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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 2  |  Page : 60-67

Epidermal growth factor receptor as an early diagnostic marker of oral squamous cell carcinoma: Marking the line between severe dysplasia and oral squamous cell carcinoma


Department of Oral and Maxillofacial Pathology, KIMS Dental College and Hospital, Amalapuram, Andhra Pradesh, India

Date of Submission08-Dec-2018
Date of Decision09-Mar-2019
Date of Acceptance03-Sep-2019
Date of Web Publication22-Oct-2019

Correspondence Address:
Sankeerti Mala
Department of Oral and Maxillofacial Pathology, KIMS Dental College and Hospital, Amalapuram, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dmr.dmr_38_18

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  Abstract 


Context: Epidermal growth factor receptor (EGFR) and its ligands are involved in the cell growth of 70% cancer cells. EGFR overexpression or dysregulation may alter intracellular signaling pathways that affect tumor cell survival and apoptosis. It is hypothesized that only those potentially malignant lesions express high levels of EGFR progress to frank malignancies during tumorigenesis which suggests that increase in receptors might be the cause of malignancy rather than the effect of malignancy. Aims: The aim of the study was to assess and correlate the expression of EGFR in varying grades of oral epithelial dysplasia, oral squamous cell carcinoma (OSCC), and normal oral mucosa and to evaluate the early rate of malignant transformation in potentially malignant lesions. Settings and Design: It was a immunohistochemical study. Subjects and Methods: Twenty formalin-fixed, paraffin-embedded blocks of various grades of oral epithelial dysplasia, 10 formalin-fixed, paraffin-embedded blocks of OSCC, and 10 normal mucosae are stained with routine hematoxylin and eosin and immunostained with EGFR by avidin–biotin method. Statistical Analysis Used: Statistical analysis was performed by one-way ANOVA and multiple comparison tests using IBM SPSS Software 20.0 20. Results: Of 40 cases analyszd, 38 cases showed EGFR expression with varied extent and intensity of staining in the cytoplasm and cell membrane of keratinocytes in basal, parabasal, deep spinous, and superficial spinous layer. The results showed a statistically significant increase (P < 0.0001) in the staining extent and intensity of EGFR expression in severe dysplasia and OSCC. Conclusions: EGFRs may act as a marker since they reflect the early changes in dysplastic lesions – predicting the rate of malignant transformation in potentially malignant disorders and thereby aiding the oral pathologist in arresting the lesion before it enters malignancy.

Keywords: Dysplasia, epidermal growth factor receptor, oral squamous cell carcinoma


How to cite this article:
Mala S. Epidermal growth factor receptor as an early diagnostic marker of oral squamous cell carcinoma: Marking the line between severe dysplasia and oral squamous cell carcinoma. Dent Med Res 2019;7:60-7

How to cite this URL:
Mala S. Epidermal growth factor receptor as an early diagnostic marker of oral squamous cell carcinoma: Marking the line between severe dysplasia and oral squamous cell carcinoma. Dent Med Res [serial online] 2019 [cited 2019 Nov 12];7:60-7. Available from: http://www.dmrjournal.org/text.asp?2019/7/2/60/269671




  Introduction Top


Cancer is the second most common cause of death after heart diseases in developed countries and the third leading cause of mortality following heart and diarrheal diseases in developing countries. Cancer of the oral cavity accounts for approximately 3% of all malignancies. Many factors are known to have etiologic roles in oral squamous cell carcinoma (OSCC) such as tobacco smoke; alcohol; phenol; viral, bacterial, and fungal infections; electrogalvanic reaction; radiation; genetics; immunosuppression; expression of oncogens; deactivation of tumor suppressor genes; and malnutrition. It is noteworthy that many OSCCs develop from potentially malignant disorders (PMDs). Correct diagnosis and timely treatment of PMDs may help prevent malignant transformation in oral lesions.[1]

During the process of tumor initiation and progression, aberrant growth signaling plays an important role in the perturbation of growth restriction and cell cycle checkpoints. One such growth factor receptor is epidermal growth factor receptor (EGFR), which is a transmembrane protein receptor made up of a glycoprotein with an intrinsic tyrosine-specific protein kinase activity that regulates cell growth in cell lines and in variety of human tumors.[2]

