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GASTROINTESTINAL CANCER SYMPOSIUM - ORIGINAL ARTICLE
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Year : 2014  |  Volume : 51  |  Issue : 3  |  Page : 358--362

Epidermal growth factor receptor gene expression evaluation in colorectal cancer patients

G Motalleb1, E Pourrahmat1, A Rashki2, A Yegane Moghadam3, M Mazaheri4, M Jahantigh5, K Sabagh6, N Sanadgol1, S Najafi1, R Talaee7,  
1 Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran
2 Department of Physiopathology, Faculty of Veterinary Medicine, University of Zabol, Iran
3 Department of Otolaryngology, Kashan University of Medical Sciences, Kashan, Iran
4 Department of Sciences, Faculty of Medicine, Zabol University of Medical Sciences, Zabol, Iran
5 Department of Pathology, Zahedan University of Medical Sciences and Health Services, Zahedan, Iran
6 Department of Plant Pathology, Faculty of Agriculture, University of Zabol, Iran
7 Department of Dermatology, Kashan University of Medical Sciences, Kashan, Iran

Correspondence Address:
A Yegane Moghadam
Department of Otolaryngology, Kashan University of Medical Sciences, Kashan
Iran
G Motalleb
Department of Biology, Faculty of Sciences, University of Zabol, Zabol
Iran

Abstract

Background: Colorectal cancer is one of the most common causes of death in the world and third and fourth most common cancer among men and women in Iran respectively. Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that shows over expression in epithelial tumors and regulates important processes in tumorigenesis. Incidence and characteristics of colorectal cancer are based on the geographic region and race. Aim: In this research work, the over expression of EGFR in formalin fixed paraffin-embedded (FFPE) colorectal cancer tumor tissue of patients was studied. Materials and Methods: Fifteen FFPE colorectal cancer tumor tissues (10 women and 5 men; 25-65 years old and stage IV) and 15 non-patients (nine women and six men; 25-65 years old) that were collected during 2006-2012. EGFR gene expression level was analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (PCR). All PCR reactions were performed in triplicate for both target gene and internal control (18s ribosomal ribonucleic acid) with the 2−ΔΔCT method. Gene expression differences in patients and controls were evaluated with t-test. Results: The results were showed EGFR gene over expression in 12 (80%) of 15 patients. There was a statistically significant difference in the prevalence of EGFR expression between patients and control (P < 0.05). Conclusion: Our results demonstrated EGFR gene over expression in colorectal cancer tumor tissue compared with controls.



How to cite this article:
Motalleb G, Pourrahmat E, Rashki A, Moghadam A Y, Mazaheri M, Jahantigh M, Sabagh K, Sanadgol N, Najafi S, Talaee R. Epidermal growth factor receptor gene expression evaluation in colorectal cancer patients .Indian J Cancer 2014;51:358-362


How to cite this URL:
Motalleb G, Pourrahmat E, Rashki A, Moghadam A Y, Mazaheri M, Jahantigh M, Sabagh K, Sanadgol N, Najafi S, Talaee R. Epidermal growth factor receptor gene expression evaluation in colorectal cancer patients . Indian J Cancer [serial online] 2014 [cited 2019 Dec 5 ];51:358-362
Available from: http://www.indianjcancer.com/text.asp?2014/51/3/358/146771


Full Text

 Introduction



Cancer is the second most common cause of death in the United States and more than 1500 people die from cancer in a day and about 16,38,910 new cases of cancer diagnosed in 2012. [1] Cancer is the third most common cause of death in Iran. [2] Colorectal cancer is the third most common cause of death in the world [2] also third and fourth most common cancer among men and women in Iran respectively. [3] The incidence of colorectal cancer has steadily increased in Iran during recent years and worryingly, is affecting younger population, compared with Western countries. [2] Incidence and characteristics of colorectal cancer are based on the geographic region and race. [4] Multiple genes in different genetic pathways are involved in the development of pathogenesis of colorectal cancer. In recent years, the colorectal cancer molecular pathogenesis study has become very important. [5] One type of tyrosine kinase receptor is epidermal growth factor receptor (EGFR) that frequently seen and expressed in epithelial tumors. [6] A transmembrane glycoprotein is EGFR (170 kDa) that contains a tyrosine kinase or intracellular tyrosine kinase domain, an extracellular ligand-binding domain and a transmembrane lipophilic segment. [7] Binding of the ligand to the ectodomain initiates receptor homo-and heterodimerization and activates the cytoplasmic tyrosine kinase, stimulating the intracellular signaling pathways. [8] Increased levels of EGFR gene expression as well as over expression of EGFR protein levels are observed in many solid tumors, including lung, head and neck, ovary, cervix, bladder, esophagus, stomach, brain, breast, endometrium, colon and pancreas. [9] EGFR regulates important tumorigenic processes, including proliferation, apoptosis, angiogenesis and invasion. [9] The majority of human epithelial cancers are marked by functional activation of growth factors and receptors of the EGFR family. Given this phenomenon, EGFR was the first growth factor receptor to be proposed as a target for cancer therapy. [10] Blocking EGFR activation would obviously represent an innovative and key strategy in patient care because this therapeutic strategy impairs crucial cellular functions linked to proliferation and survival. [11],[12] EGFR signaling can be targeted by either monoclonal antibodies or tyrosine kinase inhibitors or even by antisense approaches (antisense molecules to EGFR or targeting key regulatory regions of the EGFR). [12],[13] Although EGFR has been reported to be overexpressed in anywhere from 25% to 82% of colorectal cancers, the clinical significance of EGFR overexpression in colorectal cancer is uncertain. [14] In this research work, the EGFR gene expression rate in formalin fixed paraffin-embedded (FFPE) tissue of advanced colorectal cancer patients in Iran were studied.

