DNA microarray in DLBCL

The New England Journal of Medicineter B cells and were associated with a good outcome,whereas the activated B-cell–like subgroup expressedgenes characteristic of activated blood B cells and wereassociated with a poor outcome.Age, Eastern Cooperative Oncology Group(ECOG) performance status, tumor stage, lactatedehydrogenase level, and the number of sites of extranodaldisease also have prognostic value in diffuselarge-B-cell lymphoma, and they are included in theinternational prognostic index. 4 Although the indexis of value, it has not been used successfully to stratifypatients for therapeutic trials. 5We hypothesized that gene-expression profiles ofdiffuse large-B-cell lymphoma could be used independentlyof the international prognostic index to predictthe outcome of chemotherapy. Since the outcomesfor patients within the same subgroup of diffuselarge-B-cell lymphoma vary, 3 we sought to identifyindividual genes that could influence survival withinthese subgroups.METHODSTumor-biopsy specimens and clinical data were obtained retrospectivelyfrom 240 patients with untreated diffuse large-B-celllymphoma who had no previous history of lymphoma, accordingto a protocol approved by the National Cancer Institute institutionalreview board. A panel of eight hemopathologists confirmed thediagnosis of diffuse large-B-cell lymphoma in all patients and wereable to assign a histologic subtype to 236. Patients were selectedon the basis of the availability of tumor-biopsy specimens, withoutregard to the clinical outcome. All patients had received anthracycline-basedchemotherapy, in most cases a regimen of cyclophosphamide,doxorubicin, vincristine, and prednisone or similar regimens.Median follow-up was 2.8 years overall (7.3 years forsurvivors), and 57 percent of patients died during this period.The median age of the patients was 63 years, and 56 percent weremen. According to the Ann Arbor classification, 15 percent of patientshad stage I disease, 31 percent had stage II, 20 percent hadstage III, and 34 percent had stage IV.“Lymphochip” DNA microarrays 6 are composed of genes whoseproducts are preferentially expressed in lymphoid cells and genesthought or confirmed to play a part in cancer or immune function.These microarrays were constructed from 12,196 clones of complementaryDNA (i.e., microarray features) and were used to quantitatethe expression of messenger RNA in the tumors. 3 Establishedprocedures were followed to detect the amplification of c-rel 7 andthe translocation of bcl-2. 8Hierarchical clustering 9 was used to define subgroups of diffuselarge-B-cell lymphoma. To create an outcome variable based ongene-expression studies, we divided the patients into two groups:the preliminary group comprised 160 patients, and the validationgroup comprised 80 patients. Within the preliminary group, thesignificance of the correlation between outcome (overall survivalafter chemotherapy) and gene-expression data from individual microarrayfeatures was determined with use of the Cox proportionalhazardsmodel. Genes that were associated with a good or a badoutcome at a significance level of P

MOLECULAR PROFILING OF SURVIVAL IN DIFFUSE LARGE-B-CELL LYMPHOMAAGeneA-mybLMO2Jnk3CD10bcl -6Cyclin D2IRF -4FlipCD44Germinal-center B-cell–like Type 3 Activated B-cell–likeSubgroup of Diffuse Large-B-Cell Lymphoma0.25 0.50 1.00 2.00 4.00Relative Level of Expression (¬ median value)BOncogenicAbnormalityGerminal-centerB-cell–likeType 3no. of samplesActivatedB-cell–likec-rel amplification1700bcl-2 t(14;18)2600C1.0Probability of Survival0.80.6Germinal-center B-cell–like0.4Type 30.2Activated B-cell–likeP

The New England Journal of Medicinelarge subgroups (Fig. 1A). One had a high level ofexpression of the genes characteristic of germinalcenterB-cell–like diffuse large-B-cell lymphoma andnormal germinal-center B cells, another expressedgenes characteristic of activated B-cell–like diffuselarge-B-cell lymphoma and mitogenically activatedblood B cells, and the third, termed type 3 diffuselarge-B-cell lymphoma, did not express either set ofgenes at a high level. The heterogeneity of expressionwithin this third subgroup indicates that it mayconsist of more than one type of diffuse large-B-celllymphoma.The subgroups differed substantially with respect totwo recurrent oncogenic events. The t(14;18) translocationinvolving the bcl-2 gene and the amplificationof the c-rel locus on chromosome 2p occurred exclusivelyin germinal-center B-cell–like diffuse large-B-celllymphomas (Fig. 1B). These findings support the viewthat the various subgroups represent different diseasesthat arise as a result of distinct mechanisms of malignanttransformation. 3,11The clinical and pathological features of the threesubgroups are shown in Table 1. The most commonhistologic type of diffuse large-B-cell lymphoma —centroblastic monomorphic — was found in 66 percentof the germinal-center B-cell–like subgroupbut also in 32 percent of the activated B-cell–like subgroupand 27 percent of the type 3 subgroup. Centroblasticpolymorphic and immunoblastic subtypeswere more common in activated B-cell–like andtype 3 diffuse large-B-cell lymphomas, but they werealso observed in the germinal-center B-cell–like subgroup.Thus, these three subgroups were not strictlyrelated to a specific histologic subtype. With respectto clinical features, a significantly higher proportionof patients in the activated B-cell–like subgroupthan in the other two groups were older than 60years of age (P=0.05) and had an ECOG performancestatus of more than 1 (P=0.03), but the tumorsubgroup did not correlate with the risk groups definedon the basis of the international prognostic index(P=0.44).Overall survival after anthracycline-based chemotherapydiffered significantly among the three subgroups(P1¬ normal 57 56 50 65 0.3Age >60 yr 55 48 58 66 0.05Ann Arbor stage >II 55 53 53 59 0.68No. of extranodal sites >1 20 20 25 15 0.34ECOG performance status >1 22 19 16 33 0.03Risk group 0.44Low (score, 0–1) 37 40 41 29Intermediate (score, 2–3) 49 49 43 51High (score, 4–5) 14 11 15 19Histologic subtypeCentroblastic monomorphic 47 66 27 32

MOLECULAR PROFILING OF SURVIVAL IN DIFFUSE LARGE-B-CELL LYMPHOMATABLE 2. USE OF GENE-EXPRESSION PROFILES TO PREDICT OUTCOME FOR PATIENTS WITH DIFFUSE LARGE-B-CELL LYMPHOMA.*GENE-EXPRESSIONVARIABLENO. OFMICROARRAYFEATURES INSIGNATURE(N=7399)NO. OFMICROARRAYFEATURESSIGNIFICANTLYASSOCIATED(P

The New England Journal of Medicinehad the best prognosis, but these patients still had a36 percent risk of death within three years after treatment.The patients with activated B-cell–like diffuselarge-B-cell lymphoma, by contrast, had the worstprognosis, but the five-year survival rate of 35 percentsuggests that some patients in this subgroup may becured by chemotherapy.For these reasons, we used a Cox proportional-hazardsmodel to identify individual genes whose expressioncorrelated with the