Pathology and Microbiology, Haldia Institute of Dental Sciences and Research, Purba Medinipur, Haldia, West Bengal, India
2 Department of Oral Pathology and Microbiology, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Oral Pathology and Microbiology, Saraswati Dental College and Hospital, Lucknow, Uttar Pradesh, India
4 Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
5 Department of Oral Pathology and Microbiology, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India
6 Department of Oral Pathology and Microbiology, King's George Medical University, Lucknow, Uttar Pradesh, India

Correspondence Address:
Arpita Kabiraj,
Department of Oral Pathology and Microbiology, Haldia Institute of Dental Sciences and Research, Purba Medinipur, Haldia, West Bengal - 721 645
India
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Source of Support: None, Conflict of Interest: None

 > Abstract 

Background: Increased angiogenesis has been associated with neoplastic progression, metastasis and outcome in several studies and in a number of malignancies. Among the various host immune cells, mast cells have been implicated in tumor progression by promoting angiogenesis. The aim of this study is to examine the relationship between angiogenesis, mast cells with that of the normal oral mucosa (NOM) and oral squamous cell carcinoma (OSCC).
Materials and Methods: The study was conducted using routine haematoxylin and eosin staining procedure and included immunohistochemical staining for microvessels and toluidine blue staining for mast cells.
Results: The microvessel density (MVD) and mast cell density (MCD) of two groups (NOM and OSCC). The MVD and MCD in OSCC ranged from 59.18 to 263.31 microvessel/mm 2 and 41.65 to 193.28 cells/mm 2 respectively with mean (±standard deviation) 161.73 ± 48.27 microvessel/mm 2 and 83.59 ± 40.67 cells/mm 2. In both NOM and OSCC, the mean MCD was comparatively lower as compared to respective MVD (MCD < MVD) and comparatively lower in NOM as compared to OSCC (normal < OSCC).
Conclusion: A significant correlation is present between MCD and MVD in OSCC and also that both these entities are significantly increased in the disease process when compared to that of the NOM.

Keywords: Angiogenesis, mast cell density, mast cells, microvessel density, oral squamous cell carcinoma



How to cite this URL:
Kabiraj A, Jaiswal R, Singh A, Gupta J, Singh A, Samadi FM. Immunohistochemical evaluation of tumor angiogenesis and the role of mast cells in oral squamous cell carcinoma. J Can Res Ther [Epub ahead of print] [cited 2017 Apr 29]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=163693




 > Introduction Top


Cancer is a multifactorial disease occurring due to the combination of causal and predisposing genetic factors. Malignant neoplasm has been estimated to be amongst the principle causes of death worldwide and it is therefore, a highly serious public health matter.[1] Cancer and its great variation in the patterns of occurrences, is increasingly recognized to be a global problem and not one limited to the industrial nations.[2] The term “oral cancer” includes a diverse group of tumors characterized by proliferative lesions and tumor like masses arising from the oral tissue. It is the sixth most common cancer in the world accounting for approximately 4% of new cancer cases and 2% of all cancer deaths worldwide.[3],[4] More than 95% of the carcinomas of the oral cavity are squamous cell type, in nature. They constitute a major health problem in developing countries; representing a leading cause of death.[5] The World Health Organization (WHO) reported oral cancer as having one of the highest mortality ratios amongst all malignancies.[4] WHO expects a worldwide rising oral squamous cell carcinomas (OSCCs) incidence in the next decades.[6]

