Molecular characteristics and clinical outcomes of complex ALK rearrangements identified by next-generation sequencing in non-small cell lung cancers.

ABSTRACT

BACKGROUND: Complex kinase rearrangement, a mutational process involving one or two chromosomes with clustered rearrangement breakpoints, interferes with the accurate detection of kinase fusions by DNA-based next-generation sequencing (NGS). We investigated the characteristics of complex ALK rearrangements in non-small cell lung cancers using multiple molecular tests. METHODS: Samples of non-small cell lung cancer patients were analyzed by targeted-capture DNA-based NGS with probes tilling the selected intronic regions of fusion partner genes, RNA-based NGS, RT-PCR, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS: In a large cohort of 6576 non-small cell lung cancer patients, 343 (5.2%) cases harboring ALK rearrangements were identified. Fourteen cases with complex ALK rearrangements were identified by DNA-based NGS and classified into three types by integrating various genomic features, including intergenic (n = 3), intragenic (n = 5) and "bridge joint" rearrangements (n = 6). All thirteen cases with sufficient samples actually expressed canonical EML4-ALK fusion transcripts confirmed by RNA-based NGS. Besides, positive ALK IHC was detected in 13 of 13 cases, and 9 of 11 cases were positive in FISH testing. Patients with complex ALK rearrangements who received ALK inhibitors treatment (n = 6), showed no difference in progression-free survival (PFS) compared with patients with canonical ALK fusions n = 36, P = 0.9291). CONCLUSIONS: This study firstly reveals the molecular characteristics and clinical outcomes of complex ALK rearrangements in NSCLC, sensitive to ALK inhibitors treatment, and highlights the importance of utilizing probes tilling the selected intronic regions of fusion partner genes in DNA-based NGS for accurate fusion detection. RNA and protein level assay may be critical in validating the function of complex ALK rearrangements in clinical practice for optimal treatment decision.