BCL2 Translocation Defines a Unique Tumor Subset within the Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma
2004; Elsevier BV; Volume: 165; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63284-1
ISSN1525-2191
AutoresJaveed Iqbal, Warren G. Sanger, Douglas E. Horsman, Andreas Rosenwald, Diane L. Pickering, Bhavana J. Davé, Sandeep S. Davé, Xiao Li, Kajia Cao, Quiming Zhu, Simon Sherman, Christine P. Hans, Dennis D. Weisenburger, Timothy C. Greiner, Randy D. Gascoyne, German Ott, H. K. Müller-Hermelink, Jan Delabie, Rita M. Braziel, Elaine S. Jaffe, Elı́as Campo, James C. Lynch, Joseph M. Connors, Julie M. Vose, Jamés O. Armitage, Thomas M. Grogan, Louis M. Staudt, Wing C. Chan,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoGene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup. Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup. Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B cells with an annual incidence of ∼25,000 cases in the United States. DLBCL is a heterogeneous entity both clinically and morphologically. We have recently shown by gene expression profiling that DLBCL can be classified into two major subgroups.1Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PO Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8205) Google Scholar The germinal center B-cell-like (GCB) subgroup expresses genes characteristic of normal GC B cells and is associated with a good outcome after multiagent chemotherapy, whereas the activated B-cell-like (ABC) subgroup expresses genes characteristic of activated blood B cells and is associated with a poor clinical outcome. Nonetheless, considerable molecular heterogeneity exists within each subgroup. A small number of DLBCL cases are unclassifiable and do not express the GCB or ABC signature genes at a high level.2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3263) Google Scholar More recently, primary mediastinal large B-cell lymphoma (PMBL) has been identified as a distinct subgroup of DLBCL that can be distinguished by gene expression profiling from GCB- and ABC-DLBCL.3Rosenwald A Wright G Leroy K Yu X Gaulard P Gascoyne RD Chan WC Zhao T Haioun C Greiner TC Weisenburger DD Lynch JC Vose J Armitage JO Smeland EB Kvaloy S Holte H Delabie J Campo E Montserrat E Lopez-Guillermo A Ott G Muller-Hermelink HK Connors JM Braziel R Grogan TM Fisher RI Miller TP LeBlanc M Chiorazzi M Zhao H Yang L Powell J Wilson WH Jaffe ES Simon R Klausner RD Staudt LM Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma.J Exp Med. 2003; 198: 851-862Crossref PubMed Scopus (931) Google Scholar, 4Savage KJ Monti S Kutok JL Cattoretti G Neuberg D De Leval L Kurtin P Dal Cin P Ladd C Feuerhake F Aguiar RC Li S Salles G Berger F Jing W Pinkus GS Habermann T Dalla-Favera R Harris NL Aster JC Golub TR Shipp MA The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin's lymphoma.Blood. 2003; 102: 3871-3879Crossref PubMed Scopus (738) Google Scholar The t(14;18)(q32;q21) is a characteristic feature of follicular lymphoma and is considered to be the initiating event in lymphomagenesis. The t(14;18) is the result of an error during the process of VDJ recombination, leading to deregulation of the expression of the anti-apoptotic gene BCL2 by bringing it into proximity of the immunoglobulin heavy chain (IgH) gene enhancer.5Bakhshi A Jensen JP Goldman P Wright JJ McBride OW Epstein AL Korsmeyer SJ Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18.Cell. 1985; 41: 899-906Abstract Full Text PDF PubMed Scopus (1010) Google Scholar, 6Tsujimoto Y Cossman J Jaffe E Croce CM Involvement of the bcl-2 gene in human follicular lymphoma.Science. 1985; 228: 1440-1443Crossref PubMed Scopus (1620) Google Scholar, 7Magrath I Molecular basis of lymphomagenesis.Cancer Res. 1992; 52: 5529s-5540sPubMed Google Scholar, 8Yang E Korsmeyer SJ Molecular thanatopsis: a discourse on the BCL2 family and cell death.Blood. 1996; 88: 386-401Crossref PubMed Google Scholar In our initial study of 35 cases of DLBCL, we correlated BCL2 translocation data with gene expression profiles and showed that the t(14;18) defines a unique subset of DLBCL within the GCB subgroup.9Huang JZ Sanger WG Greiner TC Staudt LM Weisenburger DD Pickering DL Lynch JC Armitage JO Warnke RA Alizadeh AA Lossos IS Levy R Chan WC The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile.Blood. 