OSCC develops from the combined influences of an individual's genetic predisposition and exposure to environmental carcinogens and is thought to be a multistep process.[7] The various changes that occur include the aberrant expression and function of molecules regulating cell signaling, growth, survival, motility, angiogenesis and cell cycle control.[8] During the process of its growth, OSCCs invade and metastasize and hence new blood vessel formation is critical as such. Adequate amount of blood supply is created by stimulating endothelial cell proliferation and new blood vessel formation. This angiogenesis is an essential part of solid tumor formation.[7] Angiogenesis, the growth of new blood vessels from preexisting ones, is one of the essential phenotypes of tumor formation and is also important in a number of normal physiologic processes including growth and development, wound healing and reproduction.[9] The process typically involves sprouting, branching and differential growth of preexisting blood vessels and employment of supporting cells associated with the endothelium, including smooth muscle cells and pericytes.[10] Adequate supply of tumor cells with nutrients and oxygen and efficient drainage of metabolites is provided by a complex network of tumor blood micro vasculature.[11] The balance between positive and negative angiogenic modulators within the vascular microenvironment is the basis for angiogenesis. It requires the activities of a number of molecules, including angiogenic factors (promote proliferation and differentiation of vascular endothelial cells), extracellular matrix proteins, adhesion receptors and proteolytic enzymes. Several factors have been identified, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), transforming growth factor beta (TGF-β), hepatocyte growth factor, angiogenin, interleukin-8, angiopoietins etc.[12] Several biological markers have been investigated, studied and developed so as to implement preventive and prognostic measures for patients with OSCC. von Willebrand factor (vWF), a large multimeric glycoprotein encoded by a gene on chromosome 12 is synthesized by vascular endothelial cells and megakaryocytes, circulates in human plasma at concentrations of 10 μg/ml.[13] Immunohistochemical detection of anti-vWF VIII has been used extensively to quantify angiogenesis in tumors.

Mast cells were first described by Paul Ehrlich in his doctoral thesis (Ehrlich, 1878). Mast cells are phylogenetically old, highly granulated cells originating from the haematopoietic lineage. These cells complete their differentiation in peripheral tissues.[14] They are important in allergic reactions, inflammation, autoimmunity and T cell-mediated immune responses. They are located perivascularly and in close proximity to neurons.[15] Mast cells have the capability to release numerous pro-inflammatory, immunoregulatory and angiogenic molecules through different stimulation pathways.[14] They are a rich source of preformed and newly synthesized cytokines and growth factors that induce or modulate angiogenesis.[12] These cells are essentially play the role of the angiogenic phenotypes in premalignant lesions and also do contribute to new blood vessel formation during squamous epithelial carcinogenesis. The effects of mast cells on carcinogenesis are thought to be carried out through various pathways, including immunosuppression, enhancement of angiogenesis and disruption of the extracellular matrix and promotion of tumor cell mitosis.[16] Certain authors suggest that the accumulation of mast cells around the tumor margins and their release of potent pro-angiogenic and angiogenic factors may represent a tumor-host interaction which probably favors tumor progression. Among the various host immune cells, mast cells have been implicated in tumor progression by promoting angiogenesis.[17]

The aim of this study is to examine the relationship between angiogenesis, mast cells with that of the normal oral mucosa (NOM) and OSCC. This study will be enabled with the use of toluidine blue to detect the mast cell density (MCD) and an immunohistochemical marker-anti-vWF VIII to analyze the microvessel density (MVD) in OSCC.


 > Materials and Methods Top


The retrospective study was done to examine the relationship between angiogenesis, mast cells with that of the NOM and OSCC.

The study comprised of two groups. Group I (control group) contained 10 cases of NOM and Group II consisted of 30 cases of OSCC. The tissues for the control group were obtained from the formalin-fixed and paraffin-embedded blocks of the archives of the patients who had undergone minor surgical interventions previously. The samples for study group were collected from the formalin-fixed and paraffin-embedded blocks of histologically proven OSCCs from the archives of Department of Oral and Maxillofacial Pathology. There was no conflict of interest for the present study. The study was approved by the respective Ethical Committee of the institution. The paraffin blocks were sectioned on a rotary semiautomatic soft tissue microtome into three tissue sections. Two section of 5 μm thickness and one section of 3 µm thickness were taken. Five micrometer thick sections were stained with haematoxylin and eosin for the confirmation of lesion; another section of the same thickness was stained with the metachromatic dye 1% toluidine blue while 3 μm thick sections were taken on poly-L-lysine coated glass slides for immunohistochemical staining with anti-factor VIII related antigen.

Staining of different sections

Haematoxylin and eosin staining

Formalin-fixed, paraffin-embedded sections of the normal and squamous cell carcinomas were stained by haematoxylin and eosin. The samples were suspended in 10% buffer formalin for fixation variable period of time (about 24 h) depending on the volume of the specimen. After tissues were adequately fixed grossing of sample was done. Paraffin-embedded formalin-fixed tissues were processed and section by routine method.

