Analysis of Genetic Polymorphisms and Mutations on ALK Gene in Neuroblastoma in Taiwanese Population

Neuroblastoma (NB) is an embryonal malignancy derived from precursor cells of the sympathetic nervous system. Children suffer from the stage 4 disease display a poor 5-years survival (less than 40%) even following multi-modality treatments. For years, studies of neurblastoma show that MYCN gene amplification consistently associated with treatment failure. Recently, anaplastic lymphoma kinase (ALK) has been evolved as an important factor in carcinogenesis of neuroblastoma. Previous studies showed that NB with high ALK immune reactivity was associated with clinical outcome. Aberration in ALK, including ALK gene gain/ amplification (12.2%/ 1.5% in NB), ALK gene mutations (8% in NB) and others, makes ALK the second most commonly mutated gene in neuroblastoma. ALK is a receptor tyrosine kinase, mutations on the tyrosine kinase domain of ALK (eg. F1174, F1245, and R1275), would cause ALK overactivation and predispose to carcinogenesis. ALK and MYCN showed regulatoty loop: ALK was able to stimulate MYCN promoter via activation of ERK signaling, and MYCN was found to bind onto ALK promoter region to regulate ALK transcription. Targeting to ALK, by small molecular inhibitor, Crizotinib has been applied in the treatment of NSCLC harboring ALK translocation. In cell experiments and mice xenograft model, crzotinib can also inhibit the growth of NB cell lines and NB tumors and make ALK inhibitors a potential effective adjuvant therapy for high-risk NB. However, in the MYCN-transgenic mice harboring ALK F1174 mutation, Crizotinib couldn’t inhibit the development of NB tumor, point out that Crizotinib is ineffective in inhibit ALK with certain mutation within kinase domain. ALK inhibitors have limitation in treatment. The goal of this research is to inspect the situation on ALK gene expression and mutations in neuroblastoma in Taiwan. 61 NB tumor samples sand PBMCs from 34 healthy adults were analyzed, PCR amplification of ALK gene fragments spanning exon20 to exon25 and theirs neighboring intronic regions, followed by Sanger’s sequencing, In 61 tumor DNA samples, 7 (11.3%) were found to have heterozygotic ALK mutation: 3 F1174L mutation, 3 with F1245 mutation, 1 with A1274T mutation, but there is no R1275 mutation found. Moreover, we find 6 SNPs in exon20 to 25 of ALK gene, 3 SNPs are synonymous variants which located in exon20, 21 and23; the other 3 SNPs are intron variants, located in intron20 and 25. The allelic distribution of the ALK SNPs is not significantly different between NB tumors and normal PBMCs. We find that ALK mutation is associated with adverse clinical features (advanced stage, high risk, poorly differentiation, and MYCN amplification), and inferior survival (EFS: HR 4.2, p=0.0021; OS:HR 5.6, p=0.0015). Subset analysis of advanced stage, high-risk NB showed that patient harboring ALK mutation displayed a shorter 5-year survival. Relative mRNA expression of ALK and MYCN were determinate by q-RT-PCR. ALK expression was found positively correlated with MYCN gene expression (r=0.5831, p<0.0001). High ALK expression was associated with undifferentiated/ poorly differentiated NB (p=0.0012), but not with ALK mutation, ALK genetic polymorphism, nor other clinical features. We found ALK high expression correlated with worse patients’ survival (EFS: HR 2.3, p=0.0600; OS: HR 4.8, p=0.0240). In Summary, we obtain information in our research: (1) ALK gene mutation occurs in NB with adverse clinical features. Further, ALK mutation is an important factor that cause faster disease progression in advanced stage and/or high-risk NB. Detection of ALK mutational spectrum in early diagnosis, could predict patients’ prognosis and choice suitable ALK inhibitor as adjuvant therapy in neuroblastoma. (2) ALK high expression could exist in ALK WT or mutant+ tumors. ALK high expression was not necessary found in ALKMut+ tumor. (3) ALK expression is highly correlated with MYCN expression in NB tumor, (4) High ALK expression in NB tumor predicts poor clinical outcomes. (5) ALK F1174L (known Crizotinib-resistant) is a prevalent type of mutation in NB in Taiwan. Screening ALK mutation to evaluate the use of ALK inhibitor as therapentics is very important for NB patients in Taiwan.