Background: Erlotinib (Tarceva®, Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as 70% of patients show significant tumor regression upon treatment (Santarpia et. al., 2013). However, all patients relapse due to development of acquired resistance, which in approximately half of the cases is due to a secondary mutation (T790M) in EGFR (Pao et al. 2005), and in 5-10% of cases is due to amplification of MET (Bean et al. 2007). Importantly, a significant fraction of resistant tumors are still unexplained (Lin et. al., 2012). Our aim was to identify novel resistance mutations in erlotinib-resistant subclones of the NSCLC cell line, HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively). DNA libraries of each subclone and the parental HCC827 cell line were prepared in biological duplicates using the SeqCap EZ Human Exome Library v3.0 kit and whole-exome sequencing of these (100 bp paired-end) were performed on an Illumina HiSeq 2000 platform. Using a recently developed in-house analysis pipeline the sequencing data were analyzed. The analysis pipeline includes quality control using Trim-Galore, mapping and alignment using BWA, removal of PCR duplicates using Picard Tools, followed by single nucleotide polymorphism (SNP) calling using Strelka, SomaticSniper and VarScan2 together with insertion-deletion (INDEL) calling using Strelka and VarScan2. Results: The resistant and sensitive clones exhibited a significant difference in viability over a time course of 25 days when treated with erlotinib. Importantly, the resistant clones did not acquire the T790M or other EGFR or KRAS mutations, potentiating the identification of novel resistance mechanisms in these clones. For the sensitive and the 3 resistant clones, an average 93% of the exome was sequenced to a depth of on average 25x (from 23x – 26x) of which on average 49% (from 46 – 51 %) of the exome was covered by at least 20x. During SNP and INDEL calling, we filtered all somatic mutations common between the sensitive and the resistant clones, and only analyzed mutations that were acquired by the resistant subclones compared to the parental cell line. A total of 9131 resistance-associated SNVs were identified of which 270 were identified by 2 callers and 100 were identified by all 3 callers utilized, on average in the 3 subclones. From the identified SNVs, 75, 96 and 87 were predicted to be non-synonymous in the 3 resistant subclones, respectively. Ten of these SNPs, distributed in 9 genes, were common to all 3 resistant subclones. On average 284 INDELs (from 275 – 299) were identified in the 3 resistant subclones, of which on average 6 (from 3 – 8) were found in exonic regions. However, no common INDELs were found. Conclusion: We established 3 erlotinib-resistant NSCLC subclones, which did not harbor any of the common resistance mutations, potentiating the identification of novel resistance mechanisms. By exome sequencing we identified both novel and previously reported mutation SNVs and INDELs, including 10 novel non-synonymous SNVs common to the 3 resistant subclones. These SNVs might indicate a common molecular mechanism of erlotinib resistance in NSCLC and may form the basis for future medical intervention in the large number of patients with erlotinib-resistant NSCLC.
|Publikationsdato||9. okt. 2014|
|Status||Udgivet - 9. okt. 2014|
|Begivenhed||AACR Meeting Translation of the Cancer Genome - San Francisco, USA|
Varighed: 7. feb. 2015 → 9. feb. 2015
|Konference||AACR Meeting Translation of the Cancer Genome|
|Periode||07/02/2015 → 09/02/2015|