Mechanism Research Of ALK Effects On Prognosis In Primary Systemic Anaplastic Large Cell Lymphoma

BACKGROUND & OBJECTIVEAnaplastic large cell lymphoma(ALCL) is a subtype of T-cell non-Hodgkin's lymphoma characterized by the expression of CD30 antigen(also known as tumor necrosis factor).Primary systemic ALCL has a peak incidence in childhood, accounting for approximately 40%of NHL cases diagnosed in pediatric patients, whereas it accounts for＜5%of NHL in adults.The common type is composed of large,pleomorphic,atypical tumor cells with an abundant grey-blue cytoplasm.Their nuclei are large,horseshoe- or kidney-shaped,and sometimes have pseudonuclear inclusions(so-called doughnut cells).About half of these lymphomas have the t(2;5)(p23;q35) translocation and express a hybrid protein comprised of the anaplastic lymphoma kinase(ALK) and the nucleolar phosphoprotein nucleophosminutes (NPM).Additional translocations are rare in which ALK is fused to other partners. Half of ALCL do not express ALK or other recurrent translocations.Full-length ALK have been shown as novel,putative receptor tyrosine kinase (RTK) of the insulin receptor family,encoded by chromosome 2p23,and comprised of extracellular,transmembrane,and cytoplasmic domain.The most closely related kinase to ALK is leukocyte tyrosine kinase(LTK),which lacks a sequence corresponding to the amino terminal half of the ALK extracellular domain.The extracellular domain and protein kinase domain of LTK possess 50%and 78%amino acid identity,respectively,to the corresponding regions of ALK.These two kinases comprise a new subfamily of the insulin receptor family.Although ALK was originally identified in hematopoietic neoplasm anaplastic large cell lymphomas (ALCL),ALK expression was never detected in normal hematopoietic tissue. However ALK is expressed preferentially in neurons of the central and peripheral nervous systems at late embryonic stages.ALK function in normal tissue is still an enigma.In ALCL the cytoplasmic domain of ALK is fused to the amino terminal half of nucleophosmin(NPM) to generate a fusion gene product,p80NPM-ALK(Morris et al.,1994).NPM is a multifunctional protein that plays a role in protein shuttling between the cytoplasm and nucleus(Borer,at al.,1989) and in cell cycle-dependent entrosome duplication(Okuda et al.,2000).Previous studies have shown that the NPM portion of p80NPM-ALK mediates oligomerization.The t(2;5)(p23;q35) translocation creates the fusion gene NPM-ALK(p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma(ALCL).NPM-ALK acts as a docking protein for a signalosome which in turn couples the fusion protein to signalling pathways with mitogenic,antiapoptotic and possibly DNA repair capabilities.NPM-ALK induces oncogenic signals by autophosphorylation at multiple Tyr residues,followed by recruitment of a signalosome:an ensemble of proteins containing docking SH2 or PTB domains that couples the fusion protein to downstream signalling pathways. Investigations based largely on cell line data suggest that the NPM-ALK signalosome stimulates pathways regulating mitogenesis and survival,involving the phosphoinositide 3-kinase(PI 3-kinase) and Ras/MAP kinase pathways,and the STAT family of transcription factors. ALK can function as an oncogene via dysregulation of its tyrosine kinase regions in NPM-ALK(p80) much as bcr-abl tyrosine kinase.Tyrosine kinase inhibitors have been considered as antineoplastic agents.The recent studies demonstrated utilization of signal transduction inhibition as shown by STI-571,which selectively inhibits the activation of the bcr-abl tyrosine kinase for successful treatment of chronic myelogenous leukemia(CML).Therefore much attention was paid to ALK in ALCL although it is rare in human carcinoma.ALK in ALCL is known as an oncogenic protein.However,ALK protein expression is an independent predictor of survival and serves as a useful biologic marker of a specific disease entity within the spectrum of ALCL.Patients with ALK(+)ALCL are reported to have a better prognosis than patients with ALK(-)ALCL.Because the mechanisms for this survival difference are unknown and the prognostic factors that are useful for predicting survival remain unclear,we investigated the hypothesis that pathways of expansion,invasion,adhesion or apoptosis may be involved.We therefore collected the clinical features and survival time and attempted to identify the clinical and pathological factors of prognostic importance and then we assessed expression levels of the expansion,invasion, adhesion and anti-apoptotic proteins in ALCL tissue and identify them in Karpas 299 cell line using tyrosine kinase inhibitors to find a possible mechanism pathway of ALK effects to the prognosis in Anaplastic Large cell lymphoma.METHODS1 the clinical features and survival time follow-up in ALCL and the relationship between ALK and the prognosisThe study group