Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways Georg Lenza, George W. Wrightb, N. C. Tolga Emrea, Holger Kohlhammera, Sandeep S. Davea, R. Eric Davisa, Shannon Cartya, Lloyd T. Lama, A. L. Shaffera, Wenming Xiaoc, John Powellc, Andreas Rosenwaldd, German Ottd,e, Hans Konrad Muller-Hermelinkd, Randy D. Gascoynef, Joseph M. Connorsf, Elias Campog, Elaine S. Jaffeh, Jan Delabiei, Erlend B. Smelandj,k, Lisa M. Rimszal, Richard I. Fisherm,n, Dennis D. Weisenburgero, Wing C. Chano, and Louis M. Staudta,q aMetabolism Branch, bBiometric Research Branch, cCenter for Information Technology, and hLaboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892; dDepartment of Pathology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany; eDepartment of Clinical Pathology, Robert-Bosch-Krankenhaus, 70376 Stuttgart, Germany; fBritish Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 4E6; gHospital Clinic, University of Barcelona, 08036 Barcelona, Spain; iPathology Clinic and jInstitute for Cancer Research, Rikshospitalet Hospital, Oslo, Norway; kCentre for Cancer Biomedicine, Faculty Division the Norwegian Radium Hospital, N-0310, Oslo, Norway; lDepartment of Pathology, University of Arizona, Tucson, AZ 85724; mSouthwest Oncology Group, 24 Frank Lloyd Wright Drive, Ann Arbor, MI 48106 ; nJames P. Wilmot Cancer Center, University of Rochester, Rochester, NY 14642; and oDepartments of Pathology and Microbiology, University of Nebraska, Omaha, NE 68198 Edited by Ira Pastan, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved July 7, 2008 (received for review May 2, 2008) Gene-expression profiling has been used to define 3 molecular Recent studies using fluorescence in situ hybridization, con- subtypes of diffuse large B-cell lymphoma (DLBCL), termed germi- ventional comparative genomic hybridization (CGH), or low- nal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, resolution array-based CGH (aCGH) have shown that certain and primary mediastinal B-cell lymphoma (PMBL). To investigate genetic aberrations occur at different frequencies among DLBCL whether these DLBCL subtypes arise by distinct pathogenetic subtypes (4, 9–11). The t(14, 18) translocation involving BCL2 and mechanisms, we analyzed 203 DLBCL biopsy samples by high- amplification of REL was detected in 45% and 16% of GCB resolution, genome-wide copy number analysis coupled with DLBCLs, respectively, but never in ABC DLBCLs (4). Roughly gene-expression profiling. Of 272 recurrent chromosomal aberra- one-quarter of ABC DLBCLs had trisomy 3 or gain/amplification tions that were associated with gene-expression alterations, 30 of chromosome arm 3q, but these abnormalities never were de- were used differentially by the DLBCL subtypes (P < 0.006). An tected in GCB DLBCLs (9, 10). ABC DLBCLs were characterized amplicon on chromosome 19 was detected in 26% of ABC DLBCLs further by frequent gain of 18q and loss of 6q (9, 10). but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated These relatively low-resolution techniques were unable to gene in this amplicon was SPIB, which encodes an ETS family pinpoint the presumed cancer-relevant target gene in most transcription factor. Knockdown of SPIB by RNA interference was instances. To overcome these limitations, we combined aCGH toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or using high-density whole-genome microarrays with gene expres- myeloma cell lines, strongly implicating SPIB as an oncogene sion profiling. By this concerted approach, we identifiedchro- involved in the pathogenesis of ABC DLBCL. Deletion of the mosomal aberrations that were significantly more frequent in a INK4a/ARF tumor suppressor locus and trisomy 3 also occurred particular DLBCL subtype than in the others, and some of these aberrations were associated with clinical outcome. This analysis almost exclusively in ABC DLBCLs and was associated with inferior revealed oncogenic pathways that are used differentially by the outcome within this subtype. FOXP1 emerged as a potential DLBCL subtypes, reinforcing the view that they represent oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by pathogenetically distinct diseases. more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17–92 microRNA cluster and deletion of the Results and Discussion tumor suppressor PTEN were recurrent, but these events did not Definition of Recurrently Altered Minimal Common Regions (MCRs). occur in ABC DLBCL. Together, these data provide genetic evidence aCGH was performed on 203 DLBCL samples, including 74 that the DLBCL subtypes are distinct diseases that use different ABC DLBCLs, 72 GCB DLBCLs, 31 PMBLs, and 26 unclassi- oncogenic pathways. fied DLBCLs from a previously studied cohort of patients (4). We used oligonucleotide microarrays covering the entire ge- gene-expression profiling ͉ oncogenes ͉ tumor suppressor genes ͉ nome at 5-kb average spacing, allowing us to delineate copy comparative genomic hybridization