Expression of Vascular Endothelial Growth Factor in Patients With Premalignant Lesions and Squamous Cell Carcinoma of Oral Cavity

Abstract

To evaluate and compare expression of VEGF in patients of premalignant lesions and squamous cell carcinoma of oral cavity. The cross sectional observational study is undertaken at the department of otorhionolaryngology and pathology, PGIMER and Dr RML Hospital, New Delhi,from 1st Nov 2017 to 31st March 2019,with a sample size of 30 cases each of premalignant lesions and oral squamous cell carcinoma immunohistochemistry by polymer method. In the participants with oral SCC, VEGF expression of Score 1 was observed in verrucous and well differentiated tumor, Score 2 in moderately differentiated SCC & Score 3 in poorly differentiated SCC with a p value of 0.0001. The observed difference and value of proportion p, is statically significant. In this study we concluded that VEGF expression increases as the lesion progresses from premalignant lesions to oral squamous cell carcinoma and is strongly associated with lymph node status (N-staging). Thus, VEGF can be a target in chemotherapy and its therapeutic implications in the HNSCC needs further research.

Levels of Evidence 1A: Systematic review of randomized control trials.

Introduction

The oral cavity is the upper most part of the digestive tract. The oral cavity is lined with stratified squamous epithelium of varying degrees of keratinization.

Squamous cell carcinoma is 94% of all oral malignancies. Globally over 3,00,000 people are diagnosed with oral cancer each year and it is the eighth most common malignancy [1] Globally mortality rates due to oral cancer is 30% in males and 12% in females [2]. Sex ratio reveals over 3:1 preponderance in male patients [3, 4].

The high frequency of cancer of oral mucosa in south east Asia is due to use of smokeless tobacco in form of betel quid, a combination of areca nut,tobacco and lime; pan masala- a mixture of areca nut,slaked lime, catechu and condiments; khaini—a tobacco lime preparation; alcohol; poor diet and viral (HPV) infections [5, 6].

Squamous cell carcinomas of the oral cavity, are clinically preceded by precancerous lesions, often.

1. Leukoplakia with histologic evidence of hyperplasia or dysplasia and prevalence of 1.1% and 11.7% with a mean value of 2.9% [7].

2. Erythroplakia, a fiery red patch, with a prevalence of 0.02%, 0.83% [8]. Lower alveolar mucosa, gingivobuccal sulcus and floor of the mouth are the most common sites [8].

3. Oral Lichen Planus (OLP) a chronic, autoimmune, inflammatory disease which may affect oral mucosa with prevalence from 0.5 to 3% [9].

4. Oral submucousfibrosis (OSMF) a chronic and potentially malignant disorder characterized by juxtaepithelial fibrosis of oral cavity.

Angiogenesis, the formation of new blood vessel is crucial to growth, invasion and metastasis of tumors [10, 11]. It was first proposed in 1971 by Judah Folkman. Angiogenetic process is activated by factors produced by tumors to form new blood vessels through basal membrane decomposition by the protease secreted by tumor cells and to support the movement and proliferations of vascular endothelial cells [12]^. Vascular endothelial growth factor (VEGF) is most potent cytokine that stimulates angiogenesis in tumors [13]. It was first identified by Ferrara and Henzel [14]. It is a 46 kDa heparin binding homodimeric glycoprotein which functions as a mitogen for vascular endothelial cells that help in migration and organization for neovascularization of tumor micrometastasis. VEGF bind to VEGF-1 receptor and VEGF-2 receptor and plays a crucial role in tumor angiogenesis, by increasing blood vessel permeability, endothelial cell growth, proliferation, migration and differentiation [20] (Fig. 1).

Fig. 1.

Dysplasia shown with immunohistochemistry staining

A significant increase in vascularity occurs during transition from normal oral mucosa, through differing degrees of dysplasia to invasive SCC [15–17]. This increase in vascularity has been associated with tumour progression from early to late carcinoma [18] and lymph node metastasis. Controlling the tumor size by manipulating the blood vessel formation may contribute to inhibition of tumor progression in malignant tumors.

Identification of molecular marker that can predict patients with precancerous lesions at high risk of transition to malignancy will provide a much needed opportunity to target forceful therapy for effective disease management. About 1/3rd of patients develop recurrent tumors after treatment that might be due to poor monitoring in the absence of a reliable biomarker. Therefore, identification and defining a suitable diagnostic and prognostic biomarker can provide help in designing strategies for treatment of oral cancer and monitoring disease activity during follow up period [19].

