A Genome-wide Gene Expression Analysis Of Diffuse Large B Cell Lymphoma

Objective:Diffuse large B cell lymphoma (Diffuse large B cell lymphoma, DLBCL) is the most common non-Hodgkin's lymphoma worldwide. It is clinical, morphologically and genetically a heterogeneous group of tumors composed of large B-cell. We applied the method of microarray to tumor classification and identify the different expression of genes in 60 patients with diffuse large B cell lymphoma. In additional, we will verify significent genes within this set of microarray identified candidate genes in the level of mRNA and protein in 60 cases of diffuse large B cell lymphoma.Methods:(1) genome-wide gene expression analysis of Diffuse large B cell lymphoma:60 patients of DLBCL with OCT-embedded frozen tissue cell lymphoma, clinical data and follow-up data are relatively complete, Affymetrix HG U133 Plus 2.0 whole genome expression chip was selected, for diffuse large B cell whole genome microarray detection. (2) different genes and related outcomes of validation with the method of quantitative real-time PCR in the levels of mRNA:60 cases of diffuse large B cell lymphoma which have analysised by of gene expression profiles, and the other 19 cases of DLBCL OCT embedded frozen tissue from Fudan University Cancer Hospital tissue bank between January 2004 to September 2009, including six cases of GCB-DLBCL and 14 cases of non-GCB-DLBCL with the method of immunohistochemical analysis and the method of quantitative real-time PCR(SYBR green I),11 genes selected by expression of gene expression profile (GEP)were detected, of which five genes was down-regulated expression in GCB-DLBCL (CCND2, BCL2, FOXP1, IL6, STAT3), including 2 genes of JAK-STAT signaling pathway (IL6, STAT3); 2 genes was up-regulated expression in GCB-DLBCL (BCL6, TP53); 4 genes without any change with the result of gene chip analysis, but they have prognosis significant in some papers (LM02, FN1, SCYA3, BCL7A),and using the method of 2 -ΔΔCt to calculated the relative expression. We will explore a new classfication of diffuse large B cell lymphomaand followed by multiple analysis of gene chips to selectable markers and a number of possible genes associated with prognosis in contrast with the clinical data. (3) Tissue microarray associated with gene chip to analysis the pathological classification of diffuse of diffuse large B cell lymphoma: 335 cases of paraffin-embedded lymph node tissue collected in January 2000 to September 2009 in the Fudan University Cancer Hospital, including 271 cases of diffuse large B cell lymphoma, unspecified (DLBCL-NOS),23 cases of follicular lymphoma (grade 3),14 csases of Burkitt lymphoma (BL), 11 cases of T-cells/histiocyte rich diffuse large B cell lymphoma (T/HR-DLBCL),6 csases of anaplastic large cell lymphoma (ALCL),10 cases of reactive lymphoid hyperplasia in. All cases with paraffin embedded and HE staining, their patterns were observed by two pathologists according to the analysis by microscopic morphology and immunohi stochemi stry.Results:Part 1:(1) clinical characteristics of 60 patients of DLBCL by gene expression profiling analysis:60 patients with DLBCL including 34 males and 26 females, male to female ratio was about 1.3:1; age from 14 years to 86 years, mean age was 56.8 years; 59 patients have the clinical stage data and 51 patients have clinical follow-up data with the Hans classification, including 20 cases of GCB,40 cases of non-GCB (including 6 cases with EB virus infection). There are no significant between the group of different clinical features, gender and clinical stage (p>0.05), EBV infection is more common in ABC-DLBCL patients (p<0.05),6 cases of DLBCL with infection with EBV with there is not any the history of clinical immunedeficiency, and the majority (five cases) of EBV infection cases the age more than 50 years, so pathological diagnosis for the elderly diffuse large B cell EBV-positive lymphoma. the major histological type is the center cell variant, immunoblastic variant, and a few anaplastic variations. (2) microarray analysis:using the method of gene expression profile,60 cases of DLBCL has been divided into three types, through the supervised clustering and unsupervised clustering and 38 genes from literature to supervised clustering for control, so 60 cases of DLBCL can be divided into:GCB-DLBCL, ABC-DLBCL and Type3-DLBCL, including 20 cases of GCB-DLBC1 type,26 cases of ABC-DLBCL-type,14 cases of Type3-DLBCL there are total of 2,170 different genes through analysis of gene expression profile. In addition, with the analysis of GO and the method pathway (KEGG)found that JAK-STAT3 signaling pathway genes were much significant in the ABC-DLBCL than in GCB-DLBCL type. moreover, gene chip expression analysis by clustering methods in level of molecule can divided the other two types:EBV+DLBCL and EBV-DLBCL separately.Part 2:(1) 79 case of DLBCL with the method of quantitative real-time PCR analysis:The DLBCL group included 46 male and 33 female, male to female ratio of about 1.4:1 in 79 cases of DLBCL, age from 14 years to 86 years, median age of the patients with DLBCL was 58 years; 78 cases of DLBCL have clinical follow-up data,69 case of DLBCL have clinical stage data. By Hans immunohistochemical classification, there are 26 cases GCB-DLBCL types,,53 cases of non-GCB-DLBCL. There was no significant between the type of gender and clinical features (p>0.05). (2) 11 genes selected from gene expression profile,we found that CCND2, FOXP1, and clinical stage has a significant difference in prognosis (p<0.05).Part 3:(1) 335 cases of lymphoma in patients with the method tissue microarray:diffuse large B cell lymphoma, unspecified (DLBCL-NOS) 271 cases,151 cases was male, female 120 cases, male to female ratio of about 1.3:1; Age distributed in 9 to 87 years, median age was 58 years; by Hans immunohistochemical classification,65 case of GCB-DLBCL,206 cases of non-GCB-DLBCL (2) Based on immunohistochemical expression of the DLBCL, there are statistical significantin of the expression of CD10,BCL-6,MUM1 and Cyclind2 with the methods of IHC (p<0.05), In addition, there are also statistical significantin of the expression of CD10, BCL-6, Cyclind2 and CD44 (p<0.05) with the methods of GEP (p<0.05). It can Improve the accuracy of pathological diagnosis when add the addition antibody of Cyclind2 and CD44.Conclusion:(1) total 60 cases of diffuse large B cell lymphomapplication with analysis by Affymetrix gene chip expression: using unsupervised clustering, supervised clustering and cluster analysis with the significant gene selected from the literature separately, DLBCL can be divided into 3 different types in the level of molecular, that is ABC-DLBCL, GCB-DLBCL and type3. (2) gene expression profile are more accurate than the level of mRNA and protein in DLBCL classification. (3) CCND2, FOXP1 have a little significant in prognosis, but they are not the independent prognostic indicators. (4) Hans immunohistochemistry in the classification based on the increase the antibody of Cyclind2 and CD44 can improve the diagnosis accuracy of DLBCL. it more likely to belong to GCB-DLBCL when Cyclind2 and CD44 protein are positive; whereas Cyclind2 and CD44 protein are negative, it more likely to consider the possible Non-GCB-DLBCL diagnosis.