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Increased EGFR mRNA Expression Levels in Non-Small Cell Lung Cancer.

Introduction

Lung cancer is one of the most common cancers in humans and is the most common cause of death from cancer in the world. Non-small cell lung cancer (NSCLC) constitutes about 85% of lung cancers and it is divided into subgroups as squamous cell carcinoma, adenocarcinoma, large cell carcinoma and others [1]. The treatment of lung cancer is planned mainly according to the stage of the cancer the patient's performance status and comorbid diseases. A large proportion of patients with NSCLC are diagnosed at stage 3 and stage 4, and chemotherapy is usually recommended as the first-line treatment option [2]. Chemotherapy is very toxic, especially for the elderly and patients with poor performance. Therefore, in recent years there are efforts for developing targeted molecular-based drugs.

Epidermal growth factor receptor (EGFR) is a critical oncogenic factor involved in the occurrence and progress of NSCLC. EGFR is overexpressed in the majority of patients with NSCLC and is an important target in the treatment. EGFR is a member of the EGF-related tyrosine kinase receptor family [3]. There are two specific EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, which are enhanced and used in clinic for the treatment of advanced NSCLC. An objective response rate is about 10% for unselected NSCLC patients. Female patients, non-smokers, East Asians and patients with adenocarcinoma have a much higher response rate [4]. Today molecular markers, which affect the response to EGFR-TKIs, have also been identified.

Many somatic mutations were identified in the EGFR gene in NSCLC. Most of the mutations are localized in the tyrosine kinase domain (exons 18-21) of the EGFR gene. The amino acids 746-753 encoded by exon 19 and the amino acid 858 encoded by exon 21, which comprise more than 80% of all detected mutations, are two mutation hotspots. Sensitivity to EGFR-TKIs has been identified in small frameshift deletions of exon 19 and arginine for leucine substitution at 858th amino acid of exon 21 (L858R) [5-8]. This is the most important molecular mechanism in lung cancer. The seven phase-2 study made with gefitinib or erlotinib showed that response to TKIs are more than 87% and life expectancy is between 7.7 to 14 months without progression in patients with EGFR mutation positive NSCLC [9]. This period is much longer than the time that is provided by chemotherapy in unselected patient population and other target therapies (4-6 months). Gene amplification is a mechanism responsible for the overexpression of oncogenes. Increased EGFR copies have been identified in about 30% of NSCLC patients with FISH analysis and this is often associated with poor prognosis [10]. Increased EGFR copy is an effective indicator for better treatment response to EGFR-TKIs [5,7]. High EGFR copy number is often associated with EGFR somatic mutations [5,8]. EGFR mutation rates in American and European patients (10%) are too low when compared to Asian patients (30-50%). Still, a response to EGFR-TKI therapy is received in significant proportion of patients without the EGFR mutation and increased EGFR copy number might be the cause. Japanese patients with EGFR gene amplification do not benefit from gefitinib treatment and there is no known reason for this [11].

EGFR protein expression is very high (40-80%) in patients with NSCLC and is associated with poor prognosis [12]. Both positive [7,13] and a negative relationship [14] is found in the literature between the levels of EGFR protein and EGFR TKIs sensitivity. The EGFR protein level is usually associated with EGFR gene copy number [7,10]. Both IHC and FISH positive patients can benefit from EGFR-TKIs therapy.

There are significant differences in the prevalence of EGFR gene mutations in patients with lung cancer from different ethnic groups. The frequency of these mutations are 1-10% in the US and European patients [6, 15], 19-26% in Southeast Asians patients [16], and about 58% in East Asian women [17]. The studies showed that the incidence of EGFR mutations in Middle Eastern society was similar to western society. While the amplification of the EGFR gene was seen about 30% in East Asian lung cancer patients, it is below 10% in the US and Australian patients. The frequency of EGFR amplification is higher in the Middle Eastern society (16%) than the western population (6-9%) [10].