2002; 99: 2285-2290Crossref PubMed Scopus (274) Google Scholar This observation suggested that important genetic lesions are associated with a unique, identifiable gene expression profile. To substantiate and further extend this finding, we have examined 141 new cases that were part of 240 cases of DLBCL studied by cDNA microarray for molecular predictors of survival after chemotherapy.2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3263) Google Scholar These 141 cases of DLBCL with gene expression profiles, clinical data, and genetic data for BCL2 translocation were studied to determine: 1) the distribution of the t(14;18) among the subgroups of DLBCL identified by gene expression profiling; 2) whether t(14;18)-positive cases have a unique gene expression profile; and 3) whether there are differences in the tumor characteristics and clinical behavior between cases with and without the t(14;18). We studied 240 previously analyzed cases of DLBCL with clinical data and gene expression profiles determined by complementary DNA (cDNA) microarray technology.2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3263) Google Scholar A panel of hemopathologists including E Campo, ES Jaffe, G Ott, HK Müller-Hermelink, J Delabie, R Gascoyne, T Grogan, DD Weisenburger, and WC Chan confirmed the diagnosis of DLBCL and excluded the presence of follicular lymphoma in all patients. Informed consent was obtained and the Institutional Review Board of the University of Nebraska approved this study. TMAs were prepared from cases with adequate archival paraffin-embedded tissue. Hematoxylin and eosin-stained sections from each paraffin-embedded, formalin-fixed block were examined to define diagnostic areas and two to five (average, four) representative 0.6-mm cores were obtained from each case and inserted in a grid pattern into a recipient paraffin block using a tissue arrayer (Beecher Instruments, Silver Spring, MD). Five μm sections were then cut from each TMA and stained with antibodies to BCL2, BCL6, and CD10 as described.10Hans CP Weisenburger DD Greiner TC Gascoyne RD Delabie J Ott G Muller-Hermelink HK Campo E Braziel RM Jaffe ES Pan Z Farinha P Smith LM Falini B Banham AH Rosenwald A Staudt LM Connors JM Armitage JO Chan WC Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.Blood. 2004; 103: 275-282Crossref PubMed Scopus (3358) Google Scholar CD20 stains were performed to evaluate each core for involvement by tumor, and each case was evaluated independently by two pathologists (CPH, DDW) for the percentage of tumor cells stained and recorded in 10% increments. Disagreements were resolved by joint review on a multihead microscope. For each case, the core with the highest percentage of tumor cells stained was used for analysis. Cases were considered positive if 30% or more of the tumor cells were stained with an antibody.10Hans CP Weisenburger DD Greiner TC Gascoyne RD Delabie J Ott G Muller-Hermelink HK Campo E Braziel RM Jaffe ES Pan Z Farinha P Smith LM Falini B Banham AH Rosenwald A Staudt LM Connors JM Armitage JO Chan WC Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray.Blood. 2004; 103: 275-282Crossref PubMed Scopus (3358) Google Scholar Among the 240 cases studied by gene expression profiling, 129 cases were studied by interphase FISH for the presence of the t(14;18)(q32;q21). To perform FISH studies, 4-μm sections were cut from the TMA paraffin blocks and mounted on positively charged slides. The sections were dewaxed in three changes of HEM-D (Scientific Safety Solvents, Keller, TX) followed by dehydration in 95% ethanol. They were then treated with 0.2 N HCl for 15 minutes, rinsed in distilled-deionized water, and incubated in a sodium thiocyanate solution at 80°C for 15 minutes. After rinsing in phosphate-buffered saline (PBS), the sections were digested with a protease solution at 37°C for 10 minutes; postfixed in 0.95% formaldehyde solution/PBS with 0.45% MgCl2 for 5 minutes at room temperature; rinsed in PBS; and sequentially dehydrated in 70%, 80%, and 95% ethanol. For FISH, the dual-color LSI IgH Spectrum Green/LSI BCL2 Spectrum Orange Dual-Fusion Translocation Probe (Vysis, Downers Grove, IL) was used to detect the t(14;18), and the CEP 18 Spectrum Aqua probe (Vysis) was used simultaneously to evaluate the chromosome 18 copy number. The probe mixture (10 μl) was placed on the tissue sections, coverslipped, and sealed. Hybridization was performed overnight at 37°C using an automated hybridization chamber (HYBrite, Vysis) after denaturation at 75°C for 5 minutes. The slides were washed in 2× standard