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 »  Abstract
 » Introduction
 » Aim
 »  Materials and Me...
 » Results
 » Discussion
 » Acknowledgment
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LCC-A SYMPOSIUM- ORIGINAL ARTICLE
Year : 2013  |  Volume : 50  |  Issue : 2  |  Page : 102-106
 

Molecular epidemiology of epidermal growth factor receptor mutations in lung cancers in Indian population


SRL Diagnostics, Centre of Excellence: Histopathology, Lower Parel, Mumbai, Maharashtra, India

Date of Web Publication27-Aug-2013

Correspondence Address:
J Mehta
SRL Diagnostics, Centre of Excellence: Histopathology, Lower Parel, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.117019

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 » Abstract 

Background: Lung cancer is the leading cause of cancer related mortality world-wide and amongst males in India. The discovery of tyrosine kinase inhibitors holds a ray of hope for a subset of lung cancer patients, which have activating epidermal growth factor receptor (EGFR) mutations. Much of the preliminary data on frequency of EGFR mutations emanated from Western studies, which reported EGFR mutation rates of 10-15%. However, studies from Asian countries report a much higher frequency of EGFR mutations, not only in the male population, but also in females. AIM: The object of this study was to share the author's experience of EGFR mutation testing in 402 lung cancer patients as no large-scale study addressing the issue has been published from India. Materials and Methods: Formalin fixed paraffin embedded tissues were analyzed for EGFR exon 19 deletions and exon 21 point mutation by length analysis of fluorescently labeled polymerase chain reaction products on Applied Biosystems Inc. 310 genetic analyzer. Results: Out of 402 samples, 35 samples could not be analyzed because of poor deoxyribonucleic acid material. Thus of the remaining 367 cases analyzed, EGFR mutations were found in 118 patients (32%). Mutations were equally distributed between males (50%) and females (50%). Majority of the mutations were seen in adenocarcinoma subtype (90%). Exon 19 mutations accounted for 76% while exon 21 mutations accounted for 24% of the mutations. Summary: EGFR mutation frequency is higher in Indian population vis-à-vis Caucasian population, but lower than that reported in the East Asian population. A significantly higher number of males also harbor EGFR mutations.


Keywords: Caucasians, East Asian population, epidermal growth factor receptor mutations, Indian population, lung


How to cite this article:
Mehta J. Molecular epidemiology of epidermal growth factor receptor mutations in lung cancers in Indian population. Indian J Cancer 2013;50:102-6

How to cite this URL:
Mehta J. Molecular epidemiology of epidermal growth factor receptor mutations in lung cancers in Indian population. Indian J Cancer [serial online] 2013 [cited 2019 May 19];50:102-6. Available from: http://www.indianjcancer.com/text.asp?2013/50/2/102/117019



 » Introduction Top


Lung cancer is the leading cause of cancer related mortality among males in India. According to the GLOBOCAN 2008 report, [1] lung cancer accounts for 10.9% of all cancers in males and 2.2% of all cancers in females. The corresponding mortality rates for males and females are 13% and 3.3% respectively. The higher mortality rates associated with lung cancer are linked to an advanced stage of the disease at presentation. Close to half the number of patients are at least stage III at presentation. [2]

The dismal response of lung cancer to conventional modalities of treatment led to the development of epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitors (TKIs). The use of these agents in lung cancer patients, particularly those with no history of tobacco use, threw up interesting statistics in the form of higher response rates and longer median overall survivals (OS). [3],[4] Another interesting finding was the higher frequency of EGFR mutations in female patients, patients who did not smoke and in tumors with adenocarcinoma histology. [5]

The EGFR is a receptor tyrosine kinase of the v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB) family, which consists of four closely related receptors: Human epidermal growth factor receptor (HER) HER-1/ErbB1, HER-2/neu/ErbB2, HER-3/ErbB3 and HER-4/ErbB4. Upon ligand binding and receptor homo-or hetero-dimerization and activation (phosphorylation), activated EGFR signals downstream to the phosphatidylinositol 3-kinase/AKT and RAS/RAF/MAPK pathways [Figure 1]. These intracellular signaling pathways regulate key cellular processes such as proliferation and apoptosis.

Mutations in EGFR have thus become a prime focus of research in lung cancer. A variety of EGFR point mutations and deletions affect the adenosine triphosphate binding site of the receptor (the kinase domain), leading to a constitutively active and ligand-independent activated EGFR state. The most common activating mutations in the kinase domain of EGFR are exon 19 deletions that eliminate a leucine-arginine-glutamate-alanine motif and point mutations at codon 858 in exon 21, resulting in substitution of arginine for leucine (L858R). Tumors with activating mutations in EGFR are highly dependent on continued EGFR signaling for proliferation and survival, which is also precisely the reason for their exquisite sensitivity to EGFR TKIs.
Figure 1: Schematic of epidermal growth factor receptor signaling pathway

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Associations of tumor histology, patient demographics (age and gender) and effect of smoking and specific molecular alterations have been the focus of extensive studies. EGFR mutations have been found to be more common in female patients, non-smokers and patients of Asian ethnicity. [3],[5] However, these findings pertain mainly to the Caucasian population. It is estimated that approximately 10-15% of non-small-cell lung cancers (NSCLC) in the United States harbor EGFR mutations. [6] A large scale screening study of EGFR mutations in NSCLC in Spain [5] revealed a mutation frequency of 16.6%.