In this study EGFR mutation frequency and EGFR mRNA and protein expression levels were investigated in Turkish population due to different EGFR mutation, amplification and protein expression rates between communities and their effects in response to the tyrosine kinase inhibitors. This study may determine the molecular predictors in predicting the efficacy of tyrosine kinase inhibitors in the NSCLC patients in Turkish population.

Materials and Methods

Patients

The study was ethically approved by the local ethics committee of Erciyes University (accession number: 2008/10) in accordance with the ethical standards of Helsinki Declaration and supported by Erciyes University Scientific Research Department with TST-08-399 project number. Written informed consent for study tissue DNA, RNA and protein was obtained from each patient in the study. Clinical data were obtained from patients' medical records. The following criteria were used to classify smoking status: never smokers were defined as those with lifetime exposure of 100 cigarettes or less; former smoker who had stopped smoking at least 12 months before diagnosis and current smoker who had stopped smoking 1 to 12 months before diagnosis or current smoking.

Primary cancer tissue and normal tissue samples were obtained from 34 NSCLC patients who had undergone lobectomy at the Erciyes University Department of Thoracic Surgery RNA was extracted from fresh tissue samples as soon as possible. Tissue samples were collected at -80 C for DNA isolation and isolated together

L858R (exon 21) mutation analysis by mutation specific PCR

Genomic DNA was isolated using Genelute mammalian genomic DNA miniprep kit (Sigma-Aldrich) according to manufacturer's guidelines. The EGFR gene exon 21 was amplified by PCR. Two specific primer sets were designed for the wild type sequence and the L858R mutation in exon 21 by introducing a wild type or mutated nucleotide at the 3' terminal end (Table 1). A 20 ul PCR reaction mixture contained 5 ul of 10 PCR buffer 50 nmol of Mg[Cl.sub.2], 10 pmol each of sense and antisense primer 6 nmol of dNTPs, 2 U of Taq polymerase and 100 ng of sample DNA. PCR was performed on Rotorgene RealTime PCR System (Qiagen, Germany) under the following conditions: 95[degrees]C for 10 min, followed by 35 cycles of 94[degrees]C for 30s, 64[degrees]C for 30s, 72[degrees]C for 30s and final extension at 72[degrees]C for 5 min. PCR products were electrophoresed in % 2 agarose gels and visualized under UV.

EGFR exon 19 mutation analysis by direct sequencing

Sample DNA was amplified by PCR using the primers indicated in Table 1. A 20 ul PCR reaction mixture contained 5 ul of 10 PCR buffer, 50 nmol of Mg[Cl.sub.2], 10 pmol each of sense and antisense primer 6 nmol of dNTPs, 2U of Taq polymerase and 100 ng of sample DNA. PCR was performed on Rotorgene Real-Time PCR System (Qiagen, Germany) under the following conditions: 95[degrees]C for 5 min, followed by 35 cycles of 95[degrees]C for 30 s, 64[degrees]C for 45 s, 72[degrees]C for 30 s and final extension at 72[degrees]C for 10 min. DNA products producing a positive band on agarose gel were used for further steps. Amplified DNA was purified using Genomic DNA purification kit (Fermantas). 10 ng of PCR products was applied for the sequencing reaction using a Dye terminator cycle sequencing quick start kit (Beckman Coulter). EGFR exon 19 sequence analysis was performed using CEQTM 8000 Genetic Analysis System (Beckman Coulter). The reference coding sequence of EGFR was obtained from the NCBI (NCBI Reference Sequence: NG_007726.1) [18].

EGFR mRNA expression by relative quantitative reverse transcription PCR (qRT-PCR)

Total RNA was isolated using UltraClean tissue RNA isolation kit (MO BIO) following the manufacturer's protocol. The cDNA was transcripted by using Transcriptor first strand cDNA synthesis kit with random hexamers (Roche; Germany). We prepared four PCR tubes for each patient to quantify the expression of EGFR-GAPDH in tumor samples and EGFR-GAPDH in normal samples. PCR reactions in a final volume of 25 [micro]l: 2 [micro]l cDNA, 18 [micro]l distilled water 1 ul Mg[Cl.sub.2], 2 [micro]l LightCycler faststart DNA master SYBR green I mix (Roche Diagnostics) and 2 [micro]l primers for each EGFR and GAPDH gene (Table 1).