saline citrate/0.1% Nonidet P-40 for 2 minutes at 72°C and then at room temperature for 2 minutes. Nuclei were counterstained with 4,6-diamidino-2-phenylindole at a concentration of 125 ng/ml in anti-fade solution and the slides were visualized using an Olympus BX52 fluorescence microscope. Images were captured and archived using Cytovision software (Applied Imaging, Santa Clara, CA). To analyze the hybridization, a total of 50 to 100 nuclei were scored per case for the presence of the t(14;18). In normal cells, an interphase nucleus will exhibit individual red (BCL2) and green (IgH) signals. When the t(14;18) occurs, the red and green signals form two yellow fusion signals in the interphase cell. It has been established by the University of Nebraska Medical Center Human Genetics Laboratories that reactive lymphoid tissues show <2% positive cells at 2.5 SD for this FISH assay. Extracted DNA from all 240 cases was tested for the t(14;18) by the polymerase chain reaction (PCR). Amplification of the BCL2/JH translocation at the major breakpoint region (mbr) and minor cluster region (mcr) was performed as described previously.11Sharp JGBM Chan WC et al.Application of Malignant Cell Detection Techniques to Improve the Outcome of High-Dose Therapy and Transplantation for Lymphoma, Leukemia, Breast Cancer. Karger Basel, 1997Google Scholar Positive controls consisted of DNA extracted from the human B-cell lymphoma cell lines RL-7 for the mbr and DHL-16 for the mcr. Negative controls consisted of sterile water instead of DNA, and DNA from normal peripheral blood mononuclear cells obtained from healthy donors. Standard precautions were taken to guard against PCR contamination. The PCR-positive cases were added to the FISH-positive cases to form a final set of 34 cases with the t(14;18). The FISH-negative cases comprised the t(14;18)-negative group. We used the recently published Bayesian classification system to define the GCB, ABC, and unclassifiable subgroups of DLBCL.12Wright G Tan B Rosenwald A Hurt EH Wiestner A Staudt LM A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma.Proc Natl Acad Sci USA. 2003; 100: 9991-9996Crossref PubMed Scopus (847) Google Scholar We also separated cases with the PMBL gene expression profile3Rosenwald A Wright G Leroy K Yu X Gaulard P Gascoyne RD Chan WC Zhao T Haioun C Greiner TC Weisenburger DD Lynch JC Vose J Armitage JO Smeland EB Kvaloy S Holte H Delabie J Campo E Montserrat E Lopez-Guillermo A Ott G Muller-Hermelink HK Connors JM Braziel R Grogan TM Fisher RI Miller TP LeBlanc M Chiorazzi M Zhao H Yang L Powell J Wilson WH Jaffe ES Simon R Klausner RD Staudt LM Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma.J Exp Med. 2003; 198: 851-862Crossref PubMed Scopus (931) Google Scholar into a new subgroup, and examined the t(14;18) within each of these subgroups. The presence or absence of the BCL2 translocation was used to supervise the discovery of differentially expressed genes between the t(14;18)-positive and -negative cases in the GCB subgroup. The two-tailed Student's t-test was used to compare the differences in gene expression levels. Genes differentially expressed between the two subsets with a P value of 2.6 were selected for further analysis. As previously described, of the three different BCL2 clones (232714, 342181, 1336385) on the Lymphochip microarray, only clone 232714 with a more 5′ sequence could detect message with a truncated 3′ end because of translocation at the mbr. Overexpression of BCL2 as measured by clone 232714 was highly correlated with the BCL2 translocation. However, clone 232714 is located far from the 3′ end of the transcript and, therefore, may fail to measure much of the cDNA transcribed by reverse transcription reactions starting from the poly-A tail of the normal BCL2 transcript. We used the mean values measured by the BCL2 clones that are located close to the 3′ end (342181, 1336385) of the transcript to represent the gene expression level in cases in which this mean value was greater than the measurement by clone 232714. The Kaplan-Meier method was used to estimate the overall and event-free survival of the patients, and the log-rank test was used to compare the survival experiences between the t(14;18)-positive and -negative cases. Overall survival was defined as the time from diagnosis to death from any cause or, for patients remaining alive, the time from diagnosis to last contact. Event-free survival was defined as