The aim is to assess increase in VEGF expression level with oral tumor progression.

Material and Methods

Thirty cases each of oral premalignant lesions and squamous cell carcinoma of oral cavity were included in the study.

Inclusion Criteria

• Eighteen years and above.

• Clinicopathologically diagnosed cases of oral squamous cell carcinoma.

• Clinicopathologically diagnosed cases of oral premalignant lesions.

Exclusion Criteria

Cases of oral malignancy other than squamous cell carcinoma.

Previously treated cases of oral squamous cell carcinoma and premaligant lesions of oral cavity.

Procedure

The study was conducted between 1st November 2017 and 31st March 2019.

A written and informed consent was obtained from patients and procedure to be conducted was explained to the patients.

Detailed relevant history followed by general physical examination and ENT examination was carried out.

Incisional wedge biopsy under LA was taken from clinically diagnosed cases in ENT OPD.

Formalin preserved samples of the biopsies were sent to Department of Pathology.

Appropriately labelled sections on poly-l-lyine coated slides were dewaxed by placing them on hot plate at 56 ˚C for 5 min, deparaffinised in xylene and brought through descending concentration of alcohol to water (Fig. 2).

Fig. 2.

Immunohistochemistry staining for moderately differentiated squamous cell carcinoma

Antigen retrieval was done as follows:

• The slides were immersed in citrate buffer (pH 6.0).The trough was kept in a decloaker till it reached temperature of 125 degree Celsius. Decloaker was switched off and slides were removed after it cooled at room temperature.

• Sections were washed with three changes of Tris buffer (pH 7.4) for 5 min.

• Extra buffer was tapped off the slide and slide surrounding the section was dried with help of filter paper.

• Endogenous peroxide blocking was done by adding one drop of 3% hydrogen peroxide to the sections. The sections were carefully placed in moist chamber and incubated for 10 min.

• Sections were washed with three changes of Tris buffer (pH 7.4) for 5 min each.

• Incubated in background snipper (protein blocker) solution for 10 min at room temperature.

• Excess solution was drained off

• Primary antibody (VEGF) was put on glass slide for 2 h at room temperature.

• Sections were washed with three changes of Tris buffer (pH 7.4) for 5 min each.

• Incubated in Mach 2 universal HRP – polymer for 30 min at room temperature in moist chamber.

• Sections were washed with three changes of Tris buffer (pH 6.0) for 5 min each.

• DAB solution was freshly prepared by adding one drop of DAB chromogen in one ml of DAB substrate buffer. After adding the DAB solution, the staining was controlled microscopically.

• Sections were then washed in distilled water for 5 min.

• Counter staining was done by hematoxylin for 1–2 min.

• Blueing was done under tap water.

• Sections were dehydrated in alcohol and mounted in DPX.

• Invasive carcinomas were diagnosed as well-moderately- or poorly differentiated lesions.

• Immunohistochemistry was performed using monoclonal antibody to VEGF and result was expressed in terms of endothelial to epithelial density.

• Samples of glomerular capillaries tuft were used as controls.

• VEGF expression was assessed according to positive staining and staining intensity (0 = none, 1 = mild, 2 = moderate, 3 = intense) where the staining intensity was weaker than endothelial cells specimen was assigned a score of 1, staining intensity similar to that of endothelial cells was assigned a score 2 and stronger than endothelial cells was assigned a score of 3 [21].

VEGF expression was confirmed by the presence of brown stained cytoplasm in the endothelial cells.Staining of epithelial cells and inflammatory cells was also observed [21] (Fig. 3).

Fig. 3.

Immunohistochemistry showing VEGF staining for well differentiated squamous cell carcinoma

Result

The results of the study conducted is as follows:

The mean (SD; standard deviation) age of patients with premalignant lesions was 51.4 (14.1) years and patients with oral SCC was 47.8 (10.6) years. The p-value is 0.21.

Gender distribution in premalignant lesions is (M:F), (72.7%M: 27.3% F); and for oral SCC (87.5% M: 12.5% F) with p-value is 0.137.

Socio-economic distribution in premalignant lesions is 51.5% of lower, 18.2% middle and 30.3 upper economic strata and in Oral SCC, 40.6% of lower, 15.6% middle and 43.8% upper economic p-value is 0.528.

Residence in premalignant lesions, 45.5% from rural, 33.3% to semi-urban areas and 21.2% to urban areas. In Oral SCC 46.9% rural, 31.3% semi-urban and 21.8% urban areas and p-value is 0.984.

