Indian Journal of Cancer Home 

LCC-A SYMPOSIUM- ORIGINAL ARTICLE
[Download PDF]
Year : 2013  |  Volume : 50  |  Issue : 2  |  Page : 107--111

Epidermal growth factor receptor mutation subtypes and geographical distribution among Indian non-small cell lung cancer patients

A Choughule1, V Noronha1, A Joshi1, S Desai2, N Jambhekar2, S Utture2, A Thavamanni3, K Prabhash1, A Dutt3,  
1 Department of Medical Oncology, Tata Memorial Hospital, Navi Mumbai, Maharashtra, India
2 Department of Pathology, Tata Memorial Hospital, Navi Mumbai, Maharashtra, India
3 Department of Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Navi Mumbai, Maharashtra, India

Correspondence Address:
A Dutt
Department of Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Navi Mumbai, Maharashtra
India

Abstract

Background: The Medical Oncology Department at Tata Memorial Hospital, the single largest tertiary cancer care center in Asia, receives in-house registered and referral patient samples from all parts of the country. Our recent studies establish 23% EGFR mutation frequency among Indian population. Here, we extend our study and report further analysis of distribution of different types of EGFR mutations in 1018 non small cell lung cancer patient, and its co-relation with clinical parameters and geographical variations across the country. Material and Methods: This study is a retrospective analysis on all the patients who were referred for EFGR testing as a routine service over a 1.5 year period. This was part of standard care. EGFR kinase domain mutations in exon 18-21 were probed by TaqMan probe-based assays in 1018 NSCLC patients. Results and Discussion: While EGFR exon 19 mutations, the most frequent EGFR mutation, were found be higher among non smokers females, we find surprisingly higher incidence of exon 21 mutations among EGFR mutation positive male smokers of Indian ethnicity. Furthermore, as Indian population is known to be composed of a gradient admixture of Ancestral North Indian (with genetic influence from Middle Easterners, Central Asians, and Europeans harboring variant EGFR mutation frequency) and Ancestral South Indians, as a paradox our study indicates comparable EGFR mutation frequency across different geographical locations within India Conclusion: Geographically there is uniform distribution in the EGFR mutation frequency within India. Further more, while exon 19 mutations are predominant among non smokers, higher incidence of exon 21 mutations exists among EGFR mutation positive male smokers of Indian ethnicity.



How to cite this article:
Choughule A, Noronha V, Joshi A, Desai S, Jambhekar N, Utture S, Thavamanni A, Prabhash K, Dutt A. Epidermal growth factor receptor mutation subtypes and geographical distribution among Indian non-small cell lung cancer patients.Indian J Cancer 2013;50:107-111


How to cite this URL:
Choughule A, Noronha V, Joshi A, Desai S, Jambhekar N, Utture S, Thavamanni A, Prabhash K, Dutt A. Epidermal growth factor receptor mutation subtypes and geographical distribution among Indian non-small cell lung cancer patients. Indian J Cancer [serial online] 2013 [cited 2022 Jul 1 ];50:107-111
Available from: https://www.indianjcancer.com/text.asp?2013/50/2/107/117023


Full Text

 Introduction



Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide in both men and women with less than 15% chance of surviving beyond 5 years. Chemotherapy for NSCLC, which accounts for approximately 85% of lung cancer cases, remains marginally effective. [1] Although the majority of lung cancers are linked to environmental carcinogens such as tobacco smoke and environmental pollutants, it is lung cancer in never-smokers, especially adenocarcinomas (ADCs), which are most likely to respond to tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) gene. [2] Recent clinical trials have also suggested that for advanced NSCLC patients with EGFR mutant tumors, initial therapy with a TKI instead of chemotherapy may be the best choice of treatment. [1],[2]

Of the four major subtypes of EGFR mutation present in exon 18-21, the exon 19 and 21 mutations are most frequent. [3],[4],[5],[6] They are either in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the kinase domain mutations, which confer ligand-independent activation and increased activation duration compared with wildtype receptors. [3] These sensitizing mutations of exon 19 and 21 are both believed to be associated with carcinogenesis, sensitivity to tyrosine kinase drugs and with the prognosis of NSCLCs, but their exact clinical significance remains disputable. [3],[7] Significantly, there is a plethora of studies demonstrating that patients with the exon 19 deletions and L858R mutations respond very well to EGFR inhibitors in NSCLC. [5],[7]