The EGFR-induced signaling controls the gene transcription, cell cycle progression, cell proliferation and survival, adhesion, angiogenesis, migration, and invasion in tumor cells. EGFR has been reported to be expressed in a variety of human tumors of epithelial origin. Increased expression of EGFR is found in OSCC and potentially malignant lesions.[3]

It is hypothesized that only those potentially malignant lesions express high levels of EGFR progress to frank malignancies during tumorigenesis; hence, by evaluating the expression of EGFR in potentially malignant lesions and correlating the levels of EGFR in OSCC and normal epithelium, it would be possible to assess the potentiality of the potentially malignant lesions to become malignant, and hence, assess the role of EGFR as an early diagnostic marker.


  Subjects and Methods Top


The study sample consisted of a total of 40 cases comprising 10 OSCC cases, 10 cases of PMDs with mild dysplasia, 10 cases of PMDs with severe dysplasia, and 10 cases of normal oral epithelium from the retromolar area. Immunohistochemical (IHC) staining was performed with anti-EGFR by avidin–biotin method [Figure 1].
Figure 1: Intraoral view of white lesion extending from the corner of the mouth to the retromolar area on the left buccal mucosa

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Immunohistochemical staining procedure

Immunostaining was carried out on 4 μm formalin-fixed, paraffin-embedded sections. Slides were baked in a hot air oven at 65°C overnight. Sections were sequentially dewaxed through a series of xylene, graded alcohol, and water immersion steps. Antigen (Ag) retrieving was done using microwave by the slides immersed in 500 mL of citrate buffer solution (pH 6.0) for two cycles of 5 min and 15 min. Following the Ag retrieval, the endogenous peroxidase activity was blocked. The slides were incubated with the power block, followed by the application of primary antibody, and were incubated for 60 min in a humid chamber. After washing with Phosphate buffered saline (PBS), the secondary antibody was applied for 30 min and this reaction was visualized by the application of Diaminobenzidine (DAB) chromogen. Sections were counterstained with Mayer's hematoxylin for 30 s, dehydrated, and mounted.

Assessment of immunohistochemical results

In each tissue section, five representative fields were selected for the evaluation of the extent and intensity of EGFR expression in according to the modified Putti method. The evaluation of EGFR staining for both extent and intensity was done in the basal layer, suprabasal layer, deep spinous layer, and superficial spinous layer [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Figure 2: Microphotograph showing epidermal growth factor receptor extent 1, intensity 2, and expression 3 in the basal layer at ×40 in Group I subject

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Figure 3: Microphotograph showing epidermal growth factor receptor extent 3, intensity 3, and expression 6 in the basal layer ×40 in Group II subject

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Figure 4: Microphotograph showing epidermal growth factor receptor extent 3, intensity 3, and expression 6 in the deep spinous layer at ×40 in Group III (severe dysplasia) subject

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Figure 5: Microphotograph epidermal growth factor receptor extent 3, intensity 3, and expression 6 in all the layers at ×40 in showing Group IV (oral squamous cell carcinoma) subject

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Figure 6: Microphotograph showing membranous type of epidermal growth factor receptor staining pattern in the deep spinous layer

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Figure 7: Microphotograph showing both membranous type and cytoplasmic type of epidermal growth factor receptor

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Figure 8: Microphotograph showing epidermal growth factor receptor strong intensity in dysplastic islands in oral squamous cell carcinoma

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Extent of epidermal growth factor receptor

The extent of staining was graded and scored as 0 point – negative staining, 1 point – <10% cells, 2 points – 10%–50% cells, and 3 points – >50% cells.

Intensity of epidermal growth factor receptor

The intensity of staining was graded and scored as 1 point - weak staining, 2 points – moderate staining, and 3 points – strong staining.

Total score

The overall score for each specimen was obtained by adding the extent of immunoreactivity score to the intensity score.

Overall score of more than 1 or equal to 1 represents low expression of EGFR.

Overall score of more than 4 or equal to 4 represents high expression of EGFR.

The total score is calculated and analyzed using one-way anova and multiple comparison tests.