Use of alcohol/Tobacco was 30.3%/42.4% in premalignant lesions, 87.5%/96.9% in Oral SCC and p-value is 0.000/0.001.

Oral premalignant lesions are leukoplakia (84.9%), Oral Subnucous fibrosis (24.2%) Erythroplakia (3%), Lichen planus (15.2%).

Oral SCC, well differentiated tumour (34.3%); moderately differentiated tumour (56.3%); Verrucous (6.3%) and poorly differentiated tumour (3.1%).

Clinically palpable lymph nodes are absent in premalignant lesions and in Verrucous form. In OSCC 83.3% subject with moderately differentiated SCC were positive; 100% in poorly differentiated SCC, 45.5% in well differentiated OSCC. p-Value is 0.032.

Table 1 denotes the proportion of different scores of VEGF expression among those who had oral premalignant lesions and those with oral squamous cell carcinoma.The p-value is 0.0001.

Table 1.

Scores of VEGF expression among subjects with oral premalignant lesions and oral squamous cell carcinoma

Score of VEGF	Subjects with oral premalignant lesions	Subjects with oral squamous cell carcinoma
1	33 (100.0%)	0 (0.0%)
2	0 (0.0%)	13 (40.6%)
3	0 (0.0%)	19 (59.4%)

*p = 0.0001

Tables 2, denotes the proportion of different score of VEGF expression depending upon the tumour differentiation. The p-value is 0.0001.

Table 2.

Score of VEGF expression according to type of tumour differentiation among subjects with oral squamous cell carcinoma

Score of VEGF	Verrucous	Well differentiated	Moderately differentiated	Poorly differentiated
1	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
2	2 (100.0%)	11 (100.0%)	0 (0.0%)	0 (0.0%)
3	0 (0.0%)	0 (0.0%)	18 (100.0%)	1 (100.0%)

*p = 0.0001

Table 3, denotes the presence of clinically palpable lymph nodes according to score of VEGF expression, p = 0.001, that is statically significant.

Table 3.

Presence of clinically palpable lymph nodes according to Score of VEGF expression

Score of VEGF	Score 1	Score 2	Score 3
Lymph node present	0 (0.0%)	5 (38.5%)	16 (84.2%)
Lymph node absent	33 (100.0%)	8 (61.5%)	3 (15.8%)

*p = 0.0001

Table 4, denotes the T staging and scores of VEGF expression (N = 26). P = 0.32, which is not significant statistically.

Table 4.

T staging and scores of VEGF expression (N = 26)

	Score 1	Score 2	Score 3
T2	0 (0.0%)	3 (42.8%)	4 (57.2%)
T3	0 (0.0%)	0 (0.0%)	7 (100.0%)
T4a	0 (0.0%)	6 (50.0%)	6 (50.0%)

p = 0.32

Table 5, denotes, association between lymph node status (N staging) and scores of VEGF expression (N = 26). p = 0.001, that is statistically significant.

Table 5.

Association between lymph node status (N Staging) and Scores of VEGF expression (N = 26)

N Staging	Score 1	Score 2	Score 3
N0	0 (0.0%)	1 (16.7%)	5 (83.3%)
N1	0 (0.0%)	6 (42.9%)	8 (57.1%)
N2A	0 (0.0%)	2 (40.0%)	3 (60.0%)
N2B	0 (0.0%)	0 (0.0%)	1 (100.0%)

*p = 0.001

Table 6, denotes association between TNM staging and scores of VEGF expression (N = 26); p = 0.44, that is statistically not significant.

Table 6.

Association between TNM staging and Scores of VEGF expression (N = 26)

TNM staging (N = 26)	Score 1	Score 2	Score 3
II	0 (0.0%)	1 (50.0%)	1 (50.0%)
III	0 (0.0%)	2 (18.2%)	9 (81.8%)
IV A	0 (0.0%)	6 (46.2%)	7 (53.8%)

*p = 0.44

Discussion

Oral squamous cell carcinoma (OSCC) accounts for about 5% of all neoplasms of the body. Tobacco and alcohol use have been implicated in the etiology of OSCC along with premalignant lesions which also carry an increased risk for transformation into OSCC. Malignant epithelial cells of the OSCC need adequate blood supply thus tend to recruit new blood vessels by angiogenesis. VEGF is a 46 kDa heparin binding homodimeric glycoprotein which plays an important role in tumor angiogenesis, by increasing permeability of blood vessels, endothelial cell growth, proliferation, migration and differentiation. About 1/3^rd of OSCC patients develop recurrence after treatment, which might be due to poor monitoring in the absence of a reliable biomarker. Molecular biomarkers like VEGF have their role in assessing the progression of premalignant lesions into carcinoma.