For each cDNA sample, expression levels of EGFR and the reference gene (GAPDH) were analyzed using the Rotorgene Real-Time PCR System (Qiagen, Germany). Cycle conditions of the relative qRT-PCR were preincubated at 95[degrees]C for 10 min, followed by 45 amplification cycles of 95[degrees]C for 8 s, 62[degrees]C for 15 s, 72[degrees]C for 10 s, and a melting curve analysis, which ran Melt (65-95[degrees]c), at 95[degrees]C hold 0 sec, at 65[degrees]C hold 15 secs and at 95[degrees]C hold 0 sec. qRT-PCR analysis and calculation of quantification cycle (Cq) values for relative quantification were performed by the Rotorgene Real-Time PCR software (Qiagen, Germany).

The gene expression of EGFR gene was normalized with GAPDH. The relative quantification of EGFR gene was calculated using the following formula [19] and the expression ratio greater than one is considered as overexpression (positive).

Expression ratio = [TEGFR/TGAPDH]/[NEGFR/NGAPDH]

TEGFR: measured expression of EGFR gene in tumor sample

TGAPDH: measured expression of the housekeeping gene, GAPDH in tumor sample

NEGFR: measured expression of EGFR gene in normal sample

NGAPDH: measured expression of the housekeeping gene, GAPDH in normal sample

EGFR protein expression by immunohistochemistry

Formalin-fixed paraffin embedded cancer tissue sections were used for the study The immunohistochemical study was performed for protein expression and graded using Rabbit anti-human EGFR monoclonal (Clone SP9) antibody at a dilution of 1/100 (Spring Bioscience) according to the manufacturer's instructions. The slides were counterstained with hematoxylin.

IHC analysis was independently reviewed by two pathologists who were blinded to the clinical outcome data, and differences in interpretation were resolved by consensus. EGFR expression was scored based on the intensity and percentage of IHC staining cells. The staining pattern was cytoplasmic and membranous. The intensity score was defined as follows: Score 0, no staining; score +1, faint staining; score +2 moderate staining (nearly at the same staining level as observed in the normal bronchial epithelium); score3, strong staining. The total score was calculated by multiplying the intensity score and the percentage of tumor cells showing characteristic staining (0-100%) producing a total range of 0-300. For statistical analyses, scores of 1-100, 101-200, and 201-300 were considered grade 1, grade 2 and grade 3, respectively Samples that exhibited grade 3 immunostaining scored as overexpression (positive).

Statistical analysis

Statistical analyses were performed with SPSS software, version 15.0. Comparisons of EGFR mRNA expression and protein expression level within clinical patient characteristics were performed by using the Fisher exact test or the [x.sup.2] test. A p value equal or less than 0.05 was considered statistically significant. EGFR mRNA expression and protein expression were compared by kappa, using the Landis and Koch criteria.

Results

Patients

The sex, age, histopathology, pathologic stage distribution, and smoking status of the cases that diagnosed as non-small cell lung cancer with histopathological examination are listed in detail in Table 2.

L858R (exon 21) mutation assed by mutation specific PCR

The L858R mutation was detected in none of the patients.

EGFR exon 19 mutations assed by direct sequencing

The mutations in exon 19 of EGFR gene was detected in none of the patients.

EGFR mRNA expression assessed by relative qRT-PCR

Patients' EGFR expression ratios (TEGFR/ TGAPDH / NEGFR/NGAPDH) are shown in Table 3. EGFR expression ratios of the 16 patients are bigger than one.

EGFR protein expression assed by immunohistochemistry

Examples of the staining intensity values of + 1, +2, +3 used for evaluation of the measurement of EGFR protein expression in patients are shown in Figure 1. Fifteen samples that exhibited grade 3 immunostaining scored as overexpression are shown in Table 3.

Among 34 patients that exhibited, 16 (47%) had EGFR gene mRNA overexpression, 15 (44%) had EGFR protein overexpression. The statistical results of EGFR gene mRNA expression and EGFR protein expression according to clinical patient characteristics are shown in Table 4. According to the statistical results, there was no significant difference between EGFR mRNA and protein expression with clinical characteristics.