number changes with great precision. To identify chromosomal aberrations associated with gene-expression changes, we profiled iffuse large B-cell lymphoma (DLBCL) is the most common gene expression in the same samples and developed a statistical Dtype of non-Hodgkin’s lymphoma (1). DLBCL can be cured algorithm termed ‘‘Gene Expression and Dosage Integrator’’ using anthracycline-based chemotherapy regimens in only 40%– (GEDI) to merge the data sets (Fig. 1). For each case, GEDI 50% of patients, suggesting that DLBCL is a heterogeneous divides the aCGH data into intervals with statistically similar diagnostic category (2). Indeed, gene-expression profiling stud- gene dosage, and each interval is classified as an amplification, ies have distinguished 3 molecular subtypes of DLBCL known as ‘‘germinal center B-cell-like’’ (GCB) DLBCL, ‘‘activated B-cell- Author contributions: G.L. and L.M.S. designed research; G.L., N.C.T.E., H.K., S.S.D., R.E.D., like’’ (ABC) DLBCL, and ‘‘primary mediastinal B-cell lym- S.C., L.T.L., and A.L.S. performed research; A.R., G.O., H.K.M.-H., R.D.G., J.M.C., E.C., E.S.J., phoma’’ (PMBL) (3–7). GCB DLBCLs seem to arise from J.D., E.B.S., L.M.R., R.I.F., D.D.W., and W.C.C. contributed new reagents; G.L., G.W.W., W.X., normal germinal center B cells, whereas ABC DLBCLs may J.P., and L.M.S. analyzed data; and G.L., G.W.W., and L.M.S. wrote the paper. arise from postgerminal center B cells that are arrested during The authors declare no conflict of interest. plasmacytic differentiation, and PMBLs may arise from thymic This article is a PNAS Direct Submission. B cells (8). Patients with these DLBCL subtypes have signifi- qTo whom correspondence should be addressed. E-mail: [email protected] cantly different survival rates following chemotherapy (3–6), This article contains supporting information online at www.pnas.org/cgi/content/full/ leading to the current proposal that they represent distinct 0804295105/DCSupplemental. biological disease entities (8). © 2008 by The National Academy of Sciences of the USA 13520–13525 ͉ PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804295105 Downloaded by guest on September 26, 2021 Chromosomal position focus attention on more common abnormalities, we limited High resolution Amplification subsequent analysis to the 272 MCRs that occurred in 8 or more array-based Ͼ comparative Gain samples (i.e., 5% of ABC and GCB DLBCL cases) (Table S1). genomic Loss hybridization Double (aCGH) deletion Chromosomal Aberrations Associated with the DLBCL Subtype Dis- Identify tinction. To elucidate oncogenic mechanisms in the DLBCL Gene expression Minimal Common Regions profiling (MCRs) subtypes, we focused our analysis on MCRs that were differen- Correlate gene expression Associate MCRs tially represented among ABC DLBCLs, GCB DLBCLs, and with MCRs with the PMBLs. We further reasoned that such MCRs might be less DLBCL subtype distinction likely to be common copy number polymorphisms present in the (false discovery rate human population (14). { < 5%) MCR Based on a false discovery rate (FDR) of 0.05, we observed 30 MCRs associated with the subtype distinction, far in excess of Fig. 1. Schematic of the Gene Expression and Dosage Integration algorithm chance (Fig. S2 and Table S2). Some of these MCRs probably (see methods). represent similar biology, such as single and double loss of the INK4A/ARF locus, both of which were much more frequent in single-copy gain, single-copy deletion, homozygous deletion, or ABC DLBCLs (Fig. 2). is declared wild type [supporting information (SI) Fig. S1]. Next, Aberrations most characteristic of ABC DLBCL included GEDI defines MCRs of abnormal gene dosage by assembling trisomy 3, deletion of chromosome arm 6q, gain/amplification of segments that affect the same chromosomal region in different a region on chromosome arm 18q, deletion of the INK4a/ARF cases and that belong to the same dosage category (12, 13). tumor suppressor locus on chromosome 9, and gain/amplifica- Finally, the GEDI algorithm determines the degree to which tion of a 9-Mb region on chromosome 19. Aberrations prefer- each gene within an MCR is altered in expression and computes entially used by GCB DLBCLs included amplification of the a statistic, summarizing these correlations across all genes in mir-17–92 microRNA in the MIHG1 locus cluster on chromo- the MCR. some 13, gain/amplification of a 7.6-Mb region on chromosome The GEDI algorithm yielded a total of 1940 MCRs within the 12, deletion of the PTEN tumor suppressor gene on chromosome DLBCL data set, ranging in size from 5 kb, affecting a single 10, and amplification of the REL locus on chromosome 2. The gene, to more than 240 Mb, affecting a whole chromosome. Of most frequent chromosomal lesions in PMBL included amplifi- these MCRs, 719 showed a significant association with gene- cation of a telomeric region of chromosome 9p, monosomy 10, expression alterations, whereas 1221 did not.