EGFR mutation subtypes display distinct biological features concerning kinase activity, transforming potential, and sensitivity to EGFR inhibitors. In vitro studies have shown that exon 19 deleted mutants and L858R mutant receptors appear to be more sensitive to gefitinib inhibition as compared with wildtype receptors. [8] It has also been documented that patients with exon 20 harboring deletions were found to have longer survival following treatment with gefitinib or erlotinib compared with those having L858R mutations in NSCLC. [3],[7] However, Mark et al., reported no difference in survival between exon 19 deletions and L858R mutations in the absence of EGFR-targeted therapy. [8],[9] Most EGFR exon 19 insertions occur in between amino acids 767-774 of exon 20, within the loop that follows the C-helix of the kinase domain of EGFR. [10],[11],[12] It is thus imperative to determine the distribution of EGFR mutation subtype in a given population to determine the overall implication for establishing relevance for routine EGFR mutation diagnostics for NSCLC patients in clinics and emphasizes effectiveness for adoption of EGFR inhibitors as the firstline treatment in a given population.

Besides variation of individual subtypes, overall incidence of EGFR mutation (including all subtypes) varies across different ethnicity; 10%-15% in Caucasians and 20%-30% in various East Asians, we recently showed an intermediate EGFR mutation in Indian population to be of 23%. We argue that this intermediate frequency is reminiscent of an ancestral admixture of genetic influence from Middle Easterners, Central Asians, and Europeans on modern Indian population. A recent study has as well showed that Indian population is composed of a mixture of original Ancestral South India and Ancestral North Indians with genetic influence from Middle Easterners and Europeans. [13] As genetic diversity is known to influence incidence of EGFR mutations, we asked if EGFR mutation frequency varies across four different geographical locations within India. In this extensive study, our main objective was to determine the distribution of EGFR mutation subtype in NSCLC patients registered in TMH by using rapid and sensitive technique of RQ-PCR with TaqMan® primer probes, as well as to evaluate the actual prevalence of mutation types in NSCLC.

 Material and Methods



This study has been done on 1018 cases that were referred for EFGR testing from medical oncology as a routine service over a 1.5-year period. This was part of standard care; therefore, no Institutional Review Board approval was obtained. The patient characteristics including the age, gender, smoking/tobacco use, and histopathology were recorded.

Collection of patient samples

The FFPE blocks of the patients were collected from the Pathology Department, TMH. The hematoxylin and eosin-stained sections of the blocks were viewed under the microscope to confirm that the tumor region constitutes more than 75% of the tissue mass.

DNA extraction

For DNA isolation, three FFPE tissue sections of 14 μm each were taken using microtome (Leica). After deparaffinizing the sections with xylene, [14] the DNA was extracted as per the kit insert (QiaAmp FFPE tissue kit, Cat no 56404).

Mutation analysis by PCR and sequencing

Extracted DNA was amplified for the exons 18, 19, 20, and 21 using a nested-PCR method, [15],[16],[17] with a 100 ng DNA as the template as shown recently. With the above PCR products as template, second round of nested PCR amplification was carried out using a different set of primers specific for these regions. After the amplification of the regions of interest, the amplicons were then purified using the Qiaquick PCR purification kit (Qiagen). About 2.5 ng of the PCR product, along with 1.6 pmols of the forward or reverse primer was used for sequencing in the Applied Biosystems DNA Analyzer. [5] The resulting sequence was compared with the available normal sequence in the NCBI database using the NCBI BLAST and the variations observed thereupon were recorded.

Mutation analysis by TaqMan® -based real-time PCR technique

The reaction mixture was carried out in 10 μL volumes, which contains 5 μL of the TaqMan probe, [15],[16] master mix (Roche), primers at a final concentration of 9 μM and probes at a final concentration of 2 μM and the remaining volume was made up to 5 μL using PCR grade water. The instrument used for the assay was LC 480 II. The program used was, 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The detectors used were FAM/VIC at 5′ *and TAMRA/MGB at 3′ fluorescence for the wildtype and mutant, respectively. The assay sensitivity has a limit of 1% detection and each run consisted of a positive and a negative control to check the integrity of the assay.

