Esophageal Cancer and the Importance of Epidermal Growth Factor (EGFR)

Introduction
Esophageal cancer is one of the most common worldwide malignancies with the annual incidence of almost 4.4 per 100000 individuals each year. In 2000, the mortality rate was estimated to be more than 300000 patients (1). Esophageal squamous cell carcinoma (ESCC) has high prevalence in Asian countries which accounts for over 95% of the esophageal cancers. The poor prognosis of this disease can be due to the detection of patients not in the early-stages of the cancer, and also the high possibility of lymph nodes involvement even in the localized disease (2).
Esophageal carcinoma is among the 10 most common cancers in the world and it is one of the exceptional gastrointestinal malignancies which encompass two specific histopathologic types including Adenocarcinoma and Squamous cell carcinoma (3).
In developing countries such as China and Iran, ESCC is considered to be the most prevalent esophageal cancer (almost 90% of esophageal cancers), but recent studies in Iran have shown that ESCC incidence decreased in the past 20 years while the adenocarcinoma occurrence increased (4). Various issues such as life style, social, economic, and environmental factors influence on the type of esophageal cancer in every individual or its dominance in a specific geographic area.

Epidemiology
According to the studies back to 35 years ago in the city of Rasht-south western of Caspian Sea- and the surrounding area, the ESCC age-standardized incidence rates (ASRs) were 20 and 10 per 100000 men and women respectively. In contrast, in southeast of the Caspian Sea, around the city of Gonbad toward the East, it had an equal prevalence between men and women which had the highest ASRs in comparison with other parts of the world (more than 100 per 100000 cases). The unusual age-standardized incidence rate and the greatly higher incidence raise the possibility of the presence of a noticeable risk factor for this disease. Addition to the north parts of Iran, esophageal cancer has been reported in East and Central Asia such as Turkmenistan, Uzbekistan, and Kazakhstan. Recent studies of the province of Golestan showed a male/female ratio of about one and the higher rate of squamous cancer. This substantial decline can be probably due to the improvements of socio-economic conditions in the city of Gonbad and the other area of Golestan province. This decline is in concordance to the esophageal statistics in developed countries which occurs in ESCC, a main subtype of esophageal cancer in Iran (5).
According to studies conducted in Iran, the risk of ESCC in first degree relatives of patients is twice the risk of disease in individuals without family history of the disease. Hot tea and other beverages, opium, polycyclic aromatic hydrocarbons, nitrate concentrations in drinking water, Human papilloma virus type 16, inadequate nutrition, are the possible risk factors for the high prevalence of the esophageal cancer in Iran (5,6).
A recent study in Golestan province showed that mutation of BRCA2 will significantly increase the risk of ESCC. Almost 50% of the esophageal tumors have somatic mutations in TP53 mainly in men. Transition of C>T in CPG and mutation of  
T: A pair are the most common mutations in both sexes. High frequency of C>T mutations in CPG part indicate the possibility of inflammatory process involved in ESCC in Iran. High incidence of T:A mutations in Iran may propose the drug usage effects and other masculine behavior. Gene polymorphism related to ESSC is also evaluated and it considered the enzymatic polymorphism as a possible change in the incidence of cancer. The prevalence of polymorphism of 10 theoretically effectual genes in esophageal cancer has been compared among the following three Iranian ethnic groups, high risk Turkamans form Golestan province, medium risk Azeri’s from Ardabil, and low risk resident Zoroastrians of Tehran. Compared to Zoroastrians, Turkamans has contained high frequency of 4 alleles including ADH2*1, CYP2A6, CYPLAM2, and CYPLAM1, which was considered to have more carcinogen effects. Results were compatible with the effects of the mentioned alleles on population with high possibility of ESCC (5,7).
Epidermal growth factor receptor (EGFR) is one of the tyrosine kinases receptors with high expression in esophageal cancer cells. EGFR as a transmembrane protein contains an extracellular receptor, a transmembrane component, and an intercellular part. The activation of the EGFR components leads to proliferation, cell differentiation, angiogenesis, metastasis, and anti-apoptotic effects (8).
The high expression of EGFR has been detected in various malignancies such as head and neck, colorectal, breast, lung, and bladder tumors and almost in 40-70% of ESCC. Some studies reported a negative correlation between high expression of EGFR in tumor cells and the patients’ survival rate, chemotherapy resistance, and lymphatic metastasis. Conversely, high expression of EGFR is suggested to be associated with high chemotherapy response in other studies (9).

