|Year : 2016 | Volume
| Issue : 1 | Page : 44-49
Genetic analysis of EGFR mutations in non-small cell lung carcinoma: A tertiary care center experience
Reena Bharadwaj, Khushboo Dewan, Navdeep Mann
Department of Pathology and Molecular Medicine, Army Hospital Research and Referral, Delhi Cantt, Delhi, India
|Date of Web Publication||5-Feb-2016|
26, Amit Apartments, Sector - 13, Rohini, Delhi - 110 085
Source of Support: None, Conflict of Interest: None
Background: In the past decade, the association of epidermal growth factor receptor (EGFR) mutations with response to tyrosine kinase inhibitor "gefitinib" has revolutionized the treatment of lung adenocarcinomas. The research done in the West has suggested an association of EGFR mutation with factors such as female sex, nonsmoker status, and adenocarcinoma. Aim: This study was aimed to find the prevalence of EGFR mutations in lung cancer in the Indian population and its association with clinicopathological parameters. Materials and Methods: Sixty cases of non-small cell lung carcinoma (NSCLC) were studied histomorphologically and immunohistochemically for EGFR protein expression and for EGFR gene mutations by sequencing. The statistical analysis was done by calculating P value using Chi-square test. Results: Statistically significant relation of adenocarcinoma with female sex and nonsmoker status was noted. EGFR mutations were seen in 30% of NSCLC and 70% of lung adenocarcinoma cases. Statistically significant relation of EGFR mutations with female sex, nonsmoker status, and adenocarcinoma histology, particularly of the bronchioloalveolar type was noted. No relation of EGFR mutation with the age of the patient and the stage of the tumor was observed implying that EGFR mutation is an early event in the pathogenesis of lung adenocarcinoma. Exon 19 deletions were the most common type of EGFR mutations, with a higher prevalence in the male patients. Exon 21 and 18 mutations were also observed, particularly in females. No relation between EGFR mutations by sequencing and EGFR protein expression by immunohistochemistry was observed. Conclusion: In the Indian population, the prevalence of EGFR mutations in NSCLC is higher than that in the West, and it is associated with female sex, non-smoker status, and adenocarcinoma. Exon 19 deletions are the most common type of EGFR mutations. Immunohistochemistry for EGFR does not correlate with EGFR mutations and cannot be used as its substitute for deciding gefitinib treatment.
Keywords: Epidermal growth factor receptor (EGFR), gefitinib, immunohistochemistry, lung carcinoma, sequencing
|How to cite this article:|
Bharadwaj R, Dewan K, Mann N. Genetic analysis of EGFR mutations in non-small cell lung carcinoma: A tertiary care center experience. J Med Soc 2016;30:44-9
|How to cite this URL:|
Bharadwaj R, Dewan K, Mann N. Genetic analysis of EGFR mutations in non-small cell lung carcinoma: A tertiary care center experience. J Med Soc [serial online] 2016 [cited 2019 Dec 13];30:44-9. Available from: http://www.jmedsoc.org/text.asp?2016/30/1/44/175851
| Introduction|| |
Lung cancer is the most common type of cancer worldwide with an estimated 1.82 million (13%) new cancer cases and is the leading cause of cancer-related deaths (19.4%) according to the data published in 2012.  Till the last decade, surgical intervention, platinum-based chemotherapy, and radiotherapy were the principal available therapeutic options. With the description of epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas in the past decade and the response of these tumors to tyrosine kinase inhibitors, such as gefitinib and erlotinib, a new hope in making a significant difference in the survival of such patients has arisen. , The presence of EGFR gene mutations in lung carcinomas have been associated with female sex, non-smoker status, histologically adenocarcinomas, or mixed carcinoma with an adenocarcinoma component and low grade adenocarcinoma with lepidic/papillary/acinar histology according to various studies conducted in the West. ,,,, The present study was undertaken with an aim to find the prevalence of various genetic mutations in EGFR in non-small cell lung carcinomas (NSCLC) in the Indian population and the association of this mutation with various clinicopathological parameters.