the time from diagnosis to the first occurrence of relapse or death from any cause or, for patients remaining alive and relapse-free, the time from diagnosis to last contact. SAS software (SAS Institute Inc., Cary, NC) was used for the data analysis. Based on the results of our recent study of PMBL,3Rosenwald A Wright G Leroy K Yu X Gaulard P Gascoyne RD Chan WC Zhao T Haioun C Greiner TC Weisenburger DD Lynch JC Vose J Armitage JO Smeland EB Kvaloy S Holte H Delabie J Campo E Montserrat E Lopez-Guillermo A Ott G Muller-Hermelink HK Connors JM Braziel R Grogan TM Fisher RI Miller TP LeBlanc M Chiorazzi M Zhao H Yang L Powell J Wilson WH Jaffe ES Simon R Klausner RD Staudt LM Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma.J Exp Med. 2003; 198: 851-862Crossref PubMed Scopus (931) Google Scholar 12 GCB cases were reclassified as PMBL. Therefore, we classified the DLBCL cases into GCB, ABC, PMBL, and unclassifiable subgroups and examined the distribution of the t(14;18). Interphase FISH was applied to the 129 TMA cases and detected 22 cases that were positive for the t(14;18). Based on FISH analysis, t(14;18) was detected in 17% (22 of 129) of the cases of DLBCL which represented 34% (19 of 55) of the cases in the GCB subgroup. We also performed PCR assays on 240 cases studied by cDNA microarray, 26 cases were found to be positive, including 14 of the cases previously found to be positive by FISH (Figure 1). The PCR-negative cases that were not tested by FISH were excluded from further analysis because of the recognized high false-negative rate of the PCR assay. Thus, PCR analysis revealed an additional 12 positive cases and in this cohort of 141 cases, 34 DLBCL cases carried the t(14;18) translocation, and 107 lacked this abnormality (Figure 1). Review of the frequency of this anomaly in relation to the DLBCL subgroups (ie, GCB, ABC, PMBL, and unclassifiable) showed that the great majority (29 of 34) of the t(14;18)-positive cases occurred in the GCB subgroup. In contrast, only 3 cases occurred among the 23 cases in the unclassifiable subgroup, and none among the 41 ABC cases. Interestingly the t(14;18) translocation was found in 2 of the12 cases categorized by gene expression profiling as PMBL3Rosenwald A Wright G Leroy K Yu X Gaulard P Gascoyne RD Chan WC Zhao T Haioun C Greiner TC Weisenburger DD Lynch JC Vose J Armitage JO Smeland EB Kvaloy S Holte H Delabie J Campo E Montserrat E Lopez-Guillermo A Ott G Muller-Hermelink HK Connors JM Braziel R Grogan TM Fisher RI Miller TP LeBlanc M Chiorazzi M Zhao H Yang L Powell J Wilson WH Jaffe ES Simon R Klausner RD Staudt LM Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma.J Exp Med. 2003; 198: 851-862Crossref PubMed Scopus (931) Google Scholar (Figure 2). We reviewed the immunohistochemical reactions of 109 TMA samples to correlate the presence and absence of the t(14;18) translocation with the expression of three relevant proteins, namely BCL-2 (encoded by the gene deregulated by the translocation) and two markers of normal germinal center B cells (BCL6 and CD10). The protein expression of BCL2 was similar in the GCB (28 of 50, 56%) and ABC (19 of 31, 61%) subgroups, but was less frequent in the unclassifiable (6 of 19, 31%) or the PMBL subgroups (2 of 9, 22%). The difference did not reach statistical significance (P = 0.054) (Table 1). Within the GCB subgroup there was a significant difference (P < 0.0001) in BCL2 protein expression between thet(14;18)-positive cases (88%) and t(14;18)-negative cases (24%) (Table 2) indicating that the t(14;18) is highly associated with BCL2 protein expression in this subgroup. In other subgroups BCL2 was generally up-regulated by mechanisms other than the t(14;18), as frequently seen in the ABC cases.Table 1Expression of BCL2, BCL6, and CD10 in DLBCL SubgroupsProtein expressionDLBCL subgroupsP valueGCB n = 50ABC n = 31Unclassifiable n = 19PMBL n = 9BCL228 (56%)19 (61%)6 (31%)2 (22%)0.054BCL642 (84%)11 (35%)6 (31%)4 (44%)<0.0001CD1026 (52%)1 (3%)3 (16%)1 (11%)<0.0001 Open table in a new tab Table 2Expression of BCL2, BCL6, and CD10 with Regard to BCL2 TranslocationProtein expressionBCL2 translocationP valuePositive n = 28Negative n = 81BCL225 (89%)30 (37%)<0.0001BCL621 (75%)42 (52%)0.033CD1019 (67%)12 (15%)<0.0001 Open table in a new tab BCL6 protein was detected in 58% (63 of 109) of the cases of DLBCL and it was very frequently expressed in the GCB subgroup (42 of 50, 84%; P < 0.0001), but within this