|Year : 2016 | Volume
| Issue : 3 | Page : 345-348
Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms − 216 G>T, −191 C>A, (CA) n IVS1, and R521K
JH Torres-Jasso1, AR Bustos-Carpinteyro2, JR Garcia-Gonzalez3, J Peregrina-Sandoval4, JA Cruz-Ramos5, E Santiago-Luna6, JY Sanchez-Lopez3
1 División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social; Doctorado en Genética Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Nextipac, Jalisco, ; Departamento de Ciencias Biológicas, División de Ciencias Biológicas y de la Salud, Centro Universitario de la Costa, Universidad de Guadalajara, Delegación Ixtapa, Puerto Vallarta, México
2 División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social; Doctorado en Genética Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Nextipac, Jalisco, México
3 División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, México
4 Departamento de Biología Celular y Molecular, Laboratorio de Inmunobiología, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Nextipac, Jalisco, México
5 Departamento de Enseñanza y Capacitación, Instituto Jalisciense de Cancerología, Guadalajara, México
6 Departamento de Gastroenterología, Servicio de Endoscopías, Hospital de Especialidades del Centro Médico Nacional de Occidente, Instituto Mexicano del Seguro Social, México
|Date of Web Publication||24-Feb-2017|
División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social
Source of Support: None, Conflict of Interest: None
BACKGROUND: Gastric cancer (GC) is the third worldwide leading cause of cancer-related death affecting both sexes. The aberrant expression of epidermal growth factor receptor (EGFR) gene has been detected in many human epithelial malignancies and linked to advanced disease, more aggressive phenotype, and poor prognosis. AIMS: To analyze the relation that the expression of EGFR in gastric tumors holds with pathological characteristics and with the germline polymorphisms −216 G>T, −191 C>A, (CA) n IVS1, and R521K. MATERIALS AND METHODS: We studied 22 biopsies from gastric tumors obtained by endoscopy. EGFR expression was determined by relative quantification real-time polymerase chain reaction with the glyceraldehyde-3-phosphate dehydrogenase reference gene (as for messenger RNA [mRNA]) and by immunohistochemistry (IHC) (as for protein). EGFR germline polymorphisms were analyzed by sequencing, GeneScan, and restriction fragment length polymorphisms. RESULTS: EGFR mRNA expression was increased (>2-fold) in 13.6% of GC cases, decreased (<0.5-fold) in 68.2%, and normal in 18.2%; overexpression was related to well-differentiated gastric tumors, whereas underexpression was linked to moderate or poorly differentiated gastric tumors (P < 0.001). EGFR protein expression was high (IHC 2+ and 3+) in 29.4% of gastric tumors and was normal or low (score 0 to 1+) in 70.6% cases. EGFR expression, in both mRNA and protein, was not related to any EGFR polymorphism (P > 0.05). CONCLUSIONS: Most gastric tumors showed low EGFR expression (mRNA and protein), whereas EGFR overexpression was related to well-differentiated gastric tumors. Furthermore, germinal polymorphisms −216, −191, (CA) n IVS1, and R521K were not related to EGFR expression (mRNA or protein).
Keywords: Epidermal growth factor receptor gene, gastric cancer, gene expression, immunohistochemistry, polymorphism, quantitative real-time polymerase chain reaction
|How to cite this article:|
Torres-Jasso J, Bustos-Carpinteyro A, Garcia-Gonzalez J, Peregrina-Sandoval J, Cruz-Ramos J, Santiago-Luna E, Sanchez-Lopez J. Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms − 216 G>T, −191 C>A, (CA) n IVS1, and R521K. Indian J Cancer 2016;53:345-8
|How to cite this URL:|
Torres-Jasso J, Bustos-Carpinteyro A, Garcia-Gonzalez J, Peregrina-Sandoval J, Cruz-Ramos J, Santiago-Luna E, Sanchez-Lopez J. Epidermal growth factor receptor expression in gastric tumors and its relationship with the germline polymorphisms − 216 G>T, −191 C>A, (CA) n IVS1, and R521K. Indian J Cancer [serial online] 2016 [cited 2017 Jun 28];53:345-8. Available from: http://www.indianjcancer.com/text.asp?2016/53/3/345/200648
| » Introduction|| |
Gastric cancer (GC) has a high incidence and ranks as the fifth most common cancer in the world. It is the third worldwide leading cause of cancer-related death involving both sexes. Histologically, it has been classified as intestinal (50%), diffuse (33%), and the remaining are mixed or unclassified.