| Materials and Methods|| |
The study was conducted in a tertiary care hospital in Delhi from 2011 to 2013. Hematoxylin and eosin stained slides of all suspected lung carcinoma cases received during the period of study were reviewed by two pathologists and the diagnosis was made using the World Health Organization (WHO) classification. In cases where classification of lung carcinoma was not possible on morphology alone, immunohistochemical (IHC) analysis for thyroid transcription factor (TTF-1), p63, napsin A, and cytokeratin 5/6 (CK5/6) was performed. The patients who are already on treatment and cases where tumor cells comprised less than 25% of the tissue, or where the amount of tumor tissue was limited, were excluded from the study. Sixty consecutive patients diagnosed as NSCLC on transbronchial lung biopsy (TBLB)/trucut biopsy/pneumonectomy specimen were finally included in the study.
In all cases, after heat induced antigen retrieval, IHC staining for EGFR protein was performed using EGFR PharmaDx kit (K1492, Dako Denmark) according to manufacturer's instructions. Sections from normal heart muscle and skin tissue were used as a negative and positive control, respectively, for each batch of IHC performed. In addition, control cell lines provided within the kit were used as controls. The following scoring approach in the assessment of EGFR immunostaining was used: 0 for no or nonspecific staining of the tumor cells, 1+ for weak intensity and incomplete membranous staining of more than 10% tumor cells, 2+ for moderate and complete membranous staining of more than 10% of tumor cells, and 3+ for strong and complete membranous staining of more than 10% of tumor cells. Cytoplasmic staining of the tumor cells was considered as negative. For statistical analysis, a score of 0 and 1+ was treated as "negative" and a score of 2+ or 3+ was considered "positive." 
DNA extraction, PCR amplification, and sequencing
Area containing maximum amount of tumor tissue from all 60 cases was marked by pathologist on both the slide and the paraffin block. Macrodissection of the tumor tissue from paraffin blocks by trimming off excess paraffin was then done. Genomic DNA was extracted from tumor tissue using QIAamp DNA formalin-fixed paraffin embedded (FFPE) tissue kit (QIAGEN, Catalog no: 56404, Hilden, Germany) following manufacturer's instructions. Exons 18, 19, and 21 of EGFR with flanking intronic sequences were Polymerase chain reaction (PCR) amplified using intron-based primers. The primer sequence details for exon 18 were as follows: Forward 5′-AGGGCTGAGGTGACCCTTGT-3′ and reverse 5′-TCCCCACCAGACCATGAGAG-3′. The primer sequence details for exon 19 were as follows: Forward 5′-ACCATCTCACAATTGCCAGTTAAC-3′, reverse 5′-GAGGTTCAGAGCCATGGACC-3′; The primer sequence details for exon 21 were as follows: Forward 5′-TCACAGCAGGGTCTTCTCTGTTT-3′, reverse 5′-ATGCTGGCTGACCTAAAGCC-3′. The PCR amplification was performed on Gene Amp PCR system 9700 (Perkin-Elmer Corp. Foster City, CA) in a total volume of 10 μL, containing 1xTaqman buffer, Magnesium chloride 0.5 mmol/L, 800 μmol/L dNTPs, 300 μmol/L each primer, 0.3 units Taq polymerase and 10 ng of genomic DNA. DNA amplification was done for 4 min at 95°C, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and one cycle of 72°C for 7 min. All tests were performed in duplicate to ensure accurate results. The PCR product was then verified for suitability for sequencing by 2% agarose gel electrophoresis. The PCR products were then incubated using Exo-SAP IT (Amsheram Biosciences Corp., Piscataway, NJ) and then sequenced using big dye terminator sequencing reaction. The sequencing fragments were detected on the automated DNA capillary sequencer ABI 3100 PRISM (Applied Biosystems, Foster City, CA).
| Results|| |
The mean age of the 60 patients studied was 62.7 years (ranged 42-79 years) with a standard deviation of 6.5 years. Male-female ratio was 2.75 (44/16). Of the 60 patients included in the study, 41 patients (68.3%) were current smokers, 12 patients (20%) had never smoked, and 7 patients (11.7%) had quit smoking more than 5 years back. Transbronchial lung biopsy was received in 38 cases (63.3%), trucut biopsy in 20 cases (33.3%), and pneumonectomy specimens in 2 cases (3.4%). When classified according to the recent WHO classification 2004, 32 cases (53.3%) of squamous cell carcinoma, 20 cases (33.3%) of adenocarcinoma, 6 cases (10%) of bronchioloalveolar carcinoma, and 2 cases (3.3%) of large cell carcinoma were present.
