Genomic aberrations in crizotinib resistant lung adenocarcinoma samples by transcriptome sequencing

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using DeFuse. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing and digital droplet PCR were used to validate fusion genes and mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The validated fusion genes were also present in the biopsy before crizotinib by RT-PCR. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, in the presence of ALK mutation, it is less likely that the validated fusion genes identified in our study to be involved in crizotinib resistance. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.