Prevalence and profile of EGFR mutations in NSCLC: Insights from an Eastern Indian cohort

INTRODUCTION

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for more than 2.5 million new cases and 1.8 million deaths in 2022, as reported by the GLOBOCAN cancer statistics.^[1-3] Histologically, lung cancer is classified into two major types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC comprising ~85% of all cases. NSCLC includes squamous cell carcinoma (SCC), adenocarcinoma (ADC), large cell carcinoma (LCC), and aden-squamous carcinoma (ASC).^[4]

While genetic predispositions play a key role in lung cancer development, environmental exposures such as tobacco smoke, air pollution, occupational carcinogens, and infections further contribute to oncogenic mutations.^[4,5] Advances in molecular oncology have uncovered several key signalling pathways involved in tumour progression, one of the most significant being the epidermal growth factor receptor (EGFR) pathway.

EGFR, a member of the HER/ErbB family of transmembrane receptor tyrosine kinases encoded on chromosome 7, includes HER1 (ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4).^[6,7] Structurally, EGFR comprises extracellular, transmembrane, and intracellular domains.^[8] Upon ligand binding, EGFR undergoes autophosphorylation, activating downstream signalling cascades that promote cell proliferation and inhibit apoptosis. Mutations in the EGFR gene result in overactivation of these pathways, contributing to tumorigenesis, particularly in NSCLC.

Importantly, activating mutations in the EGFR gene, particularly in exons 18–21, are predictive biomarkers for responsiveness to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, which improve outcomes in advanced NSCLC.^[9] The most common mutations include deletions in exon 19 (e.g., delE746-A750) and the L858R point mutation in exon 21.^[10] These mutations are significantly more frequent in adenocarcinoma (35–45%) than in SCC (<5%) and in non-smokers and Asian populations.

EGFR-TKIs, as small-molecule inhibitors, competitively bind to the ATP-binding site of the EGFR tyrosine kinase domain, thereby blocking phosphorylation and downstream oncogenic signalling.^[11] As such, molecular testing for EGFR mutations is crucial for guiding targeted therapy in NSCLC.^[12,13]

Our study delves into the prevalence of EGFR gene mutations and their profile in NSCLC and supports their routine assessment to optimise treatment strategies.

MATERIAL AND METHODS

This observational cross-sectional study was conducted in the Department of Pathology at a tertiary care centre in Odisha over a period of 2 years, from August 2021 to September 2023. Comprehensive data, including patient history, clinical examination findings, and radiological investigations (wherever available), were collected and analysed. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks representing the most diagnostic areas of lung tumours were selected for immunohistochemistry (IHC) and molecular analysis. A total of 84 NSCLC cases classified according to the latest WHO Classification of Tumours (5th Edition), confirmed by histopathology using CT/ USG-guided biopsies and IHC, were included in the study. Cases of small-cell lung carcinoma and metastatic tumours to the lung and pleura were excluded from the study. Ethical clearance was obtained from the Institutional Ethics Committee (IEC No.: 588)

DNA was extracted from FFPE tissue sections using the cobas® DNA Sample Preparation Kit, followed by EGFR mutation analysis using the cobas® EGFR Mutation Test v2. The test targeted mutations in the EGFR gene regions, including:

• Exon 18: G719X

• Exon 19: Deletions (Ex19Del)

• Exon 20: S768I, T790M

• Exon 21: L858R, L861Q

RESULTS

Out of 84 cases of NSCLC, adenocarcinoma was the most common histological subtype, accounting for 60.7% (51 cases), followed by SCC in 38.1% (32 cases), and a single case (1.2%) of large cell carcinoma. The cohort comprised 62 males (73.8%) and 22 females (26.2%). Most male patients (62.9%) were aged >60 years, whereas the majority of female patients (63.6%) were in the 41–60-year age group. Cough was the most frequently reported symptom (77.3%), followed by breathlessness (69%). A history of smoking was present in 64.5% of male patients, while 90.9% of female patients were non-smokers. Anatomically, peripheral lung lesions were predominantly adenocarcinomas (90.9%) as shown in Figures 1, 2a-c, whereas central lesions were mainly SCC (88.9%) [Table 1, Figures 3a-c]. EGFR mutations were identified in 21.4% (18/84) of all NSCLC cases as shown in Table 2, with a markedly higher frequency in adenocarcinomas (31.4%) compared to other NSCLC subtypes (6.3%), a difference that was statistically significant (p = 0.0057) [Table 3]. EGFR mutations were more commonly observed in females and non-smokers. Among the 18 EGFR-positive cases, exon 19 mutations were most frequent (11 cases), followed by exon 21 (8 cases) and exon 18 (6 cases) [Figures 4a-d, Table 4]. Double mutations involving combinations of exons 18, 19, and 21 were detected in 7 cases [Figures 5a and b]. These findings highlight the predominance of adenocarcinoma in NSCLC, its peripheral localisation, and the strong association of EGFR mutations with non-smoking status, sex, and adenocarcinoma histology.