There is, however, a higher prevalence of EGFR mutations in Asian patients vis-à-vis Caucasian patients. Among Asian patients the incidence of EGFR mutations is approximately 30% compared with 7% among Caucasians. [7] Even higher mutation rates (ranging from 47% to 64% among various east Asian countries) were observed in the PIONEER study. [8]


 » Aim Top


There is no data reporting the incidence of EGFR mutations among Indian lung cancer patients. The PIONEER study [8] reported 22% mutation frequency in south India. Besides this study, where 72 patients were analyzed, no large-scale study has been reported from India. Thus, the aim of this study is to share the authors experience with EGFR mutation testing in 402 lung cancer patients.


 » Materials and Methods Top


From January 2010 to January 2013, 402 lung cancer patients were tested for EGFR mutations. All samples were formalin fixed paraffin embedded (FFPE) tissues. Genomic deoxyribonucleic acid (DNA) was extracted from the FFPE tissue sections using the QIAamp DNA FFPE tissue kit (Qiagen Inc, Valencia, CA) as per the manufacturer's instructions. The EGFR exon 19 deletion assay is based on length analysis of fluorescently labeled polymerase chain reaction (PCR) products. [9] Briefly, a 207-bp genomic fragment including all of exon 19 is amplified using the primers elaborated before. [9] The reverse primer is labeled with 6-FAM (carboxyfluorescein) fluorophore. The fluorescently labeled PCR products are subjected to capillary electrophoresis on Applied Biosystems Inc. 310 genetic analyzer (Applied Biosystems). The test is scored positive (mutation present) when the expected germ line product of 207-bp is accompanied by a distinct peak at 9, 12, 15, 18 or 24 bp below the normal product in the electrophoretogram.

The EGFR exon 21 L858R point mutation is detected by a PCR-restriction fragment length polymorphism assay, based on a Sau 961 restriction site created by the mutation (2573 T → G). [9] Briefly a 222-bp genomic fragment including all of exon 21 is amplified using the primers outlined. [9] If a 2573 T → G mutation is present, after digestion, a 173-bp wild type product and a 87-bp mutant PCR product is produced. The digested fluorescently labeled PCR product is analyzed by capillary electrophoresis.


 » Results Top


The age of patients ranged from 24 years to 84 years (median age 68 years) [Figure 2]. There were 242 male patients and 160 female patients. Out of 402 cases, 249 cases were negative for EGFR mutations. A further 35 cases could not be tested as the DNA was not amplifiable (housekeeping gene did not amplify). Mutations in the EGFR gene were found in 118 (32%) of the 367 patients tested (402 patients, less the 35 patients whose samples could not be tested because the DNA was of technically unsatisfactory quality) [Figure 3]. These mutations were equally distributed between male patients (59 positives, 50%) and female patients (59 positives, 50%) [Figure 4]. Adenocarcinoma formed the bulk of positive tumors (90% overall, 55 positives in males and 51 positives in females). The rest of the mutations (12 positives, 10%) were in adenosquamous carcinomas [Figure 5].
Figure 2: Age and gender - wise distribution of all patients

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Figure 3: Pie chart of all patients showing mutation frequency

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Figure 4: Gender subgrouping in mutation positive patients

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Figure 5: Histologic subtypes in mutation positive patients

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Out of the 118 mutation positive cases, 90 mutations (76%) were exon 19 deletions/insertions while 28 mutations (24%) were exon 21 point mutations (L858R) [Figure 6],[Figure 7] and [Figure 8].
Figure 6: Frequency of exon 19 and exon 21 mutations

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Figure 7: Exon 19 (18 bp deletion) mutation by length analysis of fluorescently labeled polymerase chain reaction products

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Figure 8: Exon 21 mutation

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No mutations were seen in any of the tested cases of squamous carcinoma (28 patients), small cell carcinoma (2 patients), large cell carcinoma (3 patients) and sarcomatoid carcinoma (1 patient).