  Results Top


Of 40 cases analyzed, 38 cases showed EGFR expression with varied extent and intensity of staining in the cytoplasm and cell membrane of keratinocytes in basal, parabasal, deep spinous, and superficial spinous layer. The results showed a statistically significant increase (P < 0.0001) in the staining extent and intensity of EGFR expression in severe dysplasia and OSCC.

Expression of epidermal growth factor receptor in the basal layer

The mean difference between the intensities of EGFR staining in the basal layer in Group I and Group IV was highly significant, Group II and Group IV was statistically significant, while in Group III and Group IV was not statistically significant (P < 0.0001, P = 0.002, P = 0.211, respectively).

Expression of epidermal growth factor receptor in the parabasal layer

Multiple comparisons of EGFR extent between Group I and Group III and Group I and Group IV were statistically highly significant (P < 0.0001), Group II and Group IV were statistically significant (P = 0.001), while between Group III and Group IV was insignificant.

Expression of epidermal growth factor receptor in the spinous layer

Multiple comparisons of EGFR extent and intensity between Group I and Group III and Group I and Group IV were statistically highly significant (P < 0.0001), Group II and Group IV were statistically significant (P = 0.025), while Group III and Group IV were insignificant.

Expression of epidermal growth factor receptor in the superficial spinous layer

A highly statistically significant difference was obtained in the total EGFR expression between Group I and Group III, Group I and Group IV, and Group II and Group IV (P < 0.0001), while a significant difference was obtained between Group III and Group IV (P = 0.005) [Table 1], [Table 2], [Table 3], [Table 4], [Table 5] and [Graph 1], [Graph 2], [Graph 3], [Graph 4].
Table 1: Multiple comparison of EGFR extent, intensity and expression in basal layer

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Table 2: Multiple comparison of EGFR extent, intensity and expression in parabasal layer

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Table 3: Multiple comparison of EGFR extent, intensity and expression in deep spinous layer

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Table 4: Multiple comparison of EGFR extent, intensity and expression in superficial spinous layer

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Table 5: Distribution of EGFR staining pattern in Superficial spinous Layer

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[INLINE 1][INLINE 2][INLINE 3][INLINE 4]


  Discussion Top


EGFR and its ligands are involved in the cell growth and proliferation of 70% cancer cells. EGFR overexpression or dysregulation may alter intracellular signaling pathways that affect tumor cell survival and apoptosis. It is hypothesized that only those potentially malignant lesions express high levels of EGFR progress to frank malignancies during tumorigenesis which suggests that increased receptors may be cause rather than the effect of malignancy. Hence, by finding the expression of EGFR in these potentially malignant lesions, it would be possible to assess the malignant transformation of these lesions; hence, the patient is available for early treatment.

The participants in our study were categorized into four groups as dysplasia free, PMD with mild–moderate dysplasia, PMD with severe dysplasia, and OSCC. IHC reaction for the detection of EGFR was performed using anti-EGFR monoclonal antibody by following avidin–biotin method. The EGFR extent, intensity, and expression were evaluated in the basal, parabasal, deep spinous, and superficial spinous layers of all the groups following the modified Putti method.

Group I samples stained positive with EGFR mostly in the basal and parabasal layers. Few samples showed positive staining in the deep spinous layers. The intensity varied from weak to moderate, predominantly being of weak intensity.

Group II samples showed 90% positive immunoreaction to EGFR in the basal, parabasal, and deep spinous layers. The extent varied from <10% keratinocytes to 10%–20% keratinocytes. The intensity was predominantly moderate with Cell Membranous (CMB) staining pattern in the deep spinous layer.

Group III samples also showed 90% immunoreactions to EGFR in all the layers of the epithelium. The extent and intensity in the basal, parabasal, and deep spinous were at a range of moderate to high, whereas in the superficial spinous layer, minimal positive immunoreactions were seen in only two cases, showing high expression of EGFR.

Group IV samples showed all cases of OSCC-positive immunoreactions to EGFR in all the layers of the epithelium. The extent, intensity, and expression were high in the basal, parabasal, deep spinous, and superficial spinous layers.

When comparisons have been made, the extent and intensity and expression of EGFR significantly increased in the basal layer, parabasal layer, deep spinous layer, and superficial spinous layers from Group I to Group IV.

