Journal of Cancer Research and Therapeutics

: 2012  |  Volume : 8  |  Issue : 4  |  Page : 586--590

Immunohistochemical correlation of epidermal growth factor receptor and c-erbB-2 with histopathologic grading of mucoepidermoid carcinoma

Monir Moradzadeh Khiavi1, Sepideh Vosoughhosseini1, Shirin Saravani2, Monireh Halimi3,  
1 Department of Oral Pathology, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Oral Pathology, Faculty of Dentistry, Zahedan University of Medical Sciences, Zahedan, Iran
3 Department of Pathology, Imam Reza Hospital, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence Address:
Monir Moradzadeh Khiavi
Department of Oral Pathology, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz


Objective: Mucoepidermoid carcinoma is the most common salivary gland malignancy with highly variable biologic potential that correlates with the histopathologic grade of the tumor. Therefore, identification of the histopathologic grade of the mucoepidermoid carcinoma is very important in the treatment and determination of the final prognosis. The present study was performed to survey immunohistochemically Epidermal Growth Factor ReceptorEGFR and c-erbB-2 expression in different grades of mucoepidermoid carcinoma. Materials and Methods: This retrospective study included 46 formalin-fixed, paraffin-embedded blocks of mucoepidermoid carcinoma. Based on histopathologic parameters, samples were classified into three grades. Then new sections were made and stained by immunohistochemistry (IHC) method for EGFR and c-erbB-2. Finally, EGFR and c-erbB-2 expression and their correlation with histopathologic grading were statistically analyzed by ANOVA. Nineteen samples of normal salivary gland tissue were also chosen as control group. Results: The means of EGFR and c-erbB-2 were 71%, 71%, respectively. Statistically significant correlation was found between EGFR expression and histopathologic grading of mucoepidermoid carcinoma of salivary glands (P < 0.001). There was no statistically significant correlation between histopathologic grading of salivary gland mucoepidermoid carcinoma and c-erbB-2 expression (P = 0.60). Conclusion: There is a parallelism between an increase in EGFR expression and increase in the histopathologic grading of salivary gland mucoepidermoid carcinoma. Therefore, the biologic behavior of salivary gland mucoepidermoid carcinoma can be determined by EGFR expression and it is a useful technique for determination of tumor grades and probably their prognosis.

How to cite this article:
Khiavi MM, Vosoughhosseini S, Saravani S, Halimi M. Immunohistochemical correlation of epidermal growth factor receptor and c-erbB-2 with histopathologic grading of mucoepidermoid carcinoma.J Can Res Ther 2012;8:586-590

How to cite this URL:
Khiavi MM, Vosoughhosseini S, Saravani S, Halimi M. Immunohistochemical correlation of epidermal growth factor receptor and c-erbB-2 with histopathologic grading of mucoepidermoid carcinoma. J Can Res Ther [serial online] 2012 [cited 2020 Oct 31 ];8:586-590
Available from:

Full Text


Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy with variable biologic potential that is correlated with the histological grade of the tumor. [1],[2],[3] This tumor sometimes acts like a malignant tumor and sometimes behaves as a benign tumor with favorable prognosis. All treatment plans are almost exclusively based on histopathologic grading. [1] Several microscopic grading systems based on a numerical score and significant microscopic parameters have been advocated to determine the grade of mucoepidermoid carcinoma. [4],[5] This tumor may be graded as low grade, intermediate grade and high grade. According to Auclair classification, the grade of this lesion is determined as a sum of five parameters. Sums that are 0-4 are classified as low grade, those that are 5-6 are considered as intermediate grade and sums 7-14 are classified as high-grade tumor. The five parameters are: intracystic component < 20% (2), neural invasion (2), necrosis (3), four or more mitoses per 10 in high-power fields (3), anaplasia present (4). [4],[6] However, sometimes, determination of tumor grades is difficult based on those parameters. Recently, several studies have been designed to detect other reliable diagnostic factors that could potentially influence treatment plans. One of them involves tumor markers produced by tumor cells, which help intheir identification. [1]

Epithelial growth factor receptor (EGFR) is a member of ErbB receptor family, also known as the Type I receptor tyrosine kinases (RTKs), which is a 170-kDa transmembrane glycoprotein. [7]