History of smoking was available in 198 of the 367 patients tested for EGFR mutations (402 cases overall minus the 35 cases, which could not be tested because the DNA was unamplifiable). Out of the 198 cases in which history of smoking was available, 58 were smokers, 130 were non-smokers and 10 were ex-smokers [Figure 9]. Out of the 118 cases that showed EGFR mutations, 14 patients were smokers (1 female patient, 13 male patients), 44 were non-smokers (27 female patients, 17 male patients) and 2 were ex-smokers (both male patients) [Figure 10]. No information was available in the remaining 58 patients who harbored EGFR mutations.
Figure 9: Gender distribution among smokers

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Figure 10: Smoking frequency in mutation positive patients

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 » Discussion Top


The data was analyzed in this study and that published in the literature clearly show the great discrepancy in the frequency of EGFR mutations between Indian and Caucasian populations. There also appears to be a difference in the frequency of EGFR mutations between Indian patients and data reported for patients from Far East Asia. This is not surprising, given that Asians and Caucasians have different genetic susceptibilities to lung cancer as evidenced by candidate polymorphisms and genome wide association studies. [7],[10],[11],[12],[13] Large epidemiologic studies carried out in Asia have concluded that Asian ethnicity is a favorable prognostic factor for OS of NSCLC patients and this is independent of the smoking status. [14] It would, therefore, be worthwhile to keep in mind these differences in mutation frequencies and clinical response among different ethnic populations while formatting local guidelines for treatment and reflex testing for EGFR mutations. It should also be borne in mind that the Male:Female ratio of EGFR mutations is not as skewed as it is generally believed to be.

Following treatment guidelines for lung cancer laid down in the West may not be the most appropriate management strategy for our patients. Obtaining and collating robust data from our country will help aid in developing the best treatment protocols for our patients.

The frequency of EGFR mutations is significantly higher in non-smokers in the Indian population, an observation, which is consistent with that noted in lung cancer population in Far East Asia. [15] EGFR mutant lung cancers may represent a distinct class of lung cancers enriched in the non-smoking population and less likely linked directly to tobacco carcinogenesis.


 » Acknowledgment Top


The author wishes to wholeheartedly thank the excellent technical expertise provided by Ms. Dhrupal Patel.

 
 » References Top

1.Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. GLOBOCAN 2008 v2.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 10. 
Lyon, France: International Agency for Research on Cancer; 2010. Available from: http://www.globocan.iarc.fr. [Last accessed on 2013 Feb 03].  Back to cited text no. 1
    
2.Noronha V, Dikshit R, Raut N, Joshi A, Pramesh CS, George K, et al. Epidemiology of lung cancer in India: Focus on the differences between non-smokers and smokers: A single-centre experience. Indian J Cancer 2012;49:74-81.  Back to cited text no. 2
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3.Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-39.  Back to cited text no. 3
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4.Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004;101:13306-11.  Back to cited text no. 4
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5.Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009;361:958-67.  Back to cited text no. 5
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6.Dacic S, Shuai Y, Yousem S, Ohori P, Nikiforova M. Clinicopathological predictors of EGFR/KRAS mutational status in primary lung adenocarcinomas. Mod Pathol 2010;23:159-68.  Back to cited text no. 6
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7.Sekine I, Yamamoto N, Nishio K, Saijo N. Emerging ethnic differences in lung cancer therapy. Br J Cancer 2008;99:1757-62.  Back to cited text no. 7
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8.Yang PC, Shi Y, Siu-kie JA, Srinivasan S, Cornelio GH, Tsai CM, et al. Molecular epidemiological prospective study of EGFR mutations from Asian patients with advanced lung adenocarcinoma (PIONEER). J Clin Oncol 2012;30(Suppl): Abstr 1534.  Back to cited text no. 8
    
9.Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn 2005;7:396-403.  Back to cited text no. 9
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10.Nomura M, Shigematsu H, Li L, Suzuki M, Takahashi T, Estess P, et al. Polymorphisms, mutations, and amplification of the EGFR gene in non-small cell lung cancers. PLoS Med 2007;4:e125.  Back to cited text no. 10
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11.Amos CI, Wu X, Broderick P, Gorlov IP, Gu J, Eisen T, et al. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat Genet 2008;40:616-22.  Back to cited text no. 11
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12.Wang Y, Broderick P, Webb E, Wu X, Vijayakrishnan J, Matakidou A, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet 2008;40:1407-9.  Back to cited text no. 12
    
13.Yang P, Li Y, Jiang R, Cunningham JM, Li Y, Zhang F, et al. A rigorous and comprehensive validation: Common genetic variations and lung cancer. Cancer Epidemiol Biomarkers Prev 2010;19:240-4.  Back to cited text no. 13
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14.Ou SH, Ziogas A, Zell JA. Asian ethnicity is a favorable prognostic factor for overall survival in non-small cell lung cancer (NSCLC) and is independent of smoking status. J Thorac Oncol 2009;4:1083-93.  Back to cited text no. 14
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15.Zhou W, Christiani DC. East meets West: Ethnic differences in epidemiology and clinical behaviors of lung cancer between East Asians and Caucasians. Chin J Cancer 2011;30:287-92.  Back to cited text no. 15
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]

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