The Correlation Of GC-type DLBCL With Acquired Somatic Mutations In Non Coding Region Of BCL6

Diffuse large B-cell lymphoma(DLBCL) is one of the most common non-Hodgkin's Lymphoma(NHL) subtypes according to recent WHO classification of hematopoietic and lymphoid tumors(2008).However,DLBCL is characterized clinically by its heterogeneity,including the variable response to therapy and the highly variable prognosis,so the heterogeneity of DLBCL has become a hot spot focused by reasearchers.Ig gene rearrangment and SHM are not only two biological steps of germinal center reaction,but also risk factors of lymphomagenesis,the latter could contribute to many genetic abnormalities,including structural alterations(eg,balanced and unbalanced translocations),numerical changes(eg,aneuploidy),and single DNA point mutations,which disrupt the stability of B cell,lead to proliferation of B Cell or apoptosis blocked.Somatic hypermutation is a critical mechanism for the generation of diversity in the antibody repertoire.This process takes place in antigen-experienced B cells within the germinal center(GC),presumably the so-called centroblasts,and specifically targets rearranged Ig genes.During the germinal center reaction,the IgV-associated somatic hypermutation contribute to the IG molecular with high affinity.This process is easy to be disrupt by internal and external factors.Despite its high specificity for rearranged Ig genes,the somatic hypermutation mechanism was also shown to target non-Ig gene,such as the BCL-6,PIM1,c-MYC,PAX5,RhoH/TTF,and it targets BCL-6 in the normal GCB cells as well.BCL-6 is very special as a non-Ig gene but displays features of the IgV-associated somatic hypermutation mechanism,although the precise mechanism of BCL-6 SHM and its significance are poorly understood.In order to investigate BCL-6 SHM and its significance of tumorigenesis,we study the correlation between the mutation of 5' noncoding region of BCL-6 gene and GCB subtype in diffuse large B-cell lymphoma,and subtyping for the DLBCL cases.According to the WHO classification(2008),Gene expression profiling of diffuse large B-cell lymphoma(DLBCL) has revealed biologically and prognostically distinct subgroups:germinal center B-cell-like(GCB),activated B-cell-like(ABC).but in the condition of routine work,we can use immunohistochemical stain to divide 2 major subgroups of DLBCL.But one has to keep in mind that the results of the immunohistochemical approach are highly variable,for the precision of method is limitied by both internal and external factors.In order to overcome it,we need a marker to improve the classification.The GEP studies reveals that t(14;18) is seen most often in the germinal center subtype of DLBCL and is generally associated with a more favorable prognosis.Huang et al reported that the t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile.Therefore,we examined our DLBCL patients with both t(14;18) detection and IHC to determine the subtype,and further to analyse the correlation between the mutation of 5' noncoding region of BCL-6 gene and GCB subtype in diffuse large B-cell lymphoma.The t(14;18)(q32;q21) is believed to play a crucial role in the pathogenesis of follicular lymphoma because it deregulates the expression of the antiapoptotic gene BCL2 by bringing it into proximity of the immunoglobulin heavy chain gene enhancer. The t(14;18)(q32;q21),with its associated BCL2 gene rearrangement,has also been detected in up to one third of cases of primary diffuse large B-cell lymphoma(DLBCL). Conventional cytogenetic karyotyping,FISH,and polymerase chain reaction techniques can reveal this genetic rearrangement,and nest-PCR is highly sensitive,easy to perform,and thus well suited for routine analysis of t(14;18) translocation.Our study included samples from 60 patients with DLBCL for two parts of research.In the first part,we optimized the molecular typing of DLBCL,t(14;18) detection and immunohistochemical staining were performed on samples from 60 cases with DLBCL,then divided them into GCB and non-GCB subtypes.12 of 60 expressed CD10,40 of 60 expressed BCL-6,and 43 of 60 expressed MUM1,cases were considered to be GC derived according to criteria of Alizadeh et al,about 16 of 60 of our DLBCL cases could be divided as GCB preliminarily.20 of 60 cases occured t(14;18) translocation,7 in major breakpoint region,13 in minor cluster region.Using the minimally acceptable criteria,26 of 60 cases were probably germinal centre derived.In the second part,Polymerase chain reaction(PCR),single-strand conformation polymorphism(SSCP) and direct DNA sequencing were used to identify mutations in the 5' noncoding region of the BCL-6 gene.BCL6 mutations were detected in 20%of DLBCL cases(12/60),with a significantly higher frequency in the GC subgroups (34.6%) than in the non-GC subgroups(8.8%).BCL6 mutations in +363 and +469 sites happened frequently.Clear correlation between BCL6 mutation and GC subgroup was observed.In conclusion,a combination of immunohistochemical staining and t(14;18) translocation detection improved the molecular typing of DLBCL,it is significantly important for the prognosis,and the result need to be comfirmed by further studies.Our study also suggested that BCL6 mutations were detected in 20%of DLBCL cases,with a significantly higher frequency in the GCB subgroups than in the ABC subgroup.It is possible that BCL6 is important in the initial phase of transformation of DLBCL.BCL6 mutations may exert its oncogenic effects in the occurrence and development of DLBCL,The estabilishment of methods provide basis for further investigation.