The epidermal growth factor receptor (EGFR) gene plays a crucial role in the growth, differentiation, and motility of both normal and cancer cells.EGFR overexpression has been detected in many human epithelial malignancies including GC and has been related to advanced stages of the disease, a more aggressive phenotype, resistance to treatments, and poor prognosis.
Since the relationship of the polymorphisms −216 G>T (rs712829), −191 C>A (rs712830), (CA) n IVS1, and R521K (rs2227983) with overexpression of the EGFR gene in gastric tumors remained unknown, the aim of the present study was to correlate the EGFR expression with the germline polymorphisms −216 G>T, −191 C>A, (CA) n IVS1, and R521K, as well as with the pathological characteristics of gastric tumors.
| » Materials and Methods|| |
Subjects and samples
We studied 22 patients who underwent upper gastrointestinal endoscopy at the hospital of Western National Medical Center of Mexican Institute of Social Security. All patients had a confirmed histopathological diagnosis of GC (19 gastric adenocarcinoma and 3 with gastric lymphoma). The studied patients were 3 women and 19 men with a mean age of 58.5 years (±standard deviation 16.8; range 29–86). A gastric biopsy and 5 ml of peripheral blood were taken from each patient before treatment. Biopsies were collected in sterile tubes containing 3 ml of RNAlater (Ambion-Invitrogen) solution and stored at −20°C until RNA isolation. Tumor pathological characteristics are shown in [Table 1].
|Table 1: Epidermal growth factor receptor messenger RNA expression observed in different characteristics of gastric tumors|
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Epidermal growth factor receptor expression analysis by quantitative real-time polymerase chain reaction
RNA extraction was performed from twenty cuts of 5 µm of gastric tissue obtained by a cryostat (Thermo Scientific Microm) and collected in a tube with 1 ml of TRIzol (Invitrogen, USA). Total messenger RNA (mRNA) was converted to complementary DNA (cDNA) immediately with the High Capacity cDNA Reverse Transcription Kit with random primers (Applied Biosystems, USA). EGFR expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) using the TaqMan Gene Expression Inventoried Assay for EGFR gene (Applied Biosystems, USA, Assay ID: Hs01076092_m1). Relative quantification was performed by the ΔΔCt method. Six cDNAs obtained from subjects whose pathological studies were negative for malignant or premalignant conditions were used as calibrators (controls without cancer); the age of these individuals ranged between 34 and 63 with an average of 49.3 years. All reactions were carried out by triplicate in a volume of 30 µl on a 7500 RT-PCR System (Applied Biosystems, USA) with the following standard parameters: 50°C 2 min, 95°C 10 min, and forty cycles at 95°C 15 seg, 60°C 1 min. We used three housekeeping genes for normalization, ACTB (Cat. 4333762F), B2M (Cat. 4333766T), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. 4333764F) (Applied Biosystems, USA). Differences in fold changes between GC patients and calibrators were calculated. A normal EGFR expression was considered when the mRNA levels remained between 0.50 and 2.00 folds, subexpression values were those below to 0.50 folds, and overexpression those up to 2.0 folds.
Epidermal growth factor receptor expression analysis by immunohistochemistry
We analyzed the expression of EGFR protein by immunohistochemistry (IHC) in only 17 out of 22 patients. Frozen tissue sections of 5 µm were treated with 3% hydrogen peroxide in methanol for 15 min at room temperature to eliminate endogenous peroxidase activity. After rinsing in phosphate-buffered saline (PBS), sections were incubated overnight with the specific primary polyclonal anti-EGFR antibody (Abcam, UK) in a moist chamber at 4°C. The dilution of antibody used was standardized at 1:300 in PBS buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.4). After washing with PBS, biotinylated antimouse immunoglobulin at a dilution of 1:200 was added for 45 min, followed by a PBS-washing step and incubation with avidin-biotin complex reagent for 45 min. The peroxidase reaction was developed with 0.02% 3,3'-diaminobenzidine tetrahydrochloride in a PBS buffer containing 0.06% hydrogen peroxide, for 5 min. Finally, sections were counterstained with Harris' hematoxylin. A histologist evaluated the degree of immunoreactivity. As a positive control, normal gastric tissue was stained. As a negative control, serum from a nonimmunized animal was used to replace the primary antibody. Tumor membranous staining for EGFR protein was scored using a four-grade scale (0, if no staining was observed; 1+, if more than 10% of the tumor cells had weak staining on the membrane [or cytoplasm for EGFR]; 2+, if more than 10% of the tumor cells had moderate staining on the membrane; and 3+, if more than 10% of the tumor cells had strong staining on the membrane). Criteria defining EGFR overexpression were the scores of 2+ or 3+. Scores 0 or 1+ were considered negative.