category there was no correlation with the t(14;18) translocation (Table 3). CD10 protein expression was detected overall less commonly than BCL6 (28%, 31 of 109), and the great majority of these cases (26 of 31) were in the GCB subgroup (Table 1). This accounted for the fact that CD10 expression was much commoner overall in t(14;18)-positive cases than in t(14;18)-negative cases (67% versus 15%; P < 0.0001) (Table 2).Table 3Expression of BCL2, BCL6, and CD10 in GCB DLBCL Cases According to BCL2 Translocation StatusProtein expressionBCL2 translocation in GCB DLBCLP valuePositive n = 25Negative n = 25BCL222 (88%)6 (24%)<0.0001BCL621 (84%)21 (84%)1.0CD1018 (72%)8 (32%)0.0046 Open table in a new tab To search for genes that are differentially expressed between the t(14;18)-positive and t(14;18)-negative cases in the GCB subgroup, we examined all informative genes among 7399 known or uncharacterized genes on the Lymphochip.2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3263) Google Scholar Clones with a P value of <0.01 were selected for SAM analysis.13Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9849) Google Scholar Among the 146 clones selected, 53 clones were overexpressed in the t(14;18)-positive group and 93 clones were overexpressed in the t(14;18)-negative group. The genes that were overexpressed in the t(14;18)-positive group represented a heterogeneous set including genes involved in apoptosis (BCL2, BAD, DRAK1, and TXBP151), a number of transcription factors (FLI1, HOX11, and BAP135) and genes associated with cell adhesion/migration (CD62L, cytohesin-1, profilin 2, SDF1, and CD31). Interestingly, a large number of genes overexpressed in the t(14;18)-negative group are associated with cell cycle progression and regulation (Figure 3A). These include genes that control several events in mitosis (PLK, KNSL5, TTK, P55CDC, ARK2, CENE, and CENF), genes involved in cell cycle progression (CYCLIN-A, CDC2, CDC25C, and SAK) and DNA replication (HMG-I/Y, DNA helicase, and DHFR). Some of the cDNAs were represented by multiple clones on the array and these clones were selected independently by the computer algorithm, providing confidence in the reproducibility of the experimental data and the analytical approach. BCL2 gene expression by the different cDNA clones had been adjusted to obtain a more accurate measurement as described in the Materials and Methods section. It was found to be a highly significant discriminator between the two cytogenetic groups (Figure 3B). The GCB subgroup (excluding PBML) consisted of 39 men and 26 women with a median age of 61 years (range, 24 to 88 years). The median follow-up of the surviving patients was 7.3 years (range, 0.8 to 21.8 years) in this group. Figure 4, A and B, shows Kaplan-Meier estimates of overall and event-free survival experiences, respectively, of the t(14;18)-positive and negative subsets of the patients in the GCB subgroup as defined by the Bayesian classifier. P values are based on a log-rank comparison of the outcome of the two subsets. There is no significant difference between the two cytogenetic subsets. DLBCL is the most common type of lymphoma, comprising 30 to 40% of all non-Hodgkin's lymphoma.14A clinical evaluation of the international lymphoma group classification of non-Hodgkin's lymphoma. The Non-Hodgkin's Lymphoma Classification Project.Blood. 1997; 89: 3909-3918Crossref PubMed Google Scholar The recent development of DNA microarray technology provides an opportunity to take a genome-wide approach to the study of DLBCL. We have previously identified two molecularly distinct subtypes of DLBCL indicative of different functional stages of B-cell differentiation.1Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PO Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (8205) Google Scholar, 2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3263) Google Scholar One type expressed genes characteristic of GC B cells (GCB-DLBCL), whereas the other type expressed genes normally seen during in vitro activation of peripheral blood B cells (ABC-DLBCL). In a previous study,9Huang JZ Sanger WG Greiner TC Staudt LM Weisenburger DD Pickering DL Lynch JC Armitage JO Warnke RA Alizadeh AA Lossos IS Levy R Chan WC The t(14;18) defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile.Blood. 2002; 99: 2285-2290Crossref PubMed Scopus (274) Google Scholar we found that the t(14;18)-positive
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