Haematoxylin and eosin stained tissue sections were first evaluated to confirm the histological diagnosis and the most representative areas of interest were selected using magnifications ×10 and ×40 respectively for NOM [Figure 1] and OSCC [Figure 2]. Microscopic examination was carried out on Olympus BX51 trinocular light microscope with provision for photomicrograph with Olympus E-331 single-lens reflex digital camera.
Figure 1: Photomicrograph of H and E stained section showing normal oral mucosa (×40)

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Figure 2: Photomicrograph of H and E stained section showing oral squamous cell carcinoma (×40)

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Immunohistochemical staining with anti-von Willebrand factor (Factor VIII related antigen)

The 3 µm thick paraffin sections were dried in room temperature overnight on poly-L-lysine coated glass slide. The sections were deparaffinized by warming slides on slide warmer and cleared xylene and were hydrated through descending grades of alcohol. Proteolytic enzyme trypsin (HK052, BioGenex, USA) along with the reconstitution buffer was applied on the tissue sections for 8–10 min to enable the antigens to be uncovered for binding to the relevant antibody and epitope unmasking. In humid chamber, the slides were incubated in 3% hydrogen peroxide for 10 min and then rinsed in phosphate buffer solution (PBS) for endogenous peroxidise activity. The slides were incubated in 10% casein solution for 10 min then wiped off excess casein on blotting paper. Anti-vWF (Factor VIII related antigen) mouse monoclonal antibody was applied for 60 min. The slides were rinsed in PBS and super enhancer was applied for 25 min to enhance the signals. The slides were rinsed in PBS and were incubated with polymer-horseradish peroxidase reagent (anti-mouse and anti-rabbit secondary anti-body) for 30 min. Then slides were rinsed PBS and were incubated in freshly prepared 3, 3-diaminobenzidene tetrahydrochloride (DAB) solution containing 0.03% hydrogen peroxide for 10 min. The slides were rinsed with PBS and transferred to distilled water. The slides were dipped once in Mayer's haematoxylin and then washed in running water. Slides were passed through ascending grades of alcohol, cleared in xylene and mounted with DPX.

Result of staining

An individual microvessel was defined as any brown staining endothelial cell or separate cluster of brown staining endothelial cells, clearly separated from adjacent clusters and background, with or without lumen, irrespective of size. This was carried out for both NOM [Figure 3] and OSCC [Figure 4]. Endothelial cells were considered positive for anti-factor VIII related antigen if there was intracytoplasmic DAB staining (brown).
Figure 3: Photomicrograph of anti-von Willebrand factor antibody stained microvessels in normal oral mucosa (×40)

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Figure 4: Photomicrograph of anti-von Willebrand factor antibody stained microvessels in oral squamous cell carcinomas (×40)

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Counting procedure

The immunohistochemically stained sections were first analyzed for the expression of anti-vWF (Factor VIII related antigen) antibody at low power (×10). The number of microvessel in normal and in OSCC in 10 fields at a magnification of ×40 under a oculometer grid at the “hot spots” (areas of most intense neovascularization) were counted under light microscope.

Toluidine blue staining for mast cells

Reagents for 1% toluidine blue in 1% sodium chloride solution

1% toluidine blue:

  • Toluidine blue powder - 1.0 g
  • 70% alcohol - 100.0 ml


1% sodium chloride solution:

  • Sodium chloride powder - 0.5 g
  • Distilled water - 50.0 ml
  • Sodium chloride solution has to be made fresh


Working solution:

  • 1% toluidine blue - 5.0 ml
  • 1% sodium chloride - 45.0 ml.


Method for staining mast cells with toluidine blue

Sections of 5 μm thickness were deparaffinized by warming slides on slide warmer and were cleared in xylene. Slides with tissue sections were then hydrated through descending grades of alcohol and were then kept in distilled water for 5 min. The slides were flooded with working toluidine blue solution for 1–2 min and then immediately were rinsed in distilled water. The slides were then dehydrated, cleared mounted with DPX.

Results of staining

  • Mast cells:


    • Granules - purple or bright magenta
    • Nucleus - blue






  • Background:
  • Shades of blue.