EGFR over-activation can convert a normal cell to a malignant one by providing suitable signals for anti-apoptosis, cell proliferation, angiogenesis and metastasis, which are the basic properties of malignancy. [8] It is overexpressed in many tumors; examples include head and neck cancers, glioblastoma, lung, breast, prostate, ovaries, bladder and colorectal carcinomas. [7] Among the several techniques that define aberrant EGFR protein expression in clinical use, immunohistochemistry (IHC) is probably the most appropriate method. [9]

The c-erbB-2 proto-oncogene, also known as Her-2/neu, has been located on Chromosome 17 and encodes a member of the tyrosine kinase family with growth factor receptor function, which is a 185-kDa transmembrane glycoprotein. [10] Overexpression or amplification of c-erbB-2 has been documented in different human tumors, mostly adenocarcinoma of the stomach, ovaries, breast, kidney and salivary glands, and has shown association with a poor prognosis in some tumors. [10],[11]

Although some studies have examined the frequency of EGFR overexpression in salivary gland tumors, [12],[13],[14],[15] few studies have been carried out about the correlation of this protein with grading of mucoepidermoid carcinoma. [1],[16] On the other hand, controversial results have been reported about c-erbB-2 in mucoepidermoid carcinoma, with reported rates ranging from absent to 38%. [17],[18],[19],[19] Therefore, the aim of this study is to evaluate the immunohistochemical expression of c-erbB-2 and EGFR in MEC and their correlation with histopathologic grades.

 Materials and Methods

Forty-six paraffin-embedded blocks of MEC were selected. Clinical information was obtained from the hospital records, which included patients' gender, age and location of the tumor.

The samples were microscopically reviewed by two pathologists in sections stained with hematoxylin and eosin (H&E) and the tumors were classified as low-, intermediate-, and high-grade malignancies according to Auclair et al. [4]

Subsequently, tissue slices (5 μm in thickness) were prepared from formalin-fixed and paraffin-embedded specimens and deparaffinized at 37°C for 24 h and rehydrated. Then they were put inside the microwave oven in boiling citrate buffer (PH = 6) for 5 min; subsequently, they were floated in phosphate-buffered saline (PBS) (PH = 6).

Then immunostaining for the markers was performed according to the manufacturer's instructions: blocking the endogenous peroxides activity by 11% hydrogen peroxides for 5 min; immersion in PBS; use of the monoclonal antibody of EGFR (Dako Denmark A/S; Lot no: 00049086) and c-erbB-2 (Dako Denmark A/S; Lot no: 00048863A) separately for 20-30 min; immersion in PBS; and use of the envision solution (Dako, Denmark) for 15 min. Finally, the samples were blindly reviewed without any knowledge of the microscopic grading of tumors. The quantitative evaluation was done by determination of the percent of positive stained cells under 40×objective lens with light microscope. The tumors also were classified as negative (< 5% of positive cells), weak positive (5-50% of positive cells) and strong positive (> 50% of positive cells). [2]

As a control group, 19 samples of normal salivary gland tissue were selected and immunohistochemically studied.

Correlation between EGFR and c-erbB-2 expression with histopathologic grading was statistically analyzed by ANOVA and a post hoc test using SPSS 15 software with a P < 0.05 significance level.


The clinicopathological characteristics of the patients at the time of diagnosis are summarized in [Table 1]. Of the 46 cases, 30 (65.2%) were male and 16 (34.8%) female. The age of patients ranged from 7 to 90 years. The mean age was 41.6 years, with a standard deviation of 22.54. The mean age of the men was 43.17, while for the women it was 38.56. The two most common sites were parotid (54.3%) and palate (21.7%). Histologically 19 cases (41%) were high grade, 13 intermediate grade (28%) and 14 (31%) low grade. {Table 1}

