Identification and characterization of ALK kinase splicing isoforms in non-small-cell lung cancer.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. METHODS: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations and in addition generated constructs with full-length echinoderm microtubule-associated protein-like 4 (EML4)-ALK (E13;A20) and a splicing isoform. RESULTS: Splicing isoforms of the kinase domain of ALK-including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0)-were identified in 11.1% (30 of 270 cases) of NSCLC, and these changes coexisted with ALK rearrangements, KRAS mutations, and EGFR mutations. ALK splicing isoforms were observed with full-length EML4-ALK in crizotinib-naive and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phosphotyrosine sites. Coexpression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, whereas coexpression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. CONCLUSIONS: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be nonfunctional variants of ALK.

A novel KIF5B-ALK variant in nonsmall cell lung cancer.

BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is involved frequently in chromosomal translocations, resulting in fusion genes with different partners found in various lymphoproliferative conditions. It was recently reported in nonsmall cell lung cancer (NSCLC) that the fusion protein encoded by echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) fusion gene conferred oncogenic properties. The objective of the current study was to identify other possible ALK fusion genes in NSCLC. METHODS: Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. The authors used 5' rapid amplification of complementary DNA ends to screen for potential, novel 5' fusion partners of ALK other than EML4-ALK. Reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization analyses were used to confirm the identity of 5' fusion partners. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects. RESULTS: The authors identified a novel gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot analysis of clinical tumor tissues revealed the expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to the activation of signal transducer and activator of transcription 3 and protein kinase B and to enhanced cell proliferation, migration, and invasion. CONCLUSIONS: The discovery of the novel KIF5B-ALK variant further consolidated the role of aberrant ALK signaling in lung carcinogenesis.

The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.

BACKGROUND: The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from the chromosome inversion inv(2)(p21;p23) recently was identified in nonsmall cell lung cancer (NSCLC). The authors of this study investigated the frequency, genetic and clinicopathologic profiles of EML4-ALK in Chinese patients with NSCLC. METHODS: EML4-ALK was investigated in 266 resected primary NSCLC, including adenocarcinomas (AD), lymphoepithelioma-like carcinomas, squamous cell carcinomas, mucoepidermoid carcinomas, and adenosquamous carcinomas, by reverse transcriptase-polymerase chain reaction and was verified by sequencing. EML4-ALK protein expression was studied by immunohistochemistry. RESULTS: Thirteen tumors (4.9%) had EML4-ALK comprising 4 fusion transcript variants with fusion of the variable segments from 5' EML4 to 3' ALK and with preservation of the ALK kinase domain. The most common variant consisted of 8 tumors with variant 3 that involved EML4 exon 6. The others included 2 tumors with variant 1 (exon 13), 2 tumors with variant 2 (exon 20), and 1 tumor with the novel variant 5 (exon 18). There were 11 ADs and 2 unusual carcinomas with mixed squamous and glandular components. Immunohistochemistry demonstrated diffuse ALK fusion proteins in the tumor cell cytoplasm. EML4-ALK was associated with nonsmokers (P = .009). Tumors with the fusion gene had the wild-type epidermal growth factor receptor (EGFR) (P = .001) and v-Ki-ras2/Kirsten rat sarcoma viral oncogene homolog (KRAS) genes. Patients who had EML4-ALK-positive AD had a younger median age (P = .018) compared with patients who did not have the fusion gene. CONCLUSIONS: The EML4-ALK fusion gene was present in various histologic types of NSCLC. It occurred in mutual exclusion to EGFR and KRAS mutations and was associated with nonsmokers. The authors concluded that EML4-ALK may be useful for predicting the potential response to ALK inhibitors as a therapeutic option for patients with lung cancer.