Single-nucleotide polymorphism analysis
Genomic DNA was obtained by the salting out method from peripheral blood leukocytes of 22 patients with GC. The −216 G>T (rs712829) and −191 C>A (rs712830) polymorphisms were identified using PCR and DNA sequencing. A 446-bp fragment was amplified with the primers, forward 5'-CCT CCT CGC ATT CTC CTC CTC CT-3' and reverse 5'-TTT TCC TCC AGA GCC CGA CTC GC-3'. PCR was performed according to the AccuPrime GC-rich Polymerase (Invitrogen, USA) protocol in a thermal cycler (Techne) with the following program: Initial denaturing, 95°C for 4 min; thirty cycles of 95°C for 30 s, 67°C for 30 s, and 72°C for 30 s; final elongation, 72°C for 10 min. The PCR products were purified with Centri-Sep columns (Princeton Separations, USA), and the sequencing reaction was performed with the Big Dye Terminator v3.1 Kit (Applied Biosystems, USA) on an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems, USA).
The (CA)n IVS1 repeat was analyzed using GeneScan (Applied Biosystems, USA). The reaction was performed using the primers: Forward, 5'-GGG CTC ACA GCA AAC TTC TC-3', and reverse, 5'-AAG CCA GAC TCG CTC ATG TT-3'. After initial denaturation at 94°C for 5 min, samples were run for thirty cycles at 94°C for 1 min, 55°C 1 min, and 72°C for 1 min, with a final elongation at 72°C for 3 min. The obtained products ranged from 190 to 212 bp corresponding to 12–23 CA repeats. A total of 0.5 µl of each product was mixed with 9.35 µl of formamide and 0.15 µl of the GeneScan-500 LIZ size standard (Applied Biosystems, USA). Denatured PCR products were separated by capillary electrophoresis on an ABI-PRISM 310 Genetic Analyzer (Applied Biosystems, USA), and the results were evaluated with the GeneScan Analysis software (Applied Biosystems, USA).
Detection of the R521K polymorphism (rs2227983) was performed by PCR using the following primers: Forward, 5'-TGT TGT GAC CCA CTC TGT CTC CG-3', and reverse, 5'-CCT CCA GAA GGT TGC ACT TGT CC-3'. The reaction was performed with 200 ng of genomic DNA, 5 pM of each primer, 0.5 U of Taq polymerase, 0.017 mM of MgCl2, 1×PCR buffer, and 2 µM of deoxynucleotide triphosphates. The program comprised initial denaturation at 94°C for 4 min, followed by thirty cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min; then, the reaction was completed at 4°C for 10 min. The PCR product (158 bp) was digested with 1.25 U of BstNI enzyme (New England BioLabs, Ipswich, MA, USA). The resulting fragments were visualized by electrophoresis in 6% polyacrylamide gels and were stained with silver nitrate. The expected restriction fragments were G/G (wild homozygote) = 38 bp + 50 bp + 70 bp; G/A (heterozygote) = 38 bp + 50 bp + 70 bp + 120 bp; A/A (mutated homozygote) = 38 bp + 120 bp.
All procedures here described were in accordance with the ethical standards of the Institutional Committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000. All patients agreed to participate in the study by signing an informed consent. The National Committee on Research in Health-IMSS approved this study.
Correlations between EGFR expression - mRNA and protein - were calculated by Pearson and Spearman tests. ANOVA test was employed to analyze the EGFR expression and tumoral characteristics. Kruskal–Wallis test was performed to analyze the association of EGFR expression and −216 G>T, −191 C>A, R521K, and (CA) n IVS1 polymorphisms, the risk alleles considered in this last polymorphism were those repeats with number ≤16. The analysis was carried out with the SPSS v. 18 software (SPSS Inc., Chicago, IL, USA) considering as statistically significant, P < 0.05.
| » Results|| |
Epidermal growth factor receptor messenger RNA and protein expression
We first analyzed the reference genes GAPDH, ACTB, and B2M with the BestKeeper program (http://bioinformatics. gene-quantification.info/bestkeeper.html). The endogenous gene with the most stable expression in gastric tumor samples was GAPDH since it showed the lowest coefficient of variation (CV = 5.93) with respect to ACTB (6.58) and B2M (7.01); therefore, we present the results based on this reference gene.
