Anaplastic Large Cell Lymphoma: A Study Of The Expression Of ALK Protein, Survivin Protein, MTOR Signal Pathway And Their Clinical Significance

[Background and Objective] Anaplastic large cell lymphoma (ALCL) is arare non-Hodgkin lymphoma of T/null lineage. In 60-85% of ALCL cases, theexpression of anaplastic lymphoma kinase (ALK) fusion protein could bedetected. Recent studies have established that ALK induces oncogenesisthrough activation of PLC-γ, JAK/STAT and PI3K/AKT pathways. In theJAK/STAT pathway, activation of STAT3 may introduce the expression ofanti-apoptosis gene bcl-X_L and survivin. Survivin is a protein that involved in theregulation of cell mitosis and the inhibition of cell apoptosis. The expression ofsurvivin is associated with the invasion and poor prognosis and thus is useful instudying the tumor growth, diagnosis, treatment and prognosis. In thePI3K/AKT pathway, Activation of AKT leads to phosphorylation ofmammalian target of rapamycin (mTOR), which regulates the translation ofmRNA through activition of the downstream eukaryotic initiation factor4E-biding protein-1 (4E-BP1) and ribosomal protein S6 kinase (pTOS6K). It hasbeen reported that the up-regulation of mTOR pathway can be found in manytumors.In the first part of our study, the expression of ALK, survivin andphosphorylated mTOR, AKT, 4E-BP1, p70S6K in 81 systemic ALCL caseswere detected by using immunohistochimistry, in order to study their clinicopathological significance of these expression and the association betweentheir expression and the prognosis of ALCL.In the second part, the expression of phosphorylated 4E-BP1, p70S6K ofmTOR pathway in ALK+cell lines detected and the cell lines were treatedwith rapamycin, in order to observe whether activation of mTOR pathwaysignaling contributes to tumor cell survival in ALK+ ALCL.[Materials and Methods] In the first part, 81 systemic ALCL cases were fromthe Department of Pathology, West China Hospital. The histopathologicalclassification used in this study was new WHO classification (2001). Paraffinblocks of these cases were cut for immunostaining. EnVision method was usedfor immunostaining and the first antibodies were ALK1, CD30, CD3, CD45RO,CD20, CD79a, EMA, granzyme B, TIA1, survivin, phospho-AKT,phospho-mTOR, phospho-4E-BP1 and phospho-p70S6K. In the second part,The phosphorylation level of4E-BP1, p70S6K in ALK+ cell lines were detectedby Western blot before and after rapamycin treatment. The cell viability wasevaluated using MTT. The cell apoptosis and cell cycle were assessed by flowcytometer.[Results] Histopathologically, there were 62 cases of common variants (76.5%),2 cases of lymphohistocytic variants (2.5%) and 17 cases of small cell variants(21%) in 81 ALCL cases. Immumohistochemically, all cases were CD30 strongpositive in which 55 cases (67.9%) with CD3 and/or CD45RO positivity,whereas 26 cases (32.1%) without CD3,CD40RO,CD20 and CD79apositivities. Cytotixic markers granzyme B and TIA-1 expressed in 65 cases(80.2%). EMA expressed in 49 cases (60.5%). The Ki-67 proliferation indexranged from 1% to 90% in 81 cases, in which 54 cases (66.7%) showed lowexpression of Ki-67 (the expression rate≤60%) and 27 cases (33.3%) showedhigh expression of Ki-67 (the expression rate＞60%). ALK fusion protein wasdetected in 51 of 81 ALCLs (63%) in which 39(76.5%) with nuclear and cytoplasmic positive products, whereas 12(23.5%) with cytoplasmic positivity.The histological variants and immunophenotypes of ALCL were not correlatedwith the expression of ALK (p＞0.05). Also, the expression pattem of ALK wasno correlation with histological variants (p＞0.05).Survivin protein was detected in all of 77 performed cases in which44(57.1%) showed low expression, whereas 33(42.9%) showed high expression.The expression of survivin was no correlation with the histological variants,immunophenotypes and the ALK expression of ALCL (p＞0.05), but wascorrelation with the Ki-67 index (p＜0.05).The expression of phospho-AKT, phospho-mTOR, phospho-4E-BP1 andphospho-p70S6K were found in all of 71 performed cases. Phospho-AKT wasexpressed in 54 cases (76.1%) with nuclear and/or cytoplasmic positivity. Theexpression of phospho-AKT was correlated with the ALK expression of ALCL(p＜0.05); Phospho-mTOR was expressed in 57 cases (80.3%) with cytoplasmicpositivity. The expression of phospho-mTOR was correlated with the ALKexpression and phospho-mTOR (p＜0.05). 64 cases (90.1%) were positive forphospho-4E-BP1 and 66 cases (93%) were positive for phospho-p70S6K withnuclear and/or plasma positivity. The expression of phospho-4E-BP1 andphospho-p70S6K were correlated with the expression of phospho-mTOR(p＜0.05).Clinically, in this group there were 61 males and 20 females (M:F=3.05:1).The age range was from 4 to 74 year and averagely 33.0±18.91 years. Thepatients with ALK expression were much younger than those without thisexpression (p＜0.05). The 5 years survival rate of patients with ALK expressionwas 50%, whereas the 2 years survival rate of patients without ALK expressionwas 20%. The 5 years survival rate of patients with low survivin expression was55%, whereas 20% of the patients with high expression. Survival curves of thesetwo groups showed a statistic significant distinguishability (p＜0.05). For the ALK positive group, patients with high survivin expression had worse prognosisthan those with low expression (p＜0.05).There were no correlation betweenthe expression of phospho-AKT, phospho-mTOR, phospho-4E-BP1 orphospho-p70S6K and the patients' age, gender or prognosis.The expression of phosphorylated 4E-BP1 and p70S6K in ALK+ cell linesKarpas299 and BaF3/ATIC-ALK were detected at higher level than in ALK-cellBaF3. The expression of phosphorylated 4E-BP1 and p70S6K in ALK+cell wasdecreased after rapamycin treatment. Rapamycin inhibited the growth of ALK+cell by inducing the cell cycle arrest and apoptosis.[Conclusion]1. The ALK positive and negative ALCL might be different entities because ofclinical difference.2. Survivin is an independent prognostic marker in ALCL and does notassociate with the expression of ALK.3. Phosphorylated AKT, mTOR, 4E-BP1 and p70S6K protein show highexpression in ALCL cases especially in ALK+ cases, which suggest thatAKT/mTOR pathway is activated in ALK+ ALCL.4. ALK chimeric protein kinase activates mTOR pathway in ALK+ ALCL celllines. Inhibiting mTOR activation could inhibit the tumorous growth of ALK+cell lines. It likely contributes to the molecular pathogenesis of ALK+ALCL.5. Rapamycin could inhibit the proliferation of ALK+ cell line. It suggests thatrapamycin might be a new drug for ALK+tumors.