Relation between histological tumor type and clinical variables
Of the 16 female patients included in the study, 13 patients (81.2%) had adenocarcinoma on histology. In the male patients, squamous cell carcinoma was the most common histomorphology (70.4%). A statistically significant relation between adenocarcinoma histological subtype and female sex was noted (Chi-square = 24.80; P value <0.001) [Table 1]. All of the 12 nonsmokers included in the study revealed an adenocarcinoma on histology and the results were statistically significant (Chi-square = 35.25; P value <0.001) [Table 1].
Relation between EGFR mutation and clinicopathological variables
EGFR mutation was seen in around one-third (30%) of NSCLC cases (18/60). No statistically significant relation between age of the patient and EGFR mutation status was noted (Chi-square 0.056; P > 0.1). EGFR mutations were more commonly seen in female sex and in nonsmokers. Out of the females included in the study, 62.5% (10/16) showed EGFR mutation as compared to 18.18% (8/44) of males and the association was statistically significant (Chi-square = 10.96; P < 0.001) [Table 2]. Out of the nonsmokers included in the study, 66.7% (8/12) revealed EGFR mutations as compared to 17% (7/41) of the smoker group and the results were statistically significant (Chi-square = 11.46; P < 0.01) [Table 2]. The most common histomorphology observed in EGFR mutated tumor was an adenocarcinoma (14/18 = 77.7%). This is in comparison with EGFR nonmutated tumors where squamous cell carcinoma is the most common morphology (31/42 = 73.8%). The association of EGFR mutation with adenocarcinoma morphology is statistically significant (Chi-square = 42.22; P < 0.001) [Table 2]. All of the patients with bronchioloalveolar type of adenocarcinoma contained EGFR mutation and the results were statistically significant. No statistically significant relation between stage of the tumor and EGFR mutation status was noted (Chi-square 1.73; P > 0.1).
|Table 2: Relationship between the EGFR mutation and clinicopathological variables|
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Deletions in exon 19 including typical deletion of 15 nucleotides E746-750 and atypical deletions of EGFR were the most common, seen in 61.1% of the cases (11/18). Out of all the cases, 27.7% (5/18) had exon 21 L858R point mutations and 11.11% (2/18) had exon 18 mutations. All of the exon 21 and exon 18 mutations were seen in females and most of the exon 19 mutations (8/11 = 72.72%) were seen in males (Chi-square = 9.34; P < 0.01) [Table 3]. No statistically significant relation between type of EGFR mutation and smoking status (Chi-square = 4.72; P > 0.1) or histomorphological type (Chi-square = 1.18; P > 0.1) of tumor was noted [Table 3].
|Table 3: Relationship between the type of EGFR mutation and clinicopathological variables|
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Relation between EGFR mutation and EGFR protein expression
Of the 60 cases included in the study, 29 cases (48.3%) did not show EGFR expression and 31 cases (51.7%) revealed EGFR expression on immunohistochemistry. No statistically significant relation between EGFR mutation detected by sequencing and EGFR protein expression by immunohistochemistry was observed in this study (Chi-square 4.80; P > 0.1) [Table 4]. The sensitivity of immunohistochemistry was only 50% and specificity was 47.6% when compared to sequencing as a method for EGFR mutation detection.