Statistical analysis: The Chi-square test was performed to reveal any significant correlation between the mutation status and age, gender, and habits of the patient and tumor histology.

 Results



Total 1018 patients diagnosed with lung cancer in TMH from February 2011 to December 2012 were tested for common EGFR mutation subtypes by RQ-PCR using TaqMan primer probes in exon 18 point mutation, exon 19 in frame deletions, and exon 21 L858R point mutation. Exon 20 assay was incorporated from August 2012 onward in 215 cases. After standardization and validation with direct sequencing, the RQ-PCR test, which was found to be robust, rapid, and highly sensitive, was implemented as the routine diagnostic test.

Out of 1018, the maximum number of patients were from the west of India and lowest from the south of India, but the percentage positivity almost remained between 20% and 28% with highest in south and the west of India [Table 1]. Similar to our recent report (under communication), we observe an overall frequency of 25% incidence of EGFR mutation in this extended population study. Of note, approximately 53% mutations were in-frame deletions in exon 19, whereas 38% are missense mutations in L858R in exon 21, 6% of the mutations were found in exon 18 and 3% in exon 20, which is similar to that described in Spanish and Japanese populations. [18],[19],[20],[21] Exon 20 mutation analysis was performed in 215 patients, of whom 3% were positive. This is similar to that reported in the literature. [22] EGFR exon 20 insertions comprise approximately 4% of all EGFR mutant lung tumors [Table 2].{Table 1}{Table 2}

Our finding suggests exon 19 and 21 in ADC group of 53% and 38%, respectively, and 7% in exon 18, were similar to East Asian population with a mutation rate. In our earlier study, exon 19 mutation was present in 67% cases and exon 21 in 29% of cases, this difference could be because of the smaller sample size of 111 patients [17],[23] [Table 3].{Table 3}

Correlating with the smoking status among EGFR mutation-positive patients, exon 19 mutations were found in 62% nonsmoker males and 57% females compared with 39% smoker males. However, exon 21 mutations were found in 46% male smokers and compared with 29% male nonsmokers. These results reveal that exon 19 mutations are predominant among nonsmokers, whereas exon 21 more frequently exists among smokers [Table 4].{Table 4}

 Discussion



Despite historical evidence for decreasing admixture content of genetic influence while moving from North to South India, due to amalgamation of major ancestral populations over past thousands of years at different points of time, no significant difference was observed in the incidence of EGFR mutation across different geographical locations within India. Further study is needed to confirm these findings due to sparse representation of patients from South and Central India. However, within the cohort of different mutations, we analyzed the clinical and pathological characteristics of lung ADC patients. Our results suggest that exon 21 mutation was more common in male smokers compared with nonsmokers, whereas exon 19 shows higher mutation rate in never-smokers than smokers, irrespective of gender bias. It has been reported that exon 18 EGFR mutations are detected more frequently in younger patients, which was also noted in our study. [24] However, Kosaka et al., [7] found no association between EGFR mutations and age, whereas Huang et al., [25] found no significant association between the presence of an EGFR mutation and gender or smoking history in Chinese patients. [26]

 Conclusion



Geographically there is uniform distribution in the EGFR mutation frequency within India. Furthermore, while exon 19 mutations are predominant among nonsmokers, higher incidence of exon 21 mutations exists among EGFR mutation-positive male smokers of Indian ethnicity.

 Acknowledgment



We thank Mr. Sanjay Talole for statistical analysis and Dr. Shripad Banavali for support, A.D. is supported by an Intermediate fellowship from the Wellcome Trust/DBT India Alliance (IA/I/11/2500278), by a grant from DBT (BT/PR2372/AGR/36/696/2011), and intramural grants (Seed-In-Air 2897, TMH Plan Project 2712 and IRB 92). The fund providers had no role in study design, data collection and analysis, decision to publish, or in the preparation of the manuscript.