included 34 ALCLs accessioned at Southern Medical University affiliated Nanfang Hospital,Fouzhou General Hospital and Shenzhen Nanshan Hospital between 1999 and 2006..The diagnosis of ALCL was based on criteria specified by the WHO classification.Cases of primary cutaneous ALCL were excluded.4 patients were totally lost contact information and lost to follow-up.FISH and immunocytochemistry was used to evaluate NPM-ALK in ALCL tissues on genome and protein levels,respectively.Survival analysis was based on life tables and Kaplan-Meier.Statistical comparison of ALK and other clinical parameters was based on the crosstabs analysis (Microsoft SPSS 11.5).2 screening possible pathway through which ALK may effect on prognosis in ALCLImmunocytochemistry was used to screening possible pathway through which ALK may effect on prognosis in ALCL.Immunofluorescence was used to analyze co-localization of ALK and Hsp90αproteins.PCR-SSCP analysis was used to detect P53 mutation of 5,6,7,8 exons.Survival analysis was based on life tables and Kaplan-Meier.Statistical comparison of ALK and other intracellular pathway parameters was based on the crosstabs analysis(Microsoft SPSS 11.5).3 identify the pathway found in the 2 chapter in Karpas 299 cell lineTyrosine kinase inhibitor Herbimycin A was used to inhibit the ALK tyrosine kinase activity in Karpas 299.Immunocytochemistry,trypan blue and Flow cytometry was used to detect the tyrosine kinase activity,apoptosis,and apoptosis-related factors.P53 protein was protected by protease inhibitor PMSF. PCR-SSCP analysis was used to detect P53 mutation of 5,6,7,8 exons in cells. Western Blotting electrophoresis was used to semi-quantitative analysis.RESULTS1 Relationship between ALK and clinical condition and survival time:Patients with ALK(+)ALCL had a better prognosis than patients with ALK(-)ALCL.Clinical parameters related both ALK and prognosis are:age;CR;clinical tumor stage;IPI.ALK(+)ALCL patients were younger than ALK(-)ALCL patients that means better basic condition presented in ALK(+)ALCL patients.ALK(+)ALCL patients had earlier clinical tumor stage and lower IPI than ALK(-)ALCL patients that means inferior invasion power presented in ALK(+)ALCL patients.ALK(+)ALCL patients had higher CR rate than ALK(-)ALCL patients that means better reactiveness to chemical therapy presented in ALK(+)ALCL patients.2 Pathway screening related to ALK and prognosis:After the screening of the possible pathway ALK effects on prognosis in ALCL tissue,CD4,CD5,CD44,MMP2,TIMP1,nm23,CD56,survivin,Bax,bcl-2,P53,MDM2,Hsp90α,cytochrome C and Cleaved Caspase3 were selected in our research.Hsp90αwas co-localized with ALK in ALK(+) ALCL tissue.MDM2 over expression and ALK expression did not have significant difference.P53 did not mutate in all ALK(+) ALCL tissues and mutated in 2 ALK(-) cases of ALCL.Bax expression and ALK expression did not have significant difference.Bcl-2 negative expression and ALK expression did not have significant difference.Cytochrome C and cleaved caspase3 expression and ALK expression did not have significant difference.3 Pathway reaction to Herbimycin A in Karpas 299Cell tyrosine kinase activity of ALK decreased and apoptosis increased after Herbimycin A action in Karpas 299 cells.Cell cycle was arrested in G1/S.ALK, Hsp90αand MDM2 expression decreased after Herbimycin A action.Wild type P53 protein,Bax,Cytochrome C and cleaved caspase3 expression all increased after Herbimycin A action.4 Pathway reaction to Herbimycin A in Karpas 299 with ALK downregulation by AS RNA interferencePathway reaction to Herbimycin A in Karpas 299 with ALK downregulation by AS RNA interference was weaker than that in Karpas 299.Cell tyrosine kinase activity of ALK decreased and apoptosis increased after Herbimycin A action but were in lower degree than that in Karpas 299.CONCLUSIONALK(+) ALCL has a better prognosis than ALK(-) ALCL.The clinical prognosis factors were age,;CR;clinical tumor stage;IPI.There may be such a pathway which induces the tumorigenesis of ALCL but probably be reason of the well prognosis of ALK(+) ALCL:Chromosome translocation t(2;5) lets hybrid protein NPM-ALK over expression.Hsp90 protects ALK and ALK can execute its receptor tyrosine kinase activity and stimulates Ras pathway.Ras pathway can enhances MDM2 expression and MDM2 can inhibit wild type P53 protein.P53 protein is inhibited by overexpression MDM2 but still has tiny effect on its downstream factors.When ALK tyrosine kinase activity was inhibited by Herbimycin A,Hsp90 loses its client and expresses weaker.ALK's effect on ras pathway weakened and MDM2 expression is downregulated.Wild type P53 protein expresses after MDM2 expression is downregulated and effect of P53 on its downstream factors began manifest.Bax protein is upregulated by P53 and promotes Cytochrome C releasing from chondrosome.Caspase 3 is stimulated and apoptosis happens.