Reliability analysis of exonic-breakpoint fusions identified by DNA sequencing for predicting the efficacy of targeted therapy in non-small cell lung cancer

Background: Diverse genomic breakpoints of fusions that localize to intronic, exonic, or intergenic regions have been identified by DNA next-generation sequencing (NGS), but the role of exonic breakpoints remains elusive. We investigated whether exonic-breakpoint fusions could predict matched targeted therapy efficacy in non-small cell lung cancer (NSCLC).

Methods: NSCLC samples were analyzed by DNA NGS, RNA NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization.

Results: Using DNA NGS, kinase fusions were identified in 685 of 7148 (9.6%) NSCLCs, with 74 harboring exonic-breakpoint fusions, mostly anaplastic lymphoma kinase (ALK) fusions. RNA NGS and IHC revealed that 11 of 55 (20%) exonic-breakpoint fusions generated no aberrant transcript/protein, possibly due to open reading frame disruption or different gene transcriptional orientations. Four cases of genomic-positive but RNA/protein-negative fusions were treated with matched targeted therapy, but progressive disease developed within 2 months. Nevertheless, 44 of 55 (80%) exonic-breakpoint fusions produced chimeric transcripts/proteins, possibly owing to various alternative splicing patterns, including exon skipping, alternative splice site selection, and intron retention. Most of these genomic- and RNA/protein-positive fusion cases showed a clinical response to matched targeted therapy. Particularly, there were no differences in objective response rate (P = 0.714) or median progression-free survival (P = 0.500) between intronic-breakpoint (n = 56) and exonic-breakpoint ALK fusion subtypes (n = 11) among ALK RNA/protein-validated patients who received first-line crizotinib.

Conclusions: Exonic-breakpoint fusions may generate in-frame fusion transcripts/proteins or not, and thus are unreliable for predicting the efficacy of targeted therapy, which highlights the necessity of implementing RNA or protein assays for functional validation in exonic-breakpoint fusion cases.