Epidermal growth factor receptor
Tumors are almost the consequences of the alternation in expression of responsible proteins for regulation, proliferation, and vital activities of cells such as growth factors and their receptors which are important for normal tissues growth and also development and progression of neoplasms (10). Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) which is indentified in more than 50% of malignancies are transmembrane proteins with tyrosine kinase activity and participate in various functions including cell growth, proliferation, and differentiation (11).
According to previous studies, mutations of EGFR gene activates the cellular receptor and leads to the cell survival via inhibiting the apoptosis  pathways and affects the response rate to its EGFR  antagonists such as Gefitinib in non-small cell lung cancer, so an association has been detected between EGFR polymorphism and advanced stages of tumors (12-15).
EGFR is an oncogene contains 28 exons and spans approximately 188 kb. EGFR oncogenic function leads to the alternation of the cellular processes such as increase in cell proliferation, angiogenesis, invasion and metastasis (16). It is involved in pathphysiology of several malignancies with epithelial origin such as esophagus, lung, colon, and breast cancers (17).
It is also important in the prognosis and progression assessments of the clinical stages of the disease.  It has been reported that different forms of EGFR alleles are Influential in lung cancer however these mutations are rare in gastric cancer (18,19).
EGFR polymorphism rate is dependent on the patient’s race and is different in every population, for instance the amount of polymorphism is 2% in united state and 26% in Japan. This polymorphism is important in cancer progression, metastasis and response to receptor antagonist drugs and is known as a diagnostic and therapeutic marker and a landmark in increasing the patient’s lifespan (20).
In EGFR-family, EGFR (HER1) and HER2 are the founding members which are mostly involved in human malignancies. The over expression of mRNA and protein along with gene amplification are the major changes in EGFR which are observed in most of the head and neck cancers (70-90%) (21).
The entire gene sequencing has specific priority in genetic disorder detection of cancers and it is the only suggested method. For instance, exon content of EGFR gene should be analyzed while assessing the relation of EGFR gene mutations with ESCC.  Mutations in EGFR were firstly reported in lung cancers that had better response to tyrosine kinase inhibitors. These mutations mainly occurred in 18-21 exons of the EGFR gene of none smoker women with adenocarcinoma and also were detected in 25% of breast cancers and 15-25% of gastric tumors. In 1986, these changes were reported in SCC cell line (22).
According to the previous studies considered the changes of EGF and EGFR mRNA in ESCC patients, the over expression of EGFR and PYGO2 mRNAs were observed in ESCC correlated with the tumor progression. The over expression of EGFR and PYGO2 as two biomarkers of invasive tumors that have oncogenic function was significantly dependent on each other (23).
Fluorescence in situ hybridization (FISH)-positive was significantly correlated with the over expression of EGFR and the depth of invasion and lymph node metastasis. Based on the prior investigations, the over expression and the increased gene copy number of EGFR were common in ESCC and EGFR has been proposed as an appropriate target for the ESCC therapeutic approaches (24).  Amplification of C-erbB-2 from EGFR-family introduced this gene as a prognosis biomarker in ESCC (25).
Besides the recent improvements in radiotherapeutic techniques there is still low treatment response and survival rate. Therefore, there is a strong need to invent new methods for treating the ESCC patients (26). Because of the expression of EGFR as a therapeutic target in normal cells, its overexpression in tumor cells may be considered to distinguish between normal and malignant cells. Evidences on the EGFR role in different types of cancers have been resulted in designing agents that selectively target this receptor such as anti-EGFR monoclonal antibodies that inhibit the proliferation of overexpressed EGFR cells in both in vivo and in vitro conditions (27).
Other gene mutations activated by this receptor play a role in carcinogenic procedures and also no proper response to the EGFR inhibitors. For instance, in colorectal cancers with KRAS and BRAF gene mutations, patients do not response to Panitumum ab monotherapy as an anti-EGFR monoclonal antibody recently approved by FDA (28).
The large size and the long serum half life full-length antibodies are the barriers of tumor penetration and distribution of antibodies and are the limitating factor in their usage in the therapeutic and prognostic purposes. This clarifies the importance of single chain monoclonal antibodies in diagnostic and therapeutic methods as the most common types of recombinant antibodies easily produced in different expressional systems. These antibodies possess antigen binding sites that include variable domains of heavy and light chains (VH and VL domains) in which the two domains are connected by a flexible linker segment. Pentadeca linker is one of the most common linkers which are the polypeptides with 10 to 25 hydrophilic amino acids in length. These antibodies are mainly derived from genes separated from mouse hybrid cells and their antigen attachment ability is similar to the monoclonal antibody which they are derived from. The first monoclonal antibody was produced by Huston and colleagues based on the recombinant DNA and protein engineering, derived from monoclonal anti-digoxin antibody expressed in E. coli bacteria. These antibodies are the basis of investigations on therapeutic approaches of several malignancies such as esophageal and breast cancers. Trastuzuamb and Gefitinib are anti-EGFR antibodies mainly used in the studies on the esophageal cancer. According to previous studies the combination of anti-EGFR drugs and radiation can be effective in prognosis of this disease (29).
Several studies have suggested the requirement of future investigations on drugs which act through EGFR for the treatment of esophageal cancer cells. Kono in 2006 resulted that these drugs had anti-proliferative and cell cytotoxicity antibody-dependent effects against cancer cells (30,31).
Partial response to Gefitinib has been observed in patients with EGFR overexpression and SCC pathology which showed the over activity of the mentioned drug in treatment of ESCC cell line (1). Gotoh mentioned that although this biomarker may be effective in predicting the SCC response to chemoradiotherapy, future studies are needed for the definite conclusion (32).
Gene therapy is in progress as a potential therapeutic method in controlling the genetic process of cancer cells and treatment of metabolic diseases. There are wide varieties of approaches in the field of gene therapy which are varied from a substitution of single mutated or deleted tumor suppressor gene to the stimulation of the immune system against cancer cells (33).

Conclusion
Compared Genetic examinations on EGFR gene have contributed to the development of gene therapy methods. Several studies have been conducted on the EGFR mutations in different cancers with limited cell line. Future researches on EGFR and its prevalence in esophageal cancer can be effective in improving the anti-cancer  
therapeutic methods such as monoclonal antibodies, gene therapy, and also the prognosis of the disease.

Acknowledgement
We would like to thank Clinical Research Development Center of Ghaem Hospital for their assistant in this manuscript. This study was supported by a grant from the Vice Chancellor for Research of the Mashhad University of Medical Sciences for the research project as a medical student thesis with approval number of 900895.

Conflict of Interest
 The authors declare no conflict of interest.