A highly statistically significant difference was obtained in the total EGFR expression:

  • In the basal layer, between Group I and Group IV
  • In the parabasal layer, between Group I and Group III and Group I and Group IV
  • In the deep spinous layer, between Group I and Group III and Group I and Group IV
  • In the superficial spinous layer, Group I and Group III, Group I and Group IV, and Group II and Group IV (P < 0.0001), while a significant difference between Group III and Group IV (P = 0.005).


These results were in correlation with the previous studies of Rajeswari and Saraswathi (2012), Ribeiro et al. (2012), Sarkis et al. (2010), Bernardes et al. (2010), Hiraishi et al. (2006), and Shin et al. (1994).[4],[5],[6],[7],[8]

For cell growth and division to occur, a large variety of metabolic processes are mediated through EGFR-activated kinases. During physiologic conditions requiring cell growth and proliferation such as wound healing, the EGFR is activated by the ligands, which further activates the MAP kinases which help in cell growth and differentiation. However, in pathologic conditions, there is an aberrant increase in a number of EGFRs, nonligand stimulation, subsequent internalization, and continuous nuclear transcription for proliferation, dedifferentiation, inhibition of apoptosis, invasiveness, and lack of adhesion dependence; all of which have a major role in tumor growth and progression.[3]

These results suggest that changes in EGFR expression during tumorigenesis occurred in a very heterogeneous manner and in two stages. In the first stage, EGFR was upregulated in normal epithelium adjacent to the tumor and remained elevated through the hyperplastic and dysplastic premalignant lesions. The second stage was reflected by a large increase in EGFR expression between dysplasia and squamous cell carcinoma.

Overexpression of EGFR is considered to be a hallmark of SCC (Karnata et al., 1986; Ozanne et al., 1986; Ozawa et al., 1988; and Bergler et al., 1989) and may occur independently of c-erb-B-1 activation (Field, 1992; Saranath et al., 1992). It is possible that tumors which express excessive amount of EGFR may have enhanced proliferation in response to autocrine or paracrine production of transforming growth factor (TGF)-α (Partridge et al., 1988; Todd et al., 1991). The production of TGF-α may also have a positive feedback such as effect and may accelerate the degradation of EGFR soon after binding and internalization. Ligand binding and subsequent internalization reduces the half-life of the receptor and this may, in turn, affect the amount of the receptor protein being recycled or the amount of the available (Carpenter, 1984).[9]

The EGFR staining of cell membrane and cytoplasm is attributed to the structure of EGFR which has a transmembrane domain and a cytoplasmic domain. The above observation suggests that the high intensity thickened membranous positivity to EGFR might be due to increased number of EGFRs per unit area and formation of abnormal receptors, which may be due to gene mutation or gene rearrangements. The viral oncogene product v-erb protein which is homologous with the transmembrane and cytoplasmic domain of EGFR can also attribute to the high intensity thickened membranous postivity to EGFR. This increased number of normal and abnormal EGFRs may be responsible for the progressive increase in the cell membranous staining intensity with severe dysplastic lesions and OSCC, in contrast to the staining in normal controls.[10]

Furthermore, the increased intensity in cytoplasmic staining might indicate the increased endocytic trafficking of EGFR, i.e., increased internalization of the receptor, and subsequently, the fate of activated EGFR for lysosomal degradation (attenuation of signaling) or recycling to the plasma membrane (prolonged signaling) and also transportation to both nucleus and mitochondria. Nuclear EGFR is reported to interact with transcription regulators and modulator of DNA repair. Mitochondrial EGFR is reported to phosphorylate the cytc C involved in the regulation of apoptosis.[11]

Therefore, increased cell membranous-staining intensity which indicates dysregulation of EGFRs might aid in the early detection of dysplastic changes.

EGFR also plays a role in cell migration by a phospholipase C-gamma (PLC-γ)-dependent pathway. EGFR induced activation of PLC-Gamma stimulates cell motility by releasing gelsolin and other actin modifying proteins from the membrane. Thereby restoring its ability to bind, sever and cap polymerize actin filaments for the formation of filopodia extension which help in cell migration.[3]

Hence, overexpression of EGFR in the basal, parabasal, and deep spinous layers might indicate the increased potential of cell migration in potentially malignant lesions with severe dysplasia, and therefore, it has a role as a marker for rate of malignant transformation.

