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Lung cancer has the highest cancer associated mortality rate in many countries across the world. In contrast to western populations, approximately half of lung adenocarcinoma cases in Singapore harbour activating epidermal growth factor receptor (EGFR) mutations, with preponderance for never smokers and female gender. Although EGFR tyrosine kinase inhibitors (TKIs) confer high response rates of up to 70%, drug resistance invariably ensues - most commonly through the “acquisition” of EGFR T790M mutation. While the extent and pattern of intratumoral heterogeneity (ITH) in non-small cell lung cancer (NSCLC) were recently described, these studies examined histologically and molecularly diverse cohort of patients, majority being current or ex-smokers. Here we report ITH in eight never-smoker EGFR mutant lung adenocarcinoma cases of Asian ethnicity. All eight patients had no prior treatment history and harboured an activating EGFR mutation (5 L858R , 2 exon 19 deletion, 1 exon 20 insertion). They underwent lobectomy for Stage IA, IB NSCLC. Tumors were harvested using a systematic sectoring protocol according to standard operation procedures, with tissue banked for exome sequencing, RNA-sequencing and SNP array. A total of 46 tumor sectors (at least 4 regions from each of the 8 tumors) were subject to whole exome sequencing, with matched normal samples. With an average sequencing depth of 100x, we identified 860 somatic exonic SNVs (601 being non-synonymous) and 49 indels across all samples. The median number of SNVs per patient was 112 and per sector was 49. Notably, activating EGFR mutations were identified across all tumor sectors of all but two patients (for whom it was identified in 3 of 5 and 5 of 7 sectors respectively). In addition, we did not identify the EGFR T790M mutation in any of the sequenced tumor sectors, suggesting that, this resistance mutation is not present at detectable frequencies even as a branch or subclonal event in a treatment naïve scenario. Of 20 genes that were significantly mutated across 46 individual tumor sectors, only two overlapped with published recurrently mutated genes in NSCLC. In conclusion, we show that activating EGFR mutations are ubiquitous truncal events in 6 of 8 Asian never-smoker lung adenocarcinoma – consistent with its role as a therapeutically tractable driver gene. The T790M mutation commonly associated with TKI resistance was not detected as a subclonal event in treatment naïve patients. Further, our study reveals the unique mutation spectra of Asian EGFR mutant lung adenocarcinoma, highlighting the value of multi-region sequencing in characterising the genomic architecture of defined molecular subsets of NSCLC from different ethnic backgrounds. Citation Format: Rahul Nahar, Weiwei Zhai, Angela Takano, Alexis Jiaying Khng, Xingliang Liu, Chong Hee Lim, Audrey S.M. Teo, Cheryl Xueli Chan, Apoorva Gogna, Kiat-Hon Lim, Tina Koh, Huay Mei Poh, Yin Yeng Lee, Liang He Chen, Tong Zhang, Vidhya Gomathi Krishnan, N Gopalakrishna Iyer, Pauline Ng, Wan Teck Lim, Bing Lim, Eng-Huat Tan, Daniel S.W. Tan, Axel M. Hillmer. Intratumor heterogeneity in never-smoker Asian EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-54.

Lung cancer has the highest cancer associated mortality rate in many countries across the world. In contrast to western populations, approximately half of lung adenocarcinoma cases in Singapore harbour activating epidermal growth factor receptor (EGFR) mutations, with preponderance for never smokers and female gender. Although EGFR tyrosine kinase inhibitors (TKIs) confer high response rates of up to 70%, drug resistance invariably ensues - most commonly through the “acquisition” of EGFR T790M mutation. While the extent and pattern of intratumoral heterogeneity (ITH) in non-small cell lung cancer (NSCLC) were recently described, these studies examined histologically and molecularly diverse cohort of patients, majority being current or ex-smokers. Here we report ITH in eight never-smoker EGFR mutant lung adenocarcinoma cases of Asian ethnicity. All eight patients had no prior treatment history and harboured an activating EGFR mutation (5 L858R, 2 exon 19 deletion, 1 exon 20 insertion). They underwent lobectomy for Stage IA, IB NSCLC. Tumors were harvested using a systematic sectoring protocol according to standard operation procedures, with tissue banked for exome sequencing, RNA-sequencing and SNP array. A total of 46 tumor sectors (at least 4 regions from each of the 8 tumors) were subject to whole exome sequencing, with matched normal samples. With an average sequencing depth of 100x, we identified 860 somatic exonic SNVs (601 being non-synonymous) and 49 indels across all samples. The median number of SNVs per patient was 112 and per sector was 49. Notably, activating EGFR mutations were identified across all tumor sectors of all but two patients (for whom it was identified in 3 of 5 and 5 of 7 sectors respectively). In addition, we did not identify the EGFR T790M mutation in any of the sequenced tumor sectors, suggesting that, this resistance mutation is not present at detectable frequencies even as a branch or subclonal event in a treatment naïve scenario. Of 20 genes that were significantly mutated across 46 individual tumor sectors, only two overlapped with published recurrently mutated genes in NSCLC. In conclusion, we show that activating EGFR mutations are ubiquitous truncal events in 6 of 8 Asian never-smoker lung adenocarcinoma – consistent with its role as a therapeutically tractable driver gene. The T790M mutation commonly associated with TKI resistance was not detected as a subclonal event in treatment naïve patients. Further, our study reveals the unique mutation spectra of Asian EGFR mutant lung adenocarcinoma, highlighting the value of multi-region sequencing in characterising the genomic architecture of defined molecular subsets of NSCLC from different ethnic backgrounds. Citation Format: Rahul Nahar, Weiwei Zhai, Angela Takano, Alexis Jiaying Khng, Xingliang Liu, Chong Hee Lim, Audrey S.M. Teo, Cheryl Xueli Chan, Apoorva Gogna, Kiat-Hon Lim, Tina Koh, Huay Mei Poh, Yin Yeng Lee, Liang He Chen, Tong Zhang, Vidhya Gomathi Krishnan, N Gopalakrishna Iyer, Pauline Ng, Wan Teck Lim, Bing Lim, Eng-Huat Tan, Daniel S.W. Tan, Axel M. Hillmer. Intratumor heterogeneity in never-smoker Asian EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-25.