Distinct Genetic Aberrations in Molecular Subtypes of Diffuse Large B Cell Lymphoma Detected by Array CGH.

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category with at least three different molecular subtypes distinguishable by gene expression profiling, termed germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL). We performed array comparative genomic hybridization (aCGH) in patient samples and cell lines representing different DLBCL subtypes to determine if they utilize distinct pathogenetic mechanisms. Using an array consisting of 386, 165 oligonucleotides (NimbleGen), we performed aCGH on 203 untreated de novo DLBCL samples and 30 DLBCL cell lines, and the same samples were profiled for gene expression using Affymetrix U133 plus arrays. Patient samples included 72 GCB DLBCLs, 74 ABC DLBCLs, 31 PMBLs, and 26 unclassified DLBCLs. Following segmentation of the aCGH data into intervals with a uniform copy number, segments were combined into minimal common regions (MCRs) that were recurrently altered in more than one sample. Statistical differences in MCR frequency between DLBCL subtypes were corrected for multiple hypothesis testing using a false discovery rate (FDR) calculation. The DLBCL subtypes differed in the frequency of MCRs residing at many chromosomal loci, and we used gene expression data to define potential target genes in these MCRs. The INK4a/ARF tumor suppressor locus on 9p21 was selectively lost in ABC DLBCL: homozygous deletions of INK4a/ARF was observed in 20% of ABC DLBCLs but in only 3% of GCB DLBCLs and never in PMBLs (FDR=4.5 E-3). Among ABC DLBCLs, loss of INK4a/ARF was associated with increased proliferation rate, as measured by a proliferation gene expression signature, and adverse survival (p=0.007, log rank test). 16% of ABC DLBCL cases had gain/amplification and overexpression of SPIB, a gene on 19q13 encoding an ETS family transcription factor that is characteristically expressed in ABC DLBCL. This copy number alteration was observed much less frequently in GCB DLBCL (3%) and never in PMBL (FDR=2.6 E-2). GCB DLBCLs had recurrent amplification and overexpression of C13orf25, which encodes the mir-17-92 polycistronic cluster of microRNAs that is transcriptionally activated by c-myc and cooperates with c-myc to accelerate tumor development. C13orf25 amplification was detected in 16% of GCB DLBCLs but in only 3% of PMBLs and never in ABC DLBCL (FDR=3.8 E-3). Recurrent amplification and overexpression of JAK2 on 9p24 was observed in 35% of PMBL cases, but only in 5% of GCB DLBCLs and 4% of ABC DLBCLs respectively (FDR=6.2 E-4). In summary, aCGH revealed copy number abnormalities in DLBCL that had strikingly different frequencies in the three DLBCL subtypes, supporting the hypothesis that these subtypes represent distinct diseases that utilize different oncogenic mechanisms. Our analysis specifically implicated the INK4a/ARF locus as a tumor suppressor and SPIB as an oncogene in ABC DLBCL, the mir-17-92 microRNA cluster as an oncogene in GCB DLBCL, and JAK2 as an oncogene in PMBL.