EGFR Expression

As shown in [Table 2] and [Figure 1], on an average, 71% of cells stained with EGFR with minimum and maximum rates of 3% and 100%, respectively. There were statistically significant differences between group means as determined by one-way ANOVA (F (2,43) = 157.96, P < 0.001). Furthermore, a significant difference was seen in comparison of each group with two other groups by Post Hoc test (P < 0.001). Immunostaining expression of tumor cells with EGFR based on different histopathologic grades has been shown in [Table 3]. Example of cytoplasmic immunostaining pattern for EGFR in high-grade MEC is shown in [Figure 2].{Figure 1}{Figure 2}{Table 2}{Table 3}

c-erbB-2 Expression

It is evident from [Table 4] that minimum and maximum rates of cells staining with c-erbB-2 were 100% and 0%, respectively. The mean of c-erbB-2 stained cells was 70.5%. One-way ANOVA did not demonstrate a significant correlation between salivary gland MEC histopathologic grades and c-erbB-2 expression (F (2, 43) = 0.51, P = 0.60). Immunostaining expression of tumor cells with c-erbB-2 based on different histopathologic grades has been shown in [Table 5]. [Figure 3] and [Figure 4] show the immunostaining pattern for c-erbB-2 in MEC.{Figure 3}{Figure 4}{Table 4}{Table 5}

Finally, in the normal salivary glands, EGFR and c-erbB-2 were occasionally detected in ductal cells, but not in acinar cells [Figure 5].{Figure 5}


Various studies have investigated the correlation between different grades of mucoepidermoid carcinoma and immunohistochemical markers. Some examples of these immunohistochemical markers have been p53, Ki-67, c-erbB-2 and PCNA, with positive correlation between them and histopathologic grading. Their expression has increased according to histopathologic grade. [1],[2],[3],[15],[20,21],[22],[23],[24],[25],[26],[27] However, some of these markers (Muc4, bcl-2, p27) correlate with histopathologic grade inversely. [25],[27],[28] Furthermore, studies have reported contradictory results regarding correlation between expression of some immunohistochemical markers such c-erbB-2 with histopathologic grades. [11],[12],[13],[17],[18],[25],[29] On the other hand a few studies have been done about correlation of EGFR with grading of mucoepidermoid carcinoma. [1],[16] Therefore, the aim of the present study was to evaluate the immunohistochemical expression of c-erbB-2, EGFR in MEC and their correlation with histopathologic grades.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor of the ErbB family, which is abnormally activated in many epithelial tumors. [7] Various mechanisms due to the aberrant activation of the receptor are observed in malignancies. These mechanisms include mutation, receptor overexpression, ligand-independent activation and ligand-dependent receptor dimerization. [30] A high frequency of EGFR overexpression has been reported in cancers of the head and neck region. [12] Generally, it is accepted that the overexpression of EGFR is common in salivary gland carcinomas, although in an earlier study with a small sample size, the incidence of positive EGFR staining was found to be 25% in mucoepidermoid carcinoma. [13] In the present study the mean of EGFR expression was 71%. Our findings confirm the results of studies carried out by Hoyek-gebeily et al., (75%), Shang et al., (67.4%) and Gibbons et al., (77%) regarding high EGFR overexpression in MEC. [1],[12],[14] Hoyeck-Gebily found a positive correlation between histopathologic grading of MEC and EGFR expression, which is consistent with our results with a similar correlation (P = 0.001). [1]

A review of literature shows an obvious controversy regarding the c-erbB-2 expression in MEC. Shrestha et al., Shang et al., and Sugano et al., in their studies on MEC found c-erbB-2 expression in only 4.5%, 4.3% and 0% of the cases, respectively. [12],[18],[31] However, Weed et al., Cho et al., and Press et al., reported c-erbB-2 overexpression in 25%, 36% and 38% of the cases studied. [11],[17],[28] Furthermore, Ito et al. in their study on salivary gland tumors found that high-grade MEC showed a higher percentage of ErbB-2. [32] The expression of c-erbB-2 in our samples was 70.5% in general, which is almost in accordance with the results reported by Suzuki (77%) and Lopes (77.7%). [2],[19] Although Lopes recognized the correlation of c-erbB-2 expression with low-grade mucoepidermoid carcinoma [2] that is completely consistent with our study, Suzuki's study showed association of higher histopathologic grading with increased expression of c-erbB-2. [19] Cho and Nguyen showed high c-erbB-2 expression in high-grade tumors compared with low-grade tumors. [11],[24] These findings contradict the results of our study. There may be many reasons for the observed differences, including methodology of the immunohistochemical procedures, patient selection, scoring and the definition of c-erbB-2 expression.