 Materials and Methods



Fifteen colorectal cancer (10 women and 5 men) and 15 normal colorectal (9 women and 6 men) FFPE blocks were obtained. The FFPE tumor and normal blocks were reviewed for quality and tumor content by a pathologist. Microdissection and deparaffinization of tissue samples were carried out as followed. Briefly, the samples were cut into 5-10 sections (corresponding to about 30 mg of tissue), depending on the size of the embedded tissue sample and placed in RNase-free, 2.0 mL Eppendorf tubes. Sections were deparaffinized by incubation in 1 mL of xylene (Merck KGaA, Darmstadt, Germany ) at 37°C for 15 min. The samples were then centrifuged, the supernatant was removed and fresh xylene was added for a second incubation. After deparaffinization and centrifugation, sections were washed with 1 mL 98-100% ethanol (Merck, Germany), air-dried for several minutes. The Qiagen RNeasy FFPE Kit (Qiagen, Valencia, CA, USA) was used to extract total ribonucleic acid (RNA) from samples according to the manufacturer's protocol. RNA was quantified using a spectrophotometer (Eppendorf, Germany) and OD 260/280 nm ratios >1.8 was obtained for all samples, indicating high purity. All solutions, including ethanol/water solutions, were made using diethylpyrocarbonate treated water. 1000 ng of total RNA was reverse transcribed using the 2-steps real-time-polymerase chain reaction (RT-PCR) kit (Vivantis, Malaysia) according to the manufacturer's protocol. EGFR oligonucleotide primers was used as described before [15] and 18s ribosomal ribonucleic acid (18s rRNA) primers was designed by Primer 3 software (Whitehead Institute/MIT Center for Genome Research, USA) as shown in [Table 1]. PCR was performed in a total volume of 25 μL containing 2 μL of reverse-transcribed complementary deoxyribonucleic acid (cDNA). After an initial incubation at 95°C for 5 min, the reaction mixtures were subjected to 40 cycles of amplification using the following protocols: 95°C for 30 s, 62°C for 50 s and 72°C for 50 s, followed by a final extension step at 72°C for 10 min.{Table 1}

Quantitation of EGFR and internal reference gene (18s rRNA) was carried out using an EvaGreen dye-based method in Hot Tag EvaGreen quantitative PCR (qPCR) master mix (Vivantis, Malaysia). Real-time PCR was carried out by a Rotor Gene 3000 from Corbett Research. PCR reactions were prepared in a final volume of 20 μL, with a final concentration of 1 Χ Hot Tag EvaGreen qPCR master mix and cDNA. The Cycle conditions were: 95°C for 15 min, followed by 40 cycles at 95°C (15 s), 62°C (30 s) and 72°C for 20 s. Each measurement was performed in triplicate and the threshold cycle (CT), the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined. [16] A standard curve for EGFR and internal reference gene was included for assay validation. In addition, a melting curve analysis was performed for the EvaGreen assay. To compare the RNA expression of target genes among different specimens, normalization based on internal reference gene expression was performed and the averages of the normalized CT values (ΔCT) were calculated as described before. [17] Relative messenger RNA (mRNA) expression of a target gene within a specimen was calculated as 2−ΔΔCT , where ΔCT = CT (target gene) - CT (internal reference gene). This calculation assumes that all PCR reactions are working with 100% efficiency. All PCR efficiencies were measured and found to be >98%; therefore, this assumption introduces minimal error into the calculations. [17] qPCR products was sent to South Korea by Gene Fanavaran company and were sequenced by ABI (capillary system) 3730 XL, USA. Triplicate experiments were performed and results are presented as mean ΁ standard error. The t-test and one-way ANOVA was used to calculate the statistical significance between groups. P <0.05 was considered to be significant. All statistical analysis was carried out using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The number of included samples was supervised by a pathologist. The colorectal cancer tissue samples were signed the informed consent.