In our study, the following additional features were found

  1. Three cases of histopathologically diagnosed potentially malignant lesions with mild–moderate dysplasia showed a high expression of EGFR. It may suggest that these lesions have inherent potential of early progression of dysplasia/transformation rate. However, it may also be either due to biopsy from improper area or interobserver bias
  2. All cases of OSCC showed a high expression of EGFR in all the layers of the epithelium, while one case of invasive carcinoma showed low expression of EGFR in the superficial spinous layer
  3. Strong intensity of EGFR was seen in the periphery of the epithelial dysplastic islands in OSCC
  4. Both cell membranous and cytoplasmic staining was seen in OSCC, while only cell membranous staining was seen in potentially malignant lesions with severe dysplasia.



  Conclusions Top


Therefore, our study supports the earlier reports in which the EGFR expression showed a progressive increase from normal oral epithelium to grades of dysplasia and a high expression in OSCCs. Thus, overexpression of EGFR can be considered as a hallmark of OSCC, and EFGR can be used as an early diagnostic marker to assess the malignant transformation in potentially malignant lesions.

Acknowledgment

I am highly thankful to my Guide and Mentors: Dr. Vanita Rathod, HOD Department of Oral Pathology, Rungta College of Dental science and Research, Dr. Siddharth Pundir, Reader, Department of Oral Pathology, Rungta College of Dental science and Research, and Dr. Sudhanshu Dixit, Reader, Department of Oral Pathology, Rungta College of Dental science and Research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Mortazavi H, Baharvand M, Mehdipour M. Oral potentially malignant disorders: An overview of more than 20 entities. J Dent Res Dent Clin Dent Prospects 2014;8:6-14.  Back to cited text no. 1
    
2.
Lin Y, Liu Z, Jiang J, Jiang Z, Ji Y, Sun B. Expression of intracellular domain of epidermal growth factor receptor and generation of its monoclonal antibody. Cell Mol Immunol 2004;1:137-41.  Back to cited text no. 2
    
3.
Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor recptor: Mechanisms of activation and signaling. Exp Cell Res 2003;284:31-53.  Back to cited text no. 3
    
4.
Ribeiro DC, Gieber-Netto FO, Sousa SF, Bernardes VD, Guimaraes-Abreu MH, Aguiar MC. et al. Immunohistochemical expression of EGFR in oral leukoplakia: Association with clinicopathological features and cellular proliferation. Med Oral Patol Oral Cir Bucal 2012;17:739-44.  Back to cited text no. 4
    
5.
Sarkis A, Abdullah BH, Abdul Majeed BA, Talabani NG. Immunohistochemical expression of epidermal growth factor receptor in oral squamous cell carcinoma in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis. Head Neck Oncol 2010;2:13.  Back to cited text no. 5
    
6.
Bernardes VF, Gleber-Netto FO, Sousa SF, Silva TA, Aguiar MC. Clinical significance of EGFR, Her-2 and EGF in oral squamous cell carcinoma: A case control study. J Exp Clin Cancer Res 2010;29:40.  Back to cited text no. 6
    
7.
Hiraishi Y, Wada T, Nakatani K, Negoro K, Fujita S. Immunohistochemical expression of EGFR and p-EGFR in oral squamous cell carcinomas. Pathol Oncol Res 2006;12:87-91.  Back to cited text no. 7
    
8.
Shin DM, Ro JY, Hong WK, Hittelman WN. Dysregulation of epidermal growth factor receptor expression in premalignant lesions during head and neck tumorigenesis. Cancer Res 1994;54:3154-9.  Back to cited text no. 8
    
9.
Nithyanand AK. Expression of Epidermal Growth Factor Receptor and P53 Oncoprotein In Premalignant And Malignant Lesions. Simon Fraser University; 1994.  Back to cited text no. 9
    
10.
Rajeswari MR, Saraswathi TR. Expression of epithelial growth factor receptor in oral epithelial dysplastic lesions. J Oral Maxillofac Pathol 2012;16:183-8.  Back to cited text no. 10
  [Full text]  
11.
Tomas A, Futter CE, Eden ER. EGF receptor trafficking: Consequences for signaling and cancer. Trends Cell Biol2014;24:26-34.  Back to cited text no. 11
    


    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]



 

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