Distribution of Age, Gender Socio Economic Status and Residence

There is no difference in the mean age of participants in the two groups, and carcinomas are associated with old age (70). Gender distribution across the two groups is similar. The socio economic distribution of the participants across the two groups is similar. The increased prevalence in rural areas can be attributed to lack of awareness and health facilities, poverty and ignorance.

Alcohol and Tobacco Use

We found statistical difference in the proportion of participants with alcohol and tobacco use among the two groups. In those with premalignant lesions, alcohol use was present in 30.3% of the participants whereas this proportion was extremely high (87.5%) in those with oral squamous cell carcinoma. This difference in proportion of alcohol use among both groups was found to be statistically significant (p = 0.0001). Further, in those with premalignant lesions, tobacco use was present in 42.4% of the participants whereas this proportion was extremely high (96.9%) in those with oral squamous cell carcinoma found to be statistically significant (p = 0.0001).

Association of VEGF and Lymph Node Metastasis

In our study we found that in patients with premalignant lesions, no clinically palpable cervical lymph nodes were found while those with oral squamous cell carcinoma, 65.6% of the participants had clinically palpable cervical lymph nodes and in the remaining 34.4%, no cervical lymph nodes were palpable. The observed difference was found to be statistically significant (p = 0.0001). It was seen in OSCC group that amongst those with clinically palpable lymph nodes 83.3% of patients had moderately differentiated squamous cell carcinoma and 100% in that of poorly differentiated squamous cell carcinoma while 45.5% of the patients with well differentiated squamous cell carcinoma had clinically palpable cervical lymph nodes and none in those of Verrucous form and the premalignant group.The observed difference was found to be statistically significant (p = 0.032). Peasron chi square test was done and a strong significant association was found (p = 0.001) between VEGF expression and lymph node status (N staging).

Acharya et al. [22] in their study found that occurrence of lymph node metastasis is significantly and positively associated with histological features of oral squamous cell carcinoma like nuclear polymorphism (p = 0.02) and degree of keratinization (p = 0.04) [22]. In our study we found that patients with score 1 (n = 33) of VEGF expression, had no clinically palpable lymph nodes. In those with score 2 (n = 13) of VEGF expression, around 38.5%had palpable lymph nodes and among those with score 3 (n = 19) of VEGF expression, 84.2% had clinically palpable lymph nodes. This shows that with increased grade of VEGF expression, the probability of having palpable lymph nodes increases. The observed differences in proportions was statistically significant (p = 0.0001).

Tumor cells has been found to induce angiogenesis and lymphangiogenesis by expressing angiogenesis factor VEGF and the lymphangiogenic factors VEGF-C and VEGF-D in OSCC, VEGF predominantly induces tumor angiogenesis, whereas VEGFC and VEGF-D induce lymphangiogenesis [23]. A significant association between elevated VEGF circulating levels and clinical stage, and a possible relationship between the VEGF-C and/or VEGF-D expression levels and the development of lymphatic tumor spread has been seen in patients with OSCC. Anatomically, the buccal mucosa extends from the line of attachments of the upper and lower alveolar ridges to the pterygomandibular raphe. The lymphatics of this region drain into submental and submandibular lymph nodes; as a result, there is a high propensity for lymphatic spread through the rich lymphatic drainage. In oral squamous cell carcinoma (OSCC), the limited anatomic barriers within the buccal space provide essentially no resistance to tumor spread [24]. This is essentially the reason why we observed high proportions of participants with squamous cell carcinoma to have clinically palpable lymph nodes. The lymphatic system is more important than the vascular system in metastasis of oral squamous cell carcinoma. In HNSCCs, presence of lymph node metastasis at time of diagnosis is an indication of poor prognosis.

TNM Staging and VEGF Expression

In our study out of a sample size of 26 patients a total of 7.7% (n = 2) had TNM stage II, 42.3% (n = 11) had TNM stage III and 50.0% (n = 13) had TNM stage IVA. There was no statistical difference in the score of VEGF expression depending upon TNM stage (p = 0.44). Among those with TNM II stage, 50.0% had score 2 and 50.0% had score 3 of VEGF expression. Among those with TNM stage III, 18.2% had score 2 and 81.8% had score 3 of VEGF expression. Among those with TNM stage IVA, 46.2% had score 2 and 53.8% had score 3 of VEGF expression. Using pearson chi square test no significant association was found between TNM staging and expression of VEGF.