EGFR mRNA expression ranged from 0.05 to 4.48-fold differences relative to controls; the mean was 0.788-fold. Low EGFR mRNA expression was observed in 15 (68.2%) gastric tumors, overexpression in 3 (13.6%), and a normal expression in 4 (18.2%). Meanwhile, EGFR-IHC analysis showed a score between 0 and 1+ in 70.6% of the samples, 23.5% had a score of 2+, and 5.9% had a score of 3+. Spearman's rho test showed no significant correlation between EGFR expression values obtained by quantitative RT-PCR or by IHC (P > 0.05).
Epidermal growth factor receptor expression levels and clinical-pathological characteristics of the tumor
We observed differences in the EGFR mRNA expression levels among the grades of differentiation of the tumor. Well-differentiated tumors (G1) had higher EGFR- mRNA expression (3.51-fold) than controls; conversely, moderate (G2) or poorly (G3) differentiated tumors had lower values than controls (0.38- and 0.46-fold, respectively) (P < 0.001) [Table 1].
When comparing EGFR mRNA levels among histological types of gastric tumors, those of the mixed type had the highest EGFR mRNA expression (2.4-fold higher than controls); however, these differences were not statistically significant (P > 0.05) [Table 1]. The placement of the stomach tumor (cardia, fundus, corpus, or antro pilorico) was not related to EGFR mRNA levels (P > 0.05) [Table 1].
EGFR protein expression was not related to any clinical-pathological characteristic of the tumor (P > 0.05).
Epidermal growth factor receptor expression levels and germline polymorphisms
None of the −216 G>T, −191 C>A, (CA) n IVS1, or R521K polymorphisms was associated to changes in EGFR expression of gastric tumors (mRNA or protein) (P > 0.05) [Table 2].
|Table 2: Epidermal growth factor receptor messenger RNA expression in gastric tumors among the genotypes of−216 G>T, −191 C>A, R521K (G>A), and (CA) n IVS1 germline epidermal growth factor receptor polymorphisms|
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| » Discussion|| |
The gastric tumors here studied showed a high level of expression of EGFR protein (IHC 2+ or 3+) in 29.4% of the cases, which is consistent with values reported in the literature for GC cases: From 2% to 58%.
By the other hand, EGFR mRNA overexpression was observed in 13.6% of gastric tumors, which was similar to those reported by Angelescu et al. (27.3%, P = not significant). Quantitative studies on EGFR mRNA expression in gastric tumors are scarce, and they depend on the use of standards or controls and endogenous genes for normalization, in our study, GAPDH showed the highest stability of the three endogenous genes we analyzed. Furthermore, EGFR mRNA overexpression was related to well-differentiated gastric tumors; however, it was previously related to the poorly differentiated gastric tumors.
Regarding the relationship of EGFR expression and the EGFR germline polymorphisms, we did not observe any association in spite of reports showing the association of mutated allele T of the −216 G>T polymorphism with higher EGFR expression in experimental conditions.,, Nevertheless, reports on fewer than 16 (CA) repeats of the (CA) n IVS1 polymorphism have shown conflicting results regarding its relationship with EGFR overexpression in cell lines., As for the polymorphisms −191 C>A and R521K, association between these single-nucleotide polymorphisms and EGFR expression in cell lines has not been previously observed. The differences between EGFR expression observed by us - under in vivo conditions - and those observed by others ,,, -under experimental conditions- are probably due to factors that may, direct or indirectly, influence the EGFR expression such as estrogens, progesterone, and glucocorticoids.,
| » Conclusions|| |
The germinal polymorphisms −216, −191, (CA) n IVS1, and R521K were not related to EGFR expression; furthermore, most gastric tumors showed low EGFR expression (mRNA and protein), and EGFR overexpression was related to well-differentiated gastric tumors. Additional studies are required to clarify the prognostic role of the EGFR overexpression in gastric tumors.
This research was supported by the National Council of Science and Technology (CONACyT), Mexico (Grant #68669; SALUD-2007). The first author received a studentship from CONACYT. We thank for the assistance with statistical analysis to Rogelio Troyo, and for the assistance with the review of the manuscript to Lourdes Carbajal.
Financial support and sponsorship
The study was supported by National Council of Science and Technology (CONACyT), Mexico (Grant #68669; SALUD-2007).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]