Therefore, our findings may suggest lack of association among immunoexpression of c-erbB-2 and histopathologic grade of mucoepidermoid carcinoma. However, the association between EGFR and histopathologic grades is obvious. As a result, immunoexpression of EGFR is a useful technique for determination of tumor grading and probably its prognosis.


This research has been supported by Research Council of Tabriz university of medical sciences under the Grant 88426. The authors would like to thank the Council for assistance to carrying out this study.


1Hoyek-Gebeily J, Nehme E, Aftimos G, Sader-Ghorra C, Sargi Z, Haddad A. Prognostic significance of EGFR, p53 and E-cadherin in mucoepidermoid cancer of the salivary glands: A retrospective case series. J Med Liban 2007;55:83-8.
2Lopes MA, da Cruz Perez DE, de Abreu Alves F, de Almeida OP, Kowalski LP. Clinicopathologic and immunohistochemical study of intraoral mucoepidermoid carcinoma. Otolaryngol Head Neck Surg 2006;134:622-6.
3Triantafillidou K, Dimitrakopoulos J, Iordanidis F, Koufogiannis D. Mucoepidermoid carcinoma of minor salivary glands: A clinical study of 16 cases and review of the literature. Oral Dis 2006;12:364-70.
4Auclair PL, Goode RK, Ellis GL. Mucoepidermoid carcinoma of intraoral salivary glands: Evaluation and application of grading criteria in 143 cases. Cancer 1992;69:2021-30.
5Brandwein MS, Ivanov K, Wallace DI, Hilee JJ, Wang B, Fahmy A, et al. Mucoepidermoid carcinoma: A clinicopathologic study of 80 patients with special reference to histological grading. Am J Surg Pathol 2001;25:835-45.
6Carlson ER, Ord RA. Text book and color atlas of salivary gland pathology, diagnosis and management. 1st ed. USA: Wiley-Blackwell; 2008. p. 153.
7Oliveira S, Van Bergen en Henegouwen PM, Storm G, Schiffelers RM. Molecular biology of epidermal growth factor receptor inhibition for cancer therapy. Expert Opin Biol Ther 2006;6:605-17.
8Sebastian S, Settleman J, Reshkin SJ, Azzariti A, Bellizzi A, Paradiso A. The complexity of targeting EGFR signaling in cancer: From expression to turnover. Biochim Biophys Acta 2006;1766:120-39.
9Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37:S9-15.
10Bernardes VF, Ramos-Jorge ML, Carmo MA, Cardoso SV, Mesquita RA, Aguiar MC. Intraoral Mucoepidermoid Carcinoma of Salivary Glands: Lack of association among clinicopathological features and immunoexpression of c-erbB-2 in 29 cases. Int J Morphol 2008;26:1005-11.
11Cho KJ, Kim JK, Lee SS, Oh KK. Mucoepidermoid carcinoma of the salivary gland- a clinico-pathologic and immunohistochemical study for c-erbB-2 oncoprotein. J Korean Med Sci 1997;12:499-504.
12Shang J, Shui Y, Sheng L, Wang K, Hu Q, Wei Q. Epidermal growth factor receptor and human epidermal growth receptor 2 expression in parotid mucoepidermoid carcinoma: Possible implications for targeted therapy. Oncol Rep 2008;19:435-40.
13Yamada K, Iwai K, Okada Y, Mori M. Immunohistochemical expression of epidermal growth factor receptor in salivary gland tumours. Virchows Arch A Pathol Anat Histopathol 1989;415:523-31.
14Gibbons MD, Manne U, Carroll WR, Peters GE, Weiss HL, Grizzle WE. Molecular differences in mucoepidermoid carcinoma and adenoid cystic carcinoma of the major salivary glands. Laryngoscope 2001;111:1373-8.