Counting procedure

The toluidine blue stained sections were first screened at low power (×10). Mast cell counting was then performed under ×40 magnification for NOM [Figure 5] and OSCC [Figure 6]. The area of the microscope field was calibrated with an ocular grid fitted inside the eyepiece with an area of 0.04 mm 2. The mast cells were counted throughout each of the tissue sections in 10 representative and consecutive grid fields (×40 magnification). The mean of 10 values was calculated and expressed as mean ± standard deviation (SD) per mm 2. The field were studied in a step ladder fashion and care was taken to prevent the overlapping of fields.
Figure 5: Photomicrograph of 1% toluidine blue stained section showing mast cells in normal oral mucosa (×40)

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Figure 6: Photomicrograph of 1% toluidine blue stained section showing mast cells in oral squamous cell carcinomas (×40)

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Statistical tests used

Various statistical tests that were used for the present study were arithmetic mean, SD, Student's t-test, Mann–Whitney U-test, Pearson correlation, simple linear regression and level of significance (P value).


 > Results Top


The present study was conducted with an objective to determine the MVD with anti-vWF (anti-factor VIII related antigen) and MCD with the help of metachromatic dye 1% toluidine blue stain and also to determine the association and correlation between them in NOM and OSCC. The study comprised of two groups. Group I comprised of 10 cases (control group) with NOM and a total of 30 proven OSCC cases were selected for Group II (study group) [Table 1] and [Figure 7].
Table 1: Group wise distribution of cases

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Figure 7: Group wise distribution of cases

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The MVD and MCD of two groups (NOM and OSCC) are summarized in [Table 2] and also shown graphically in [Figure 8] The MVD and MCD in NOM ranged from 18.71 to 99.66 microvessel/mm 2 and 8.48–86.04 cells/mm 2 respectively with mean (±SD) 55.87 ± 27.98 microvessel/mm 2 and 30.26 ± 25.47 cells/mm 2 respectively. In OSCC the values ranged from 59.18 to 263.31 microvessel/mm 2 and 41.65–193.28 cells/mm 2 respectively with mean (±SD) 161.73 ± 48.27 microvessel/mm 2 and 83.59 ± 40.67 cells/mm 2. In both NOM and OSCC, the mean MCD was comparatively lower as compared to respective MVD (MCD < MVD) and comparatively lower in NOM as compared to OSCC (normal < OSCC).
Table 2: Mean microvessel density and mast cell density (mean±SD) of two groups

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Figure 8: Mean microvessel density and mast cell density of two groups

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Comparing the mean levels of MVD and MCD in NOM, t-test revealed significantly (P < 0.05) different and lower MCD (30.26 ± 25.47) as compared to MVD (55.87 ± 27.98), (t = 2.14; P = 0.046) [Figure 9]. Similarly, comparing the mean levels of MVD and MCD in OSCC, t-test revealed significantly (P < 0.001) different and lower MCD (83.59 ± 40.67) as compared to MVD (161.73 ± 48.27), (t = 6.78; P < 0.001) [Figure 10].
Figure 9: Comparative mean of microvessel density and mast cell density in normal oral mucosa

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Figure 10: Comparative mean of microvessel density and mast cell density in oral squamous cell carcinomas

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Further, comparing the mean MVD between the two groups, t-test revealed significantly (P < 0.001) different and higher MVD in OSCC (161.73 ± 48.27) as compared to NOM (55.87 ± 27.98), (t = 6.54; P < 0.001) [Figure 11]. While comparing the mean MCD between the two groups, t-test revealed significantly (P < 0.001) different and higher MCD in OSCC (83.59 ± 40.67) as compared to NOM (30.26 ± 25.47), (t = 3.88; P < 0.001) [Figure 12].
Figure 11: Comparative mean of microvessel density of normal oral mucosa and oral squamous cell carcinomas

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Figure 12: Comparative mean of mast cell density of normal oral mucosa and oral squamous cell carcinomas

