Abstract

Constitutive expression of the NPM-ALK tyrosine kinase is a key oncogenic event in the development of anaplastic large cell lymphoma (ALCL) harboring the t(2;5)(p23;q35). The identity and function of a limited number of downstream molecules and pathways important for the oncogenic activity of NPM-ALK are known. However, the global molecular and cellular consequences of NPM-ALK overexpression are largely unknown. In this study, we used cDNA microarrays composed of 9200 unique sequences to compare the expression profiles of pediatric (age <19) NPM-ALK+ ALCLs (n=14) obtained from the Cooperative Human Tissue Network (CHTN) with pediatric NPM-ALK- large cell lymphomas (LCL) (n=10). In addition, to directly determine the transcriptional effects of NPM-ALK overexpression, we analyzed Jurkat cells transfected with a plasmid containing the NPM-ALK chimeric gene (Jurkat/NPM-ALK). The ectopic, forced overexpression of NPM-ALK was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis, which showed that the transfected cells express comparable levels of NPM-ALK to that observed in the ALCL-derived cell lines SUDHL-1 and Karpas 299. The transcriptional profile of Jurkat/NPM-ALK cells was compared to that of Jurkat/LacZ cells. Microarray analysis of NPM-ALK+ ALCL tumors demonstrated 156 genes and ESTs which were differentially expressed by greater than 1.5-fold using a cut-off of p <0.05 compared to NPM-ALK- LCLs. P-values were obtained using the unequal variance t-test. Of these, 62 genes were overexpressed and 94 genes were underexpressed in the NPM-ALK+ ALCLs compared with NPM-ALK- LCLs. Comparison of the transcription profiles of Jurkat/NPM-ALK cells and Jurkat/LacZ cells showed 530 genes (341 upregulated and 189 downregulated) that were differentially expressed by greater than 2-fold. We identified genes that belong to diverse cellular functional groups such as cytokine receptors, kinases, phosphatases, and genes involved in ubiquitin regulation, adhesion and invasion, apoptosis, proliferation and T-cell activation. A subset of these genes were also expressed in NPM-ALK+ ALCL tumors. Genes that were consistently overexpressed in NPM-ALK+ ALCLs compared with NPM-ALK- LCLs included TIMP-1 (p< 0.001), IL-2Rγ (p<0.034) IL-1R2 (p<0.01) and CYP1B1 (p<0.00077). Real-time quantitative RT-PCR was performed to validate a subset of the differentially expressed genes in pediatric ALCL tissue samples as well as the transfected Jurkat cells. Additionally, immunohistochemical analysis of tissue microarrays consisting of 17 cases of ALK+ ALCLs not included in the cDNA microarray analysis was also performed to validate the cDNA microarray findings. This study reveals for the first time, the global transcriptional consequences of NPM-ALK overexpression and provides further insights into the multiple pathogenetic cellular pathways influenced by transformation in NPM-ALK+ ALCL.

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