The level of agreement for EGFR protein expression determined by immunohistochemical analysis and EGFR mRNA expression determined by qPCR demonstrate a K of 0.348 (Table 5). A K of 0.348 indicates a fair level of agreement according to Landis and Koch criteria (values < 0 as indicating no agreement, 0-0.20 as slight, 0.21-0.40 as fair, 0.41-0.60 as moderate, 0.61-0.80 as substantial, and 0.81-1 as almost perfect agreement) [20].

Discussion

There are significant differences in the prevalence of EGFR gene mutations in patients with lung cancer from different ethnic groups. The studies showed that the incidence of EGFR mutations in Middle Eastern society was similar to western society. The frequency of EGFR amplification is higher in the Middle Eastern society than the western population [6,10,15,17]. EGFR gene mutation, amplification and expression studies in the literature in NSCLC patients and our study are shown in Table 6.

In the majority of publications, EGFR mutations were identified by sequence and mutation specific PCR + gel electrophoresis; gene amplification by FISH and qPCR and EGFR protein expression was evaluated by IHC method. EGFR protein expression positivity (with IHC method) varies between 40-80% of NSCLC tumors as shown in Table 6. Different results may be because of different detection methods used in measuring EGFR protein. Different antibodies, different scoring systems and different protocols were used in different laboratories. In this study the EGFR mRNA and protein expression were measured by qRT-PCR and IHC methods, respectively

In this study for determining mRNA expression, the EGFR expression in tumor tissue was compared with normal tissue in the same patient (TEGFR/TGAPDH/ NEGFR/ NGAPDH). This method led to the identification of the exact expression results for each patient because EGFR expression normally seen in non-tumor tissue of the patients were excluded (background expression). The differences between initial mRNA amounts of different patients are balanced with the usage of GAPDH as an internal control. This analysis method was used in the study of Brabender et al. [21] for the evaluation of EGFR and HER2-neu mRNA expression in patients with NSCLC, Mafune et al. [22] for expression evaluation in squamous cell esophagus carcinomas and Bong et al. [23] for identifying the expression of colorectal carcinoma.

The frequency of EGFR molecular pathologies change among societies. Evaluation of the usage of molecular pathology targeted drugs in different societies or development of molecular pathology targeted drugs are needed. In our study, EGFR gene changes, which are common in the etiology of NSCLC, are planned to be investigated in the NSCLC patients of Turkish society for predicting the effectiveness of TKIs and for determining the molecular changes that can be used for the response to EGFR therapy In our study about 80% of EGFR gene mutations in the literature were checked and no mutation detected.

The reason for this negativity may be the number of our patients, only three female patients, and three non-smoker patients, two of whom have tandoori story more than 20 years. With this study the average mutation rate of the Turkish population (267 case, 58 cases have mutated) is 22% between 0-48% [24-29]. The frequencies of these mutations are 1-10% in America and Europe [6, 15]. EGFR mutations are rare in Middle Eastern patients, similar to the rates in the Western society (%3). Significantly increased EGFR amplification rates (15%) have shown the need to determine the changes of gene copy number in response to anti-EGFR therapy in the Middle East patients [30].

Increased EGFR mRNA and protein expression levels were stated in patients with EGFR mutations when compared to patients without gene mutations [31]. Many publications have shown that EGFR mutations are associated with increased EGFR gene copy number and EGFR expression. EGFR gene mutations and increased amplification were found to be the best parameter in determining clinical progression in the Korean patients treated with erlotinib [32] and in Japanese patients treated with gefitinib [5]. In addition, researchers have suggested investigating the effects of these molecular markers in different ethnic groups. Similar assessment could not be done in our study due to absence of mutations.