15Monteiro LS, Bento MJ, Palmeira C, Lopes S. Epidermal growth factor receptor immunoexpression evaluation in malignant salivary gland tumours. J Oral Pathol Med 2009;38:508-13.
16Lujan B, Hakim S, Moyano S, Nadal A, Caballero M, Diaz A, et al. Activation of the EGFR/ERK pathway in high-grade mucoepidermoid carcinomas of the salivary glands. Br J Cancer 2010;103:510-6.
17Press MF, Pike MC, Hung G, Zhou JU, Ma Y, George J, et al. Amplification and Overexpression of HER-2/neu in Carcinomas of the Salivary Gland: Correlation with Poor Prognosis. Cancer Res 1994;54:5675-82.
18Sugano S, Mukai K, Tsuda H, Hirohashi S, Furuya S, Shimosato Y, et al. Immunohistochemical study of c-erbB-2 oncoprotein overexpression in human major salivary gland carcinoma: An indicator of aggressiveness. Laryngoscope 1992;102:923-7.
19Suzuki M, Ichimiya I, Matsushita F, Mogi G. Histological features and prognosis of patients with mucoepidermoid carcinoma of the parotid gland. J Laryngol Otol 1998;112:944-7.
20Cardoso WP, Denardin OV, Rapoport A, Araújo VC, Carvalho MB. Proliferating cell nuclear antigen expression in mucoepidermoid carcinoma of salivary glands. Sao Paulo Med J 2000;118:69-74.
21Giannoni C, El-Naggar AK. Ordoñez NG, Tu ZN, Austin J, Luna MA, Batsakis JG. C-erbB-2/neu oncogene and Ki-67 analysis in the assessment of palatal salivary gland neoplasms. Otolaryngol Head Neck Surg 1995;112:391-8.
22Hicks J, Flaitz C. Mucoepidermoid carcinoma of salivary glands in children and adolescents: Assessment of proliferation markers. Oral Oncol 2000;36:454-60.
23Hoyek-Gebeily J, Nehmé E, Aftimos G, Sader-Ghorra C, Sargi Z, Haddad A. Mucoepidermoid carcinoma of salivary glands: The prognostic value of tumoral markers. Rev Stomatol Chir Maxillofac 2007;108:482-8.
24Nguyen LH, Black MJ, Hier M, Chauvin P, Rochon L. HER2/neu and Ki-67 as prognostic indicators in mucoepidermoid carcinoma of salivary glands. J Otolaryngol 2003;32:328-31.
25Okabe M, Inagaki H, Murase T, Inoue M, Nagai N, Eimoto T. Prognostic significance of p27 and ki-67 expression in mucoepidermoid carcinoma of the intraoral minor salivary gland. Mod Pathol 2001;14:1008-14.
26Salehinezhad J, Ghafarzadegan K, Saghravanian N, Mohtasham N. Evaluation of Ki-67 experssion and AgNOR counts in mucoepidermoid carcinoma of salivary gland. J Mashhad Dent Sch 2004;28:53-60.
27Yin HF, Okada N, Takagi M. Apoptosis and apoptotic-related factors in mucoepidermoid carcinoma of the oral minor salivary glands. Pathol Int 2000;50:603-9.
28Weed DT, Gomez-Fernandez C, Pacheco J, Ruiz J, Hamilton-Nelson K, Arnold DJ. MUC4 and ERBB2 expression in major and minor salivary gland mucoepidermoid carcinoma. Head Neck 2004;26:353-64.
29Kernohan NM, Blessing K, King G, Corbett IP, Miller ID. Expression of c-erbB-2 oncoprotein in salivary gland tumours: An immunohistochemical study. J Pathol 1991;163:77-80.
30Mendelsohn J, Baselga J. Epidermal growth factor receptor targeting in cancer. Semin Oncol 2006;33:369-85.
31Shrestha P, Huang JW, Tsujr T, Shinozaki F, Maeda K, Sasaki K, et al. Rare expression of the c-erbB-2 oncoprotein in salivary gland tumors: An immunohistochemical study. J Oral Pathol Med 1992;21:477-80.
32Ito FA, Ito K, Coletta RD, Graner E, de Almeida OP, Lopes MA. Salivary gland tumors: immunohistochemical study of EGF, EGFR, ErbB-2, FAS and Ki-67. Anal Quant Cytol Histol 2009;31:2.