Distribution of Tumor Differentiation

In our study, 18 (56.2%) moderately differentiated SCC was the most prevalent followed by 11 (34.3%) well differentiated SCC and 2(6.2%) Verrucous then 1(3.1%) poorly differentiated SCC.

VEGF Expression: Tumor Progression and Differentiation

We found in this study that in all the participants with premalignant lesions, VEGF expression of score 1 was observed. In those with oral squamous cell carcinoma, 40.6% of the participants had VEGF expression of score 2 and in the remaining 59.4%, VEGF expression of score 3 was observed. VEGF expression of score 2 was observed in all the Verrucous and well-differentiated tumor differentiation types and VEGF expression of score 3 was observed in all the poorly differentiated and moderately differentiated tumour types. The proportion of different scores of VEGF expression depending upon the tumour differentiation was found statistically significant (p = 0.0001).This denotes that poorly differentiated tumor had higher score of VEGF expression.

VEGF is a highly potent angiogenic agent that increases vessel permeability and enhance endothelial cell growth, proliferation, migration, and differentiation. Progression of tumor from early to late carcinoma is also closely associated to vascularity and thus VEGF expression.

In moderately differentiated carcinomas (MDSCC), 88.8% cases were VEGF positive whereas in poorly differentiated squamous cell carcinomas (PDSCC), 100% were VEGF positive [23]. In the present study, an increased expression of VEGF was observed in different histologic grades of squamous cell carcinoma with well-differentiated OSCC showing the lowest mean VEGF percentage and intensity scores and poorly differentiated OSCC, the highest. Results of the study suggested that VEGF was present in elevated levels in dysplasias and OSCCs when compared with normal oral mucosal specimens.

Sheelam et al. [25] (2018) in their study evaluated the role of cell proliferation and vascularity in potentially malignant disorders and OSCC using a sample size of 60 patients (10 oral lichen planus, 10 oral leukoplakia,10 oral submucous fibrosis and 30 OSCC) by immunohistochemistry using polymer method and assessing the area of VEGF staining scored as 0, no stained cells in any microscopic field, 1, < 25% of tumor cells stained positively, 2, 25–50% of tumor cells stained positively, 3, 50–75% of tumor cells stained positively and 4, > 75% of tumor cells stained. They found increase in vascularity (VEGF expression) in the following order oral lichen planus (1.4), leukoplakia (1.9), oral submucous fibrosis (2.4) and OSCC (3.43). This difference was found to be statistically significant (p = 0.000).

VEGF: Therapeutic Implications

VEGF and Epidermal growth factor receptor (EGFR) and their signaling pathways are essential for tumor growth and dissemination, and have led to their emergence as critical targets for therapy.

A fundamental approach to inhibit angiogenesis during tumorigenesis is the disruption of VEGF/ VEGFR-2 pathways [26]. This could suppress tumors’ growth by limiting their blood supply; by changing their morphology, the vascular wall structure, and rendering VEGF and VEGFR-2-expression in tumor vasculature to a more normal pattern and thus improving drug penetration in tumors, and also by blocking VEGF autocrine pathway and thus reducing uncontrolled neoplastic cell proliferation. Currently, many VEGFR-2 inhibitors are undergoing preclinical and clinical evaluation.

Conclusion

In comparison to other studies, our study was done by immunohistochemistry analysis which is a less sensitive method as compared to RT-PCR and ELISA for estimation of VEGF expression. In the present study normal controls were not included hence correlation of expression of VEGF with disease progression could not be done which was done in the other studies.Thus further research is required for a better understanding of the VEGF expression and its association with OSCC and its various parameters. However, our study concluded that VEGF expression increases as the lesion progresses from premalignant lesion to OSCC and thus VEGF can be a target in chemotherapy and its therapeutic implications in HNSCC should be explored.

Funding

No funding sources.

Compliance with Ethical Standards

Conflict of interest

None of the authors declare conflicts of interest.

Ethical approval

Ethical clearance was obtained from Institutional Ethical Committee.

Human and animal rights

The Research is involving human participants but no animals.

Informed consent

Informed and written consent was taken from all human participants.