References

1Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497-500.
2Lynch 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.
3D'Angelo SP, Pietanza MC, Johnson ML, Riely GJ, Miller VA, Sima CS, et al. Incidence of EGFR exon 19 deletions and L858R in tumor specimens from men and cigarette smokers with lung adenocarcinomas. J Clin Oncol 2011;29:2066-70.
4Jackman DM, Yeap BY, Sequist LV, Lindeman N, Holmes AJ, Joshi VA, et al. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin Cancer Res 2006;12:3908-14.
5Riely GJ, Pao W, Pham D, Li AR, Rizvi N, Venkatraman ES, et al. Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res 2006;12:839-44.
6Zhang Y, Sun Y, Pan Y, Li C, Shen L, Li Y, et al. Frequency of driver mutations in lung adenocarcinoma from female never-smokers varies with histologic subtypes and age at diagnosis. Clin Cancer Res 2012;18:1947-53.
7Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: Biological and clinical implications. Cancer Res 2004;64:8919-23.
8Gazdar AF. Activating and resistance mutations of EGFR in non-small-cell lung cancer: Role in clinical response to EGFR tyrosine kinase inhibitors. Oncogene 2009;28(Suppl 1):S24-31.
9Kumar A, Petri ET, Halmos B, Boggon TJ. Structure and clinical relevance of the epidermal growth factor receptor in human cancer. J Clin Oncol 2008;26:1742-51.
10Arcila ME, Nafa K, Chaft JE, Rekhtman N, Lau C, Reva BA, et al. EGFR exon 20 insertion mutations in lung adenocarcinomas: Prevalence, molecular heterogeneity, and clinicopathologic characteristics. Mol Cancer Ther 2013;12:220-9.
11Suda K, Onozato R, Yatabe Y, Mitsudomi T. EGFR T790M mutation: A double role in lung cancer cell survival? J Thorac Oncol 2009;4:1-4.
12Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: Preclinical data and clinical implications. Lancet Oncol 2012;13:e23-31.
13Reich D, Thangaraj K, Patterson N, Price AL, Singh L. Reconstructing Indian population history. Nature 2009;461:489-94.
14Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, et al. Measurement of gene expression in archival paraffin-embedded tissues: Development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol 2004;164:35-42.
15Endo K, Konishi A, Sasaki H, Takada M, Tanaka H, Okumura M, et al. Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay. Lung Cancer 2005;50:375-84.
16Molina-Vila MA, Bertran-Alamillo J, Reguart N, Taron M, Castellà E, Llatjós M, et al. A sensitive method for detecting EGFR mutations in non-small cell lung cancer samples with few tumor cells. J Thorac Oncol 2008;3:1224-35.
17Noronha V, Prabhash K, Thavamani A, Chougule A, Purandare N, Joshi A, et al. EGFR Mutations in Indian Lung Cancer Patients: Clinical Correlation and Outcome to EGFR Targeted Therapy. PLoS One 2013;8:e61561.
18Cortes-Funes H, Gomez C, Rosell R, Valero P, Garcia-Giron C, Velasco A, et al. Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients. Ann Oncol 2005;16:1081-6.
19Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-57.
20Pan-Chyr Yang, Y.S., Joseph Siu-kieAu, et al., Molecular epidemiological prospective study of EGFR mutations from Asian patients (pts) with advanced lung adenocarcinoma. J Clin Oncol 2012. 30(suppl; abstr 1534).
21Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339-46.
22Jang TW, Oak CH, Chang HK, Suo SJ, Jung MH. EGFR and KRAS mutations in patients with adenocarcinoma of the lung. Korean J Intern Med 2009;24:48-54.
23Noronha 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.
24Sun MH, Yang F, Shen L, Zhang L, Chen Y, Cai X, et al. Detection of epidermal growth factor receptor mutations in non-small-cell lung carcinoma by direct sequencing and correlations with clinicopathological characteristics and sample types. Zhonghua Bing Li Xue Za Zhi 2011;40:655-9.
25Huang SF, Liu HP, Li LH, Ku YC, Fu YN, Tsai HY, et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 2004;10:8195-203.
26Li M, Zhang Q, Liu L, Liu Z, Zhou L, Wang Z, et al. The different clinical significance of EGFR mutations in exon 19 and 21 in non-small cell lung cancer patients of China. Neoplasma 2011;58:74-81.