ALK kinase domain is aberrantly expressed but not mutated in Chinese non-small cell lung cancer from male smokers with squamous cell carcinoma

Abstract

Anaplastic lymphoma kinase (ALK) can mediate transformation through activation by various genetic mechanisms in different tumors. In anaplastic large-cell lymphomas, t(2;5) chromosomal translocation resulting in fusion with nucleophosmin (NPM), and in non-small cell lung carcinomas (NSCLC), inv(2)(p21p23) chromosomal inversion resulting in fusion with echinoderm microtubule-associated protein-like 4 (EML4), respectively, have been shown to activate the ALK kinase domain leading to downstream signaling events that govern cell growth and proliferation. More recently, neuroblastomas have been found to contain germline and somatic ALK mutations clustering in the ALK kinase domain and some of them are predicted to be activating mutations. In adult humans, ALK is mainly expressed in neural tissues and not in the lung. The effects of aberrant ALK expression in lung cancers are also unknown. In our previous study on EML4-ALK, the fusion transcript has been found in around 5% of NSCLC but the prevalence of aberrant ALK mRNA expression and ALK kinase domain mutations have not been investigated. This study aims to investigate the expression frequency and mutational profile of ALK kinase domain in 266 NSCLC from Chinese patients including 13 cases previously found to show EML4-ALK fusion and 24 lung cancer cell lines. ALK kinase domain mRNA expression was screened by reverse transcription polymerase chain reaction and mutations were screened by direct sequencing. ALK kinase domain transcripts were found in 86 of 266 (32.3%) tumors and 11 of 24 NSCLC cell lines (45.8%). Statistical analysis showed that ALK kinase domain mRNA expression was significantly associated with male (51/132, Chi-square test, P = 0.029), ever-smoker (50/125, Chi-square test, P = 0.012) and squamous cell carcinoma (18/34, Fisher\#8217;s exact test, P = 0.036). No significant association was found with other clinicopathological characteristics including age, tumor size, differentiation, pathological stage, EGFR and KRAS mutation status. Mutations of the ALK kinase domain were not found in any sample except a silent mutation, C3633A which has recently been reported in neuroblastoma, in the cell line HCC366. All of the EML4-ALK cases showed wild-type ALK kinase domain sequence. On the other hand, two shorter splicing forms of the ALK kinase domain transcripts were found to co-exist with the normal splicing form. The two shorter splicing forms had either deletion of the entire exon 23 or exon 27 of ALK. This study shows that ALK kinase domain mutations are uncommon in NSCLC and activating mutations of ALK do not contribute to the oncogenic effect of EML4-ALK. Further investigation to verify and identify the mechanism of ALK kinase domain expression in male smokers with squamous cell carcinoma is worthwhile.