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Correlating the levels of MCD and MVD in NOM, correlation analysis revealed a significant (P < 0.001) and positive correlation (r) between MCD and MVD (r = 0.93, P < 0.001) and determination (r2) of correlation coefficient (i.e., strength of association) was found to be 86.48% (r2 = 0.8648) [Figure 13]. Similarly, correlating the levels of MCD and MVD in OSCC, correlation analysis revealed a significant (P < 0.001) and positive correlation (r) between MCD and MVD (r = 0.64, P < 0.001) and determination (r2) of correlation coefficient was found to be 41.02% (r2 = 0.4102) [Figure 14].
Figure 13: Correlation and regression between mast cell density and microvessel density in normal oral mucosa

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Figure 14: Correlation and regression between mast cell density and microvessel density in oral squamous cell carcinomas

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Correlating the levels of MCD and MVD in all samples (normal + OSCC), correlation analysis revealed a significant (P < 0.001) and positive correlation (r) between MCD and MVD (r = 0.78, P < 0.001) and determination (r2) of correlation coefficient was found to be 60.28% (r2 = 0.6028) [Figure 15].
Figure 15: Correlation and regression between mast cell density and microvessel density in normal oral mucosa and oral squamous cell carcinomas

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 > Discussion Top


OSCC is a malignant neoplasm arising from the dysplastic epithelium of the oral cavity.[17] The development of OSCC is a process requiring the accumulation of multiple genetic alterations including activating mutations or amplification of oncogenes promoting cell survival and proliferation, as well as inactivation of tumor suppressor genes involved in the inhibition of cell proliferation.[7]

Angiogenesis (formation of new microvasculature), is an important component in many biological processes, both in physiological conditions and in pathological conditions.[17] It has long been known to aid in progression and metastasis of malignant tumors.[18] Angiogenesis is thus thought to be the outcome of an imbalance between positive and negative angiogenic factors produced by both tumor and host cells.[19] Several stimulators, like FGFs, TGF-β, tumor necrosis factor-alpha and VEGF and inhibitors for example angiotensin, platelet factor IV and thrombospondin-1 have been associated with the process of angiogenesis.[18]

Among the various host immune cells, mast cells have been implicated in tumor progression by promoting angiogenesisas they are considered to be an important source of several pro-angiogenic and angiogenic factors, such as histamine, heparin, chymase, basic FGF (bFGF), VEGF, TGF-β etc.[17] Accumulation of mast cells around the tumor margins and their release of potent pro-angiogenic and angiogenic factors may represent a tumor-host interaction which probably favors tumor progression.[14]

Thus, the present study was conducted in order to determine the MVD and MCD in OSCC and NOM and to evaluate the association between both the entities in the disease process.

Tumors require a blood supply for growth and dissemination. It is well accepted paradigm that tumors recruit new blood vessels by secreting growth factors from tumor cells.[20] The newly recruited microvasculature comprise a complex network that provides adequate oxygen and metabolic distribution to tumor cells. Tumor angiogenesis is driven by positive and negative regulators of endothelial growth and sustained tumor growth requires a positive balance between tumor cell proliferation and cell death or apoptosis. This theory serves as a basis of potential target for therapy.[21] It has been shown in studies that the initiation of angiogenesis appears concomitantly with a decrease in tumor cell apoptosis, while levels of tumor cell proliferation remain constant, thus leading to net tumor growth. Among the host cells, which produce and release pro-angiogenic and angiogenic factors are mast cells.[22]

The density of mast cells in a tissue has been studied using histochemical stains like toluidine blue, alcian blue and immunohistochemically using mast cell tryptase, heparin, chymase and carboxypeptidase A.[17] In the present study 1% toluidine blue stain was used to identify and count mast cells. MCD was found to be significantly higher among the OSCC group (83.59 ± 40.67) as compared with the NOM group (30.26 ± 25.47) as shown in [Figure 8] and [Figure 12].