 Results



[Figure 1] shows the RNA yield and we obtained an average of 250 μg (49.7-596.6 μg) of RNA, with OD260/280 ratios from 1.8 to 2. FFPE tissue RNA extraction is difficult due to RNA is heavily degraded, nucleic acids are cross-linked to proteins and base modifications are introduced during the fixation process. [15],[18],[19] So most of the FFPE tissue RNA extracted was smeary. PCR was carried out to ensure the correct synthesis of cDNA for 18s rRNA and EGFR genes [Figure 2]. [Figure 2] shows a dedicated band of desired size and no additional bands. qPCR efficiency rates for 18s rRNA and EGFR genes were equal 1 (with R2 value of 0.98 and 0.99 respectively) [Figure 3]a and b. [Figure 3] shows the melting peaks for qPCR products with EGFR and 18a rRNA genes. Due to EvaGreen dye binds to double stranded DNA in a sequence-independent way, non-specific amplification should be avoided for reliable real-time PCR quantification. Non-specific PCR products are usually ruled out by melting peak analysis. The melting process of double-stranded DNA causes a sharp reduction in the fluorescence signal around the melting temperature (Tm) of the PCR product. [20] Single and sharply defined melting curves with narrow peaks were obtained for PCR products of the analyzed genes. The results showed single and sharp curves with narrow peaks in PCR products. EGFR gene expression analysis was performed on 15 corresponding metastatic tumors and normal colorectal cancer tissue. We then calculated relative amounts of EGFR, mRNA in relation to 18s rRNA as a housekeeping gene in FFPE tumor tissue and compared the results with those from the corresponding FFPE control tissue [Figure 4] and [Figure 5]. There was a significant difference in the prevalence of EGFR expression between patients and control (P < 0.05). The results showed EGFR gene over expression in 80% of the patients (12/15). EGFR gene over expression was calculated using the livac (2−ΔΔCT ) formula and there was 2.46 times higher in patients compared with control. There was no significant difference in the prevalence of EGFR expression with gender between patients and control (P > 0.05). The DNA sequencing analysis showed that EGFR and 18s rRNA was amplified [Figure 6]. Aligned sequences in gene bank showed 97% and 96% similarity for 18s rRNA and EGFR genes respectively.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}

 Discussion



In this research study, EGFR gene over expression in FFPE colorectal cancer tumor tissue by RT-qPCR was showed. Our results are agreed with Azuma et al. [21] They assessed a total of 45 cases of metastatic colorectal cancer (mCRC) by laser capture micro dissection/real-time RT-PCR technology and showed EGFR gene over expression in 95.6% of patients (43/45) but did not find EGFRvIII gene expression in mCRC. In contrast, Larysz et al. [22] showed EGFR gene over expression in brain gliomas by qPCR. The EGFR belongs to the ErbB family of receptor tyrosine kinases. [23] This gene located on small arms of chromosome 7. EGFR mediates signaling by activating the mitogen activated protein kinase or MAPK and phosphatidylinositide 3-kinases or PI3K signaling cascades. [24] The expression of this gene is more frequent in black patients suggesting that some genetic factors may affect its expression. [25] EGFR is now recognized as an important target for investigation because of the prognostic significance of its expression in a wide variety of tumors and its possible implication for selective therapies. A positive correlation between EGFR status and many human carcinomas has been shown. [26] One of the major goals of translational research is the identification of molecular markers that could predict the biological behavior of individual tumors and guide treatment strategies. [27] In recent years, interest has grown in understanding the relationship between the biological characteristics of cancer and the association of these characteristics with the clinical outcomes of the disease. [28] Several distinct mechanisms, which can be quantitative (over expression of an unaltered product) or qualitative (production of a modified product), have been described for the conversion of proto-oncogenes to active oncogenes. Quantitative forms of oncogene activation include multiplication (gene amplification) or translocation to an active chromatin domain that brings a growth-regulatory gene under the control of a different promoter, causing inappropriate expression of the gene. Qualitative forms include either point mutations or the production of a novel product from a chimeric gene. [29] Over the last decade, much interest has been focused on the EGFR signaling network because of its role in tumor development and aggressiveness. Although EGFR mutations are rare events in colorectal cancer, aberrant activation of the EGFR signaling network frequently occurs through alterations of downstream elements in the signaling cascade as well as aberrant activation of the EGFR signaling network can also result from the over expression of HER family members. [27] In vitro studies have shown that EGFR can regulate differential sensitivity to different classes of chemotherapy agents. [30] EGFR inhibitors have been used to block EGFR activity and thereby, increase the radio sensitivity of tumor cells. [28] In order to identify patients need to be treated with anti-EGFR, the status of these genes in tumor samples be examined. Therefore, an accurate and repeatable method for the determination of EGFR gene expression is required. Since, there have been few reports demonstrating mRNA expression of EGFR, there is as yet no consensus on the importance of measuring mRNA as a biomarker. [21] Unfortunately, we did not access to the patients' files treatments and for this reason do not know which drugs patients used in order to discuss about that. This is the first report of EGFR gene over expression in colorectal cancer patients in Iran and further study is needed to find principle and mechanism of the EGFR gene activation and up regulation.

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