Amador et al. found that head and neck cancer cells with increased EGFR mRNA expression were more susceptible to erlotinib [33]. Taro et al. studied with 28 advanced NSCLC patients and detected increased EGFR mRNA expression levels in patients with EGFR mutations when compared to patients without mutations, but it was statistically insignificant [8]. EGFR mRNA expression was shown to be a major biomarker in response to gefitinib and progression free survival [34]. The percentage of mRNA expression positivity in NSCLC patients was indicated as 22-34 % [21, 34]. Our study shows 44% positivity, which is higher than in the literature. Significantly increased EGFR mRNA expression rates (44%) suggest that mRNA expression changes should be addressed in determining the response to anti-EGFR therapy in the Turkish community.

EGFR mRNA expression by qRT-PCR, EGFR gene dosage by qPCR, and EGFR copy number by FISH in patients with gefitinib treated non-small cell lung cancer were analyzed in order to determine the association with treatment outcome, clinical, and biological features [34]. EGFR mRNA expression was higher in responders to gefitinib compared to non-responders. EGFR mRNA expression was higher in FISH-positive patients and in patients with positive EGFR immunostaining, but not in patients with EGFR mutations. EGFR gene dosage did not predict response to gefitinib and was not associated with EGFR mutation status, FISH positivity mRNA expression and EGFR protein expression [34].

In the literature, Dacic et al., reported gene amplification correlated with protein expression, and it seems that gene amplification is a mechanism for protein overexpression in a subset of squamous cell carcinomas of the lung [35]. Although Hirsch et al. [10] found EGFR protein overexpression in all tumors with gene amplification, Dacic et al. demonstrated that protein expression does not necessarily require gene amplification, suggesting that other mechanisms such as gene mutation and transcriptional or posttranscriptional factors might have a role.

In our study, no correlation was found between clinic-pathological characteristics of patients and the expression levels of mRNA and protein. EGFR mRNA and protein overexpression were detected in 44% and 47% of patients, respectively The agreement between EGFR mRNA and protein expression indicated a fair level (k = 0.348). EGFR protein overexpression was found in 67% of patients who increased mRNA expression. Although the remaining 33% of the patients increased EGFR mRNA expression, the increase in the protein expression was not detected. The reasons for this may be grading differences in the absence of objective evaluation criteria in IHC method, tumor cell contamination in the normal tissue or normal cell contamination in the tumor tissue. mRNA can also be impaired or translation can be blocked due to post-transcriptional events such as miRNA mechanisms.

In this study, even though approximately 85% of the mutations indicated in the literature were studied, no mutation detected. The mutation rates in European and Middle Eastern societies are about 1-10% and lower than East Asia community [6, 15]. The EGFR protein expression is between 16-80% in other communities and 46-54% [29, 36, 37] in Turkish society; we found a ratio of 47% in our study consistent with the literature. EGFR gene amplification has been identified in many different rates such as 9-45% by FISH and 7-51% by qPCR. EGFR mRNA expression was found 22-34% in the literature, which is lower than our positive rate of 44%. Based on the hypothesis that in general, increased gene amplification is associated with increased gene expression, our positive mRNA expression rates are higher than amplification rates measured by FISH and qPCR and higher when compared to European society, but shows compliance with East Asian societies.

In conclusion, markedly increased EGFR mRNA expression ratios in the absence of activating mutations show that identifying an increase in the EGFR mRNA expressions for the prediction of response to EGFR tyrosine kinase inhibitors may be significant in Turkish population. Further studies of patients treated with EGFR inhibitors would be necessary to estimate the influence of EGFR mRNA expression on the response to therapy in Turkish population.

Ethics Committee Approval: The study was ethically approved by the local ethics committee of Erciyes University (accession number: 2008/10) in accordance with the ethical standards of Helsinki Declaration.

Informed Consent: Informed consent was obtained from patients who participated in this study.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept--S.T Design--S.T, Y.O.; Supervision--Y.O.; Materials--O.O., OK.; Data Collection and/or Processing--S.T; Analysis and/or Interpretation --S.T, S.T, H.A.; Literature Review--S.T; Writing--S.T; Critical Review--S.T., Y.O.;

Conflict of Interest: The authors have no conflict of interest to declare.