|Table 4: Relationship between the EGFR mutation by sequencing and EGFR protein expression on immunohistochemistry|
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| Discussion|| |
Mutations in the EGFR gene have been reported to be associated with response of lung cancer patients to tyrosine kinase inhibitor "gefitinib." ,,,,, The feasibility of targeted therapy using monoclonal antibodies in EGFR mutated lung adenocarcinoma needs to be known in the Indian population. In this study, mutations in the EGFR gene were present in 30% of NSCLC and 70% of adenocarcinoma. The prevalence of EGFR gene mutations in NSCLC cases has been reported to range from 3% to 40% by various researchers worldwide. ,,,,,, The wide variation in the range can be explained to some extent on geographical grounds. The prevalence is <10% in the United States and >40% in Japan. , This difference is probably because the ratio of nonsmokers to smokers in India is higher than that in the United States but lower it is lower than that in Japan.  The EGFR mutations were seen in 70% of adenocarcinomas in this study. Other researchers have reported EGFR mutations in 40-55% of adenocarcinomas. ,,
EGFR mutations were found to be associated with female sex, nonsmokers, and adenocarcinoma histology in this study. Higher prevalence of EGFR mutations in females, nonsmokers, and patients with adenocarcinomas have been reported independently by various researchers. ,, This is in concordance with the observation of a good clinical response to gefitinib in lung carcinoma occurring in females, nonsmokers, and patients with adenocarcinoma on histology. , Seventeen out of 18 NSCLC that showed mutations in EGFR gene were adenocarcinoma and bronchioloalveolar carcinoma put together. The incidence of EGFR mutations was particularly related to bronchioloalveolar type adenocarcinoma. A similar observation was made by Miller et al. who also found a better response of bronchioloalveolar carcinoma (BAC) to gefitinib.  A single case diagnosed as squamous cell carcinoma on TBLB showed EGFR mutation in this study. This case possibly highlights the problem of failure to identify adeno component in adenosquamous carcinoma with limited availability of tumor tissue. Kosaka et al. similarly identified EGFR mutation in only a single nonadenocarcinoma out of 277 surgical resection specimens of NSCLC that happened to have an adenosquamous histomorphology. 
In this study, inframe deletions around codons 746-750 in exon 19 were the most common EGFR mutations seen in 61.1% (11/18) of the cases, point mutations in exon 21 (L858R) observed in 27.7% cases with 11.11% cases of exon 18 mutations. Previous studies have reported inframe deletions in exon 19 in 26-79%, point mutations in exon 21 (L858R) in 13-47%, and <10% mutations in exon 18. ,,,,, It is important to know the nature of the mutation as EGFR exon 19 deletions have been reported to be more susceptible to gefitinib than tumors with exon 21 point mutations.  Statistically significant relation of mutations in exon 18/exon 21 with female sex and exon 19 with male sex was observed. Tokumo et al. had also reported an association of male sex with exon 19 mutations and female sex with exon 21 mutations. 
No statistically significant relation between EGFR mutations and stage of the disease was found in this study, suggesting the possibility that EGFR mutation is an early event in the pathogenesis of lung adenocarcinoma. The same inference was also drawn from the study by Tang et al. who had tested the presence of EGFR mutation in histologically normal bronchial epithelium from lung adenocarcinoma and found that normal bronchial epithelium present within the tumor frequently contained the same EGFR mutation as seen in the tumor tissue. 
IHC EGFR expression was seen in 52% of the patients in this study that correlates well with previous studies. , No association between EGFR expression and EGFR mutations were present in this study as in the study by Li et al. 
| Conclusion|| |
In this study, 30% of NSCLC and 70% of lung adenocarcinomas carried mutations in EGFR gene. The incidence of EGFR mutations in this study is higher when compared to patients in the United States but is comparable with Japanese population. Amongst exons 18, 19, and 21 of EGFR gene studied, mutations in exon 19 were the most common of all EGFR mutations. Lung adenocarcinoma from nonsmoker and female patients are more likely to contain mutation in the EGFR gene and therefore may show better response to gefitinib. Since no correlation between the presence of EGFR mutations and the stage of disease was found in this study, we suggest that EGFR mutation is an early event that plays an important role in the pathogenesis of lung adenocarcinomas. The present study did not demonstrate any association between EGFR expression by immunohistochemistry and EGFR mutation and, therefore, we do not recommend EGFR immunohistochemistry to screen for EGFR mutations in NSCLC.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]