Abstract 2251: Intra-tumor heterogeneity in non-small-cell lung cancer (NSCLC) carrying EGFR mutations
Authors: Rachiglio Anna Maria , Lambiase Matilde , Fenizia Francesca , Morabito Alessandro , Rocco Gaetano , Galetta Domenico , Ludovini Vienna , Vincenzi Bruno , Barletta Emiddio , Botti Gerardo , Normanno Nicola 
Published: July 2016

Abstract
Introduction. Driver mutations of the epidermal growth factor receptor (EGFR) are usually detected in 10% to 15% of Caucasian non-small cell lung cancer (NSCLC) patients. Evidence suggests that a fraction of EGFR mutant NSCLC might have intra-tumor heterogeneity which could potentially affect response to EGFR tyrosine kinase inhibitors (TKIs). We performed next generation sequencing (NGS)-based analysis of a large cohort of Italian EGFR mutant NSCLC to assess the level of intra-tumor heterogeneity.
Materials and methods. Genomic DNA from EGFR mutant NSCLC samples as assessed with routine diagnostic methods (N. 181; 141 tissue specimens and 40 cytology samples), was retrospectively analyzed with the Ion AmpliSeq Colon and Lung Cancer Panel using Ion Torrent semiconductor sequencing. The panel assesses over 500 somatic mutations in 22 genes at a sensitivity of 2%. Variants with allelic frequency &gt;2% and &lt;5% were confirmed by droplet digital PCR (ddPCR), if material was available.
Results. Analysis of EGFR mutant samples with NGS revealed the presence of two different hotspot EGFR mutations in 17/181 cases (9.4%). In 11 cases a EGFR sensitizing mutation and the p.T790M resistance mutation were detected; 6 tumors carried two different EGFR activating mutations at different allelic frequency. In addition, the presence of at least one variant in genes other than the EGFR was observed in 84/181 cases (46.4%), with TP53 being the most frequent mutant gene in EGFR mutant NSCLC (32/181; 17.7%). In 42 samples (23.2%) hotspot mutations were found in genes other than the EGFR, such as KRAS, NRAS, BRAF, ERBB2, PIK3CA or MET, which might cause primary resistance to EGFR targeting drugs. In 29 samples the additional mutations were at an allelic frequency &gt;5%, and in 13 at a frequency &gt;2% and &lt;5%. All mutations at low allelic frequency for which material was available were confirmed by ddPCR (N.5). The allele frequency of the driver EGFR mutation and of the additional mutations were usually different, suggesting the sub-clonal origin of some driver mutations. In particular, in 15/42 cases the EGFR mutant allele frequency was lower as compared with the additional driver mutation.
Conclusion. These preliminary data suggest that a subgroup of EGFR mutant tumors have intra-tumor heterogeneity and are likely to carry tumor clones with different molecular profile. Follow-up data are being collected to assess whether this phenomenon might affect the activity of first line EGFR TKIs.
Citation Format: Anna Maria Rachiglio, Matilde Lambiase, Francesca Fenizia, Alessandro Morabito, Gaetano Rocco, Domenico Galetta, Vienna Ludovini, Bruno Vincenzi, Emiddio Barletta, Gerardo Botti, Nicola Normanno. Intra-tumor heterogeneity in non-small-cell lung cancer (NSCLC) carrying EGFR mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2251.