Financial Disclosure: The study was supported by Erciyes University Scientific Research Department with TST-08399 project number

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Sener Tasdemir (1), Serpil Taheri (2), Hilal Akalin (3), Olgun Kontas (4), Omer Onal (5), Yusuf Ozkul (3)

(1) Department of Medical Genetics, Ataturk University School of Medicine, Erzurum, Turkey

(2) Department of Medical Biology, Erciyes University School of Medicine, Kayseri, Turkey

(3) Department of Medical Genetics, Erciyes University School of Medicine, Kayseri, Turkey

(4) Department of Pathology, Erciyes University School of Medicine, Kayseri, Turkey

(5) Department of Thoracic Surgery, Erciyes University School of Medicine, Kayseri, Turkey

Received: October 04, 2015

Accepted: October 26, 2015

Available Online Date: January 25, 2017

Correspondence to: Sener Tasdemir

E-mail: senertasdemir@gmail.com

DOI 10.5152/eurasianjmed.2016.0237

Caption: Figure 1. Representative immunohistochemical staining intensity (left to right +1, +2, +3) for EGFR. Upper row: Adenocarcinoma, lower row: Squamous carcinoma
Table 1. Primers for exon 21 PCR, exon 19 sequence analysis
and expression of EGFR gene

                      Sense

Exon 21-Wild type     5'-gcttggtgcaccgcgacctg-3'
Exon 21-L858R         5'-gcttggtgcaccgcgacctg-3'
Exon 19               5'-gcaatatcagccttaggtgcggctc-3'
EGFR                  5'-ctcagccacccatatgtacc-3'
GAPDH                 5'-acaacagcctcaagatcatcag-3'

                      Antisense

Exon 21-Wild type     5'-cgcacccagcagtttggcgA-3'
Exon 21-L858R         5'-cgcacccagcagtttggcgC-3'
Exon 19               5'-catagaaagtgaacatttaggatgtg-3'
EGFR                  5'-ggtctcgggccattttgg-3'
GAPDH                 5'-tcttctgggtggcagtgatg-3'

Table 2. Clinical patient characteristics

                      n       %
Sex
  Male                31      91
  Female              3       9
Age
  <39                 2       6
  40-49               5       15
  50-59               11      32
  60-69               8      23.5
  >70                 8      23.5
Smoking
  Never smoker       3 *      9
  Former smoker       11      32
  Current smoker      20      59
Histology
  Adenocarcinoma      10      29
  Squamous          23 (a)    67
  Large cell          1       3
Pathologic stage
  IA-IB               16      47
  IIA-IIB             12      35
  IIIA-IIB            6       18
Total                 34     100

*; two patients have tandoori story >20 years

(a); one patient has squamous + small cell carcinoma histology
Never smoker: who had smoked fewer than 100 cigarettes in
their lifetime. Former smoker: who had stopped smoking at
least 12 months before diagnosis. Current smoker: who had
stopped smoking 1 to 12 months before diagnosis or current
smoking

Table 3. Clinical patient characteristics and study results

Case   Sex       Smoking       Age      Histology

1       M     Former smoker    54       Squamous
2       F    Never smoker *    50       Squamous
3       M    Current smoker    38    Adenocarcinoma
4       M    Current smoker    55    Adenocarcinoma
5       M     Former smoker    66       Squamous
6       M    Current smoker    48    Adenocarcinoma
7       M    Current smoker    75       Squamous
8       M     Former smoker    55    Adenocarcinoma
9       M    Current smoker    47       Squamous
10      M     Former smoker    66       Squamous
11      F    Never smoker *    72    Adenocarcinoma
12      M    Current smoker    44    Adenocarcinoma
13      M    Current smoker    63       Squamous
14      M     Former smoker    75       Squamous
15      M    Current smoker    51       Squamous
16      M    Current smoker    54       Squamous
17      M    Current smoker    57       Squamous
18      M    Current smoker    49       Squamous
19      M    Current smoker    74       Squamous
20      M    Current smoker    40       Squamous
21      M    Current smoker    56       Squamous
22      M     Former smoker    70       Squamous
23      M     Former smoker    58    Adenocarcinoma
24      M     Former smoker    73       Squamous
25      M    Current smoker    61       Squamous
26      M    Current smoker    60    Adenocarcinoma
27      M    Current smoker    58       Squamous
28      M     Former smoker    68       Squamous
29      M     Former smoker    73       Squamous
30      M    Current smoker    61      Large cell
31      M    Current smoker    50    Adenocarcinoma
32      F     Never smoker     38    Adenocarcinoma
33      M     Former smoker    68       Squamous
34      M    Current smoker    70     Squamous (a)

