Structure Aberration Of MYC Gene And Regulation Of MYC Protein By BCR Signaling In Diffuse Large B-cell Lymphoma

Part Ⅰ:Clinicopathologic analysis of diffuse large B-cell lymphoma with MYC gene structural aberrationAims:To observe structural aberrations of MYC gene and their frequencies in diffuse large B-cell lymphoma (DLBCL), to investigate the correlation of MYC protein over-expression with MYC gene aberrations, and to observe the cliniopathologic characteristics of DLBCL with MYC gene aberrations.Materials and methods:Formalin-fixed, paraffin-embedded specimens of 200 DLBCL cases from Fudan University Shanghai Cancer Center (from 2007 to 2011) were analyzed by using 10×12 tissue microarrays (TMA). Clinical information was collected through reviewing the medical records or by telephone interview. Fluorescence in situ hybridization (FISH) was employed to detect the rearrangement and copy number amplification of MYC gene. Immunostaining of CD10,BCL6, MUM1, Ki-67 and MYC was applied to 10 control cases with tonsilar reactive lymphoid hyperplasia. The germinal center B-cell (GCB)-like and non-germinal center B-cell (non-GCB)-like immunophenotypes were determined by the staining patterns of CD10, BCL6, and MUM1 according to the Hans algorithm. Results of MYC staining were denoted by IHC scoring:percent of positive cells multiply the rank of intensity (1,2,3). The highest IHC score of reactive hyperplasia of tonsil lar tissues was used as the cut-off value to define MYC protein over-expression.Results:FISH experiment was successfully conducted in 182 cases, in which 17 cases (9.3%) with MYC rearrangement were detected, with no one showing copy number amplification. The median IHC score of MYC immunostaining in reactive hyperplasia, DLBCL, MYC+and DLBCL, MYC- was 30 (from 10 to 60),120 (from 0 to 270) and 40 (from 0 to 270), respectively. The expression level of MYC protein in DLBCL, MYC+ was remarkably higher than that in DLBCL, MYC-. MYC protein over-expression was detected in a total of 84 cases (84/200,42%), among which DLBCL, MYC+ and DLBCL, MYC-accounted for 17% and 83%, respectively. There was no significant difference regarding Ki-67 proliferation index (80% versus 82.5%) as well as GCB and non-GCB distribution between DLBCL, MYC+ and DLBCL, MYC-. High IPI (IPI>2) was more frequently noted in patients with DLBCL, MYC+ compared to DLBCL, MYC-(10/17 versus 46/165,P=0.008). Based on the (R)CHOP-like treatment, DLBCL, MYC+ patients featured a notably inferior survival. There was no difference in clinicopathologic parameters and overall survival between DLBCL with MYC over-expression (DLBCL, MYC+) and DLBCL without MYC over-expression (DLBCL, MYC-).Conclusions:MYC rearrangement seems to be the prevalent form of MYC gene structural aberrations in DLBCL, which may be responsible for the over-expression of MYC protein. However, DLBCL cases with MYC over-expression are not restricted to those with MYC rearrangement, suggesting other possible mechanisms for its dysregulation. MYC rearrangement may aid in the recognition of a subset of DLBCL patients with high risk.Part Ⅱ:Regulation of MYC protein level by BCR signaling and its biological effects in DLBCLAims:To investigate the regulation of MYC protein level by BCR signaling and its biological effects in DLBCL.Materials and methods:[In vitro study] DLBCL cell lines with B-cell receptor (DLBCL, BCR+:OCI-LY8, SU-DUL-4) were used, together with DLBCL cell lines without B-cell receptor (DLBCL, BCR-:Toledo, SU-DHL-2) as the control. OCI-LY8 cell line harbored MYC gene rearrangement. pSYK, pAKT, pGSK3B and MYC in DLBCL, BCR+ and DLBCL, BCR- were detected by Western Blot and immunofluorescence assay upon BCR stimulation (anti-IgG or anti-IgM antibody) or BCR inhibition (R406). mRNA level of MYC gene was determined by real time polymerase chain reaction (RT-PCR). Apoptosis and cell cycle were detected by flow cytometry (FCM) with PI-Annexin V staining and PI staining, respectively. Cell proliferation was determined by Cell Counting Kit-8 method, [in situ study] FCM was applied for the selection of DLBCL, BCR+(experiment group) and DLBCL, BCR-(control group). Expression of pAKT and MYC in DLBCL, BCR+ and DLBCL, BCR-tissues was analyzed by Western Blot and immunohistochemistry.Results:Western Blot and immunofluorescence assays showed increased level of pSYK, pAKT, pGSK3B and MYC and decreased pMYC upon BCR stimulation in DLBCL, BCR+, and this effect could be removed by pre-incubation with R406. Alterations of MYC mRNA level were also noted when BCR was stimulated or inhibited in DLBCL, BCR+. No such effects were observed in DLBCL, BCR-, however. BCR inhibitor R406 significantly enhanced apoptotic activity, induced G1 phase arrest and inhibited cell proliferation in DLBCL,BCR+instead of DLBCL, BCR-cell lines. In situ study showed that the expression level of pSYK, pAKT and MYC in DLBCL, BCR- tissues was significantly lower than that in DLBCL, BCR+ tissues.Conclusions:The stimulation of BCR up-regulates MYC protein level via two potential mechanisms:post transcriptional editing of MYC by PI3K-AKT-GSK3B signal axis and transcriptional activation of MYC genes. BCR inhibitor inactivates BCR-PI3K-AKT-GSK3B signal axis, down-regulates MYC protein, and induces apoptosis, cell cycle and proliferation arrest. Patients with DLBCL,BCR -may not benefit from BCR inhibition therapies, due to the silence of BCR-PI3K-AKT-GSK3B-MYC signal axis.