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Figure 1: CT scan of the thorax showing a left lower lobe lung mass with a bronchus cut-off sign positive in adenocarcinoma. CT: Computed tomography.

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Figure 2: (a) Microphotograph showing squamous cell carcinoma of the lung (Haematoxylin & eosin, 400X), (b) microphotograph showing positive nuclear staining for P40 in squamous cell carcinoma of lung (Immunohistochemistry, 400X), (c) microphotograph showing negative nuclear staining for TTF1 in squamous cell carcinoma of the lung (Immunohistochemistry, 400X). TTF1: Thyroid transcription factor 1.

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Figure 3: (a) Microphotograph showing adenocarcinoma of the lung (Haematoxylin & eosin, 400X), (b) microphotograph showing positive nuclear staining for TTF1 in adenocarcinoma of the lung (Immunohistochemistry, 400X), (c) microphotograph showing negative nuclear staining for P40 in adenocarcinoma of lung (Immunohistochemistry, 400X). TTF1: Thyroid transcription factor 1.

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Figure 4: (a) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 18, 19 and 21 in the positive control, (b) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 18 in the test sample, (c) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 19 in the test sample, (d) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 21 in the test sample. EGFR: Epidermal growth factor receptor. RTPCR: Reverse transcription polymerase chain reaction, RFU: Relative fluorescence units.

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Figure 5: (a) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 18, 19, and 21 in the test sample, (b) RTPCR positive amplification graphs detecting EGFR mutations in exon sequence 18, 19, and 21 in the multiple samples. EGFR: Epidermal growth factor receptor, RTPCR: Reverse transcription polymerase chain reaction, RFU: Relative fluorescence units.

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DISCUSSION

Lung cancer remains the leading cause of cancer-related morbidity and mortality globally, with ~20 million new cases and 1.7 million deaths annually.^[1] In India, it accounts for 6.9% of all new cancers and 9.3% of cancer-related deaths.^[2]

The EGFR gene encodes a transmembrane tyrosine kinase receptor whose mutations result in abnormal signalling and oncogenesis, particularly in NSCLCs. EGFR mutation analysis plays a pivotal role in diagnosis and targeted therapy, especially in adenocarcinomas. These mutations are predictive biomarkers for responsiveness to TKIs, improving prognosis when detected early.^[14]

Due to the low frequency of mutant DNA (<5%) in NSCLC FFPE samples, conventional DNA sequencing may fail to detect mutations, necessitating highly sensitive mutation detection methods.^[15] Our study used a sensitive assay to detect EGFR mutations, confirming the necessity for optimised thermocycling conditions for FFPE specimens.

In our cohort, EGFR mutations were observed in 31.4% of adenocarcinomas and only 6.3% of SCCs, aligning with findings by Sahnane et al.^[16] and Gahr et al.,^[17] who also reported higher mutation prevalence in adenocarcinoma. The most frequent mutations in our study were deletions in exon 19 and point mutations in exon 21, consistent with findings from Unal et al.^[18] and Ichihara et al.^[19]

Gender and smoking status had significant correlations with EGFR positivity. We found a higher mutation frequency in females (31.8%) and non-smokers (25%), with statistical significance (p <0.05). Similar observations were made by Yoon et al,^[20] Sun et al.^[21] and Douillard et al. as shown in Table 5,^[22] who reported greater EGFR mutation prevalence in females and non-smokers.

Our findings also support the significance of mutation location: exon 19 and exon 21 were most frequently affected, consistent with global data.^[20,23,24] Studies by Saiyaros et al.^[4] and Yoon et al.^[20] demonstrated similar patterns, reinforcing the clinical relevance of these exons in guiding treatment.

TAKE HOME MESSAGE

NSCLC, comprising 85% of lung tumours, remains the leading cause of cancer mortality despite advances in treatment. This study highlights the clinical significance of detecting EGFR mutations in NSCLC, particularly in adenocarcinoma subtypes. EGFR mutations were predominantly observed in adenocarcinoma cases compared to squamous cell carcinoma, with a notable association among female patients and non-smokers. The most frequent mutations were found in exons 19 and 21, consistent with previous literature. Our findings reinforce the value of routine EGFR mutation testing in NSCLC cases, especially adenocarcinoma, to guide the use of EGFR-TKIs and improve patient outcomes.