Case    Pathologic stage     Expression ratio    IHCG

1              IA                  0.90           3
2             IIB                  0.03           2
3              IB                  0.76           3
4             IIIA                10.26           2
5             IIB                  4.45           3
6              IB                  0.28           2
7              IB                  0.04           1
8             IIA                  0.05           2
9             IIIA                 0.08           2
10            IIB                  0.28           2
11             IB                  1.39           3
12            IIB                  1.19           2
13            IIA                  0.64           2
14            IIIB                 0.11           3
15            IIIA                 0.04           2
16            IIA                  2.13           1
17            IIIA                 0.49           2
18             IB                 64.10           3
19             IB                 13.29           2
20             IB                  0.31           1
21             IB                 20.42           3
22             IB                  1.42           3
23             IB                  0.25           2
24            IIB                  1.37           3
25            IIIA                 0.08           1
26            IIB                  2.49           3
27             IB                 15.31           3
28            IIA                  1.01           2
29             IA                  1.93           3
30             IB                  0.83           3
31            IIB                  8.71           3
32             IB                  0.02           2
33            IIB                  0.28           3
34             IA                 17.00           2

*; have tandoori story >20 years

(a); Squamous + small cell

IHCG: Immunohistochemical grading, M: Male, F: Female
Expression ratio = TEGFR/TGAPDH / NEGFR/NGAPDH

Table 4. Characteristics of clinical patient according to EGFR
mRNA and EGFR protein expression

                                EGFR mRNA expression

Clinical characteristics     negative    positive     p

Sex
Male                          16 (52)     15 (48)    0.55
Female                        2 (67)      1 (33)
Smoking
No                            2 (67)      1 (33)     0.86
Yes                           16 (52)     15 (48)
Histology
Squamous                      12 (52)     11 (48)    0.52
Adenocarcinoma                5 (50)      5 (50)
Large cell                       1           0

Pathologic stage
IA-IB                         8 (50)      8 (50)     0.21
IIA-IIB                       5 (42)      7 (58)
IIIA-IIB                      5 (83)      1 (17)

Age
Average                         56          62       0.1

                                EGFR protein expression

Clinical characteristics      negative     positive     p

Sex
Male                          17 (55)      14 (45)     0.59
Female                         2 (67)       1 (33)
Smoking
No                             2 (67)       1 (33)     0.28
Yes                           17 (55)      14 (45)
Histology
Squamous                     13 (56.5)    10 (43.5)    0.42
Adenocarcinoma                 6 (60)       4 (40)
Large cell                       0            1

Pathologic stage
IA-IB                          7 (44)       9 (56)     0.22
IIA-IIB                        7 (58)       5 (42)
IIIA-IIB                       5 (83)       1 (17)

Age
Average                         56.5         61.5      0.19

Table 5. Correlation between expression level of EGFR
protein and mRNA

                                EGFR mRNA expression

EGFR protein expression     Negative     Positive     Total

Negative                       13            5         18
Positive                        6           10         16
                                                      (%47)
Total                          19        15 (%44)      34