However, Oliveira-Neto et al., on the contrary, found MCD to be lower in OSCC and premalignant lesions compared to normal controls. They attributed this to the migration failure of mast cells, which possibly reflect a modification in the microenvironment during tumor initiation and progression.[23]

MVD of highly vascularised tumors has been evaluated by different vascular markers in several recent studies. The most common utilized markers were VEGF, CD105, CD31, CD34 and vWF.[19] In the present study the MVD was evaluated immunohistochemically by using anti-factor VIII related antigen or vWF as it was found that factor VIII related antigen was better because of its better highlighting of vessels and its lesser nonspecific staining.[24] MVD was found to significantly higher in the OSCC group (161.73 ± 48.27) as compared to that of the normal (55.87 ± 27.98) as shown in [Figure 8] and [Figure 10], indicating that vascularity may be a reliable indicator of angiogenesis in oral lesions and that there is a close association between angiogenesis and tumor progression in oral mucosa.[25]

The definite role of mast cells and angiogenesis in OSCC is an issue of debate. The blood supply that is required for tumor growth is provided via angiogenesis as a result of tumor-mediated induction or over-expression of angiogenic factors. Mast cells have been proposed as angiogenesis promoters and the mast cell count appears to be a reliable prognostic marker in some tumors.[26]

Thus, the present study was conducted to determine the correlation between the tumor angiogenesis and the density of mast cells in OSCC. In the present study, correlation analysis revealed a significant (P < 0.001) and positive correlation (r) between MCD and MVD (r = 0.64, P < 0.001) and determination (r2) of correlation coefficient was found to be 41.02% (r2 = 0.4102) in OSCC as shown in [Figure 14].

Also, correlating the levels of MCD and MVD in NOM [Figure 13], correlation analysis revealed a significant (P < 0.001) and positive correlation (r) between MCD and MVD (r = 0.93, P < 0.001) and determination (r2) of correlation coefficient (i.e. strength of association) was found to be 86.48%.

These findings suggest that mast cells may aid in the upregulation of tumor angiogenesis as they help in the secretion of angiogenic factors, including histamine, heparin, tryptase, VEGF and bFGF. A key mediator that influences mast cell migration is mast cell growth factor which might be synthesized by endothelial cells and epidermal keratinocytes. This molecule is thought to direct the homing of mast cell precursors to epithelial tissues.[27] Tumor angiogenesis can be initiated by gathering endothelial precursor cells or available budding capillaries such as physiologic angiogenesis.[19]

On the contrary, Aroni et al.,[28] Mohseni et al.,[29] Kalra et al.,[30] in their studies found that MCD and MVD (blood vessels) were not correlated. The authors hypothesized that the lower density of mast cells was possible due to the massive degranulation of mast cells making their identification difficult.

The growth of solid tumor is dependent upon an adequate blood supply, which is achieved by generation of stroma, where the formation of new capillaries is a central event. In a particular tumor the number of microvessels and mast cells could be related to the amount of stromal component and consequently tumor having more stroma might have more mast cells and microvessels. For this reason variation in the amount of stroma and tumor cells in different counting fields might influence the objectivity of the microvessel counting procedure.

Although tumor angiogenesis is a complex event, our results support the view that mast cells are implicated in the formation of new blood vessels in tumor stroma and thus they might be involved in tumor progression. Analyzing the relationship between tumor angiogenesis and clinical outcome of patients with OSCC it can be deduced that assessment of tumor vasculature may serve as a reliable prognostic marker with which patients at risk for recurrences can be identified. Tumor vascularisation information may have a potential to develop a new strategy with anti-angiogenesis drugs.[31]

Although the number of mast cells and microvessel can be used as indicators of disease progression, further studies are essential for practical application.


 > Conclusion Top


On the basis of our study, we can thus conclude that a significant correlation is present between MCD and MVD in OSCC and also that both these entities are significantly increased in the disease process when compared to that of the NOM. The significant correlation between MCD and MVD suggests that these two factors act as indicators which demonstrate the role of mast cells in angiogenesis in OSCC and the role of angiogenesis in tumor progression. From the prognostic point of view, designing studies to determine the relationship between MCD, MVD and survival rate in oral cancers is to be encouraged. However, large scale multi-institutional studies are recommended that would provide us with a baseline data of the MCD and MVD in OSCC and may aid us in classifying the individuals with OSCC as high-risk or low-risk individuals and also in planning and developing of various adjuvants therapeutic strategies like anti-angiogenesis therapy and vascular targeting of anticancer therapy. The role of mast cells in angiogenesis in OSCC and the role of angiogenesis on tumor progression thus need to be further validated using larger samples that include recurrent cases and follow-up studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15]
 
 
    Tables

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