Table 6. Summary of selected reports on EGFR gene, protein study
in NSCLC

Author                Year   Country           Case

Paez [6]              2004   US                119
Hirsh [38]            2005   US                 82
Dacic [35]            2006   US                199
Hirsh [10]            2003   US                352
Bell [39]             2005   US                453
Shigematsu [16]       2005   US /Australia     158
Brabender [21]        2001   US/Germany         83
Marchetti [15]        2005   Italy             860
Cortez [40]           2005   Spain              83
Cappuzzo [7]          2005   US-Europe          89
Dziadziuszko [34]     2006   US-Europe          82
Taron [8]             2005   US/Europe/Asia     28
Tsao [13]             2005   Canada            325
Pinter [41]           2008   Hungary           126
Qin [42]              2005   China              41
Liang [43]            2012   China             120
Shigematsu [16]       2005   East Asia         361
Sasaki [44]           2006   Japan             575
Suziki [45]           2005   Japan             130
Yokoyoma [46]         2006   Japan             349
Sonobe [31]           2007   Japan              53
Takano [5]            2005   Japan              66
Suziki [47]           2005   Japan             181
Huang [48]            2004   Taiwan            101
Han [49]              2005   Korea              90
Ahn [32]              2008   Korea              92
Al-Kuraya [30]        2006   Saudi Arabia       47
Seyhan [36]           2010   Turkey             98
Cetin [29]            2010   Turkey
Gorgisen [24]         2013   Turkey             16
Unal [25]             2013   Turkey             48
Akca [26]             2013   Turkey             52
Bircan [27]           2014   Turkey             25
Dogan [28]            2014   Turkey             42
Gundogdu [37]         2014   Turkey             26
This study            2015   Turkey             34

                           EGFR gene              EGFR gene
                            mutation            amplification

Author                 Mutation ratio (%)    FISH (%)     qPCR (%)

Paez [6]                       13
Hirsh [38]                                       32
Dacic [35]                                       9
Hirsh [10]                                       32
Bell [39]                                                     7
Shigematsu [16]                8
Brabender [21]
Marchetti [15]                 5
Cortez [40]                    12
Cappuzzo [7]                   17                32
Dziadziuszko [34]                                           51 *
Taron [8]                      35                32
Tsao [13]                      23                45
Pinter [41]                    13                40
Qin [42]                       24
Liang [43]                                                   38
Shigematsu [16]                30
Sasaki [44]                    20
Suziki [45]                    28
Yokoyoma [46]                  29
Sonobe [31]                    32
Takano [5]                     59                            44
Suziki [47]                                      23
Huang [48]                     39
Han [49]                       19
Ahn [32]                       28                            41
Al-Kuraya [30]                 3                 15
Seyhan [36]
Cetin [29]                  4 (n=2)
Gorgisen [24]               0 (n=0)
Unal [25]                  43 (n=18)
Akca [26]                  48 (n=25)
Bircan [27]                44 (n=11)
Dogan [28]                  7 (n=2)
Gundogdu [37]
This study                  0 (n=0)

                              EGFR protein            EGFR mRNA
                               expression             expression

Author                IHK (%)     Western Blot (%)    qRT-PCR (%)

Paez [6]
Hirsh [38]
Dacic [35]               16
Hirsh [10]
Bell [39]
Shigematsu [16]
Brabender [21]                                             34
Marchetti [15]
Cortez [40]
Cappuzzo [7]             59
Dziadziuszko [34]                                          22
Taron [8]                                                  ?
Tsao [13]                57
Pinter [41]              59
Qin [42]
Liang [43]                               34
Shigematsu [16]
Sasaki [44]
Suziki [45]              56
Yokoyoma [46]
Sonobe [31]              79                                ?
Takano [5]
Suziki [47]              34
Huang [48]
Han [49]
Ahn [32]                 72
Al-Kuraya [30]           70
Seyhan [36]              52
Cetin [29]                               46
Gorgisen [24]
Unal [25]
Akca [26]
Bircan [27]
Dogan [28]
Gundogdu [37]            54
This study               47                                44

*: The average of the results obtained and specified percentage
of those, which indicated greater than the average; ?: Percent
was not given; qPCR: quantitative PCR; qRT-PCR: quantitative
reverse transcription PCR
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Title Annotation:Original Article
Author:Tasdemir, Sener; Taheri, Serpil; Akalin, Hilal; Kontas, Olgun; Onal, Omer; Ozkul, Yusuf
Publication:The Eurasian Journal of Medicine
Geographic Code:4EUGE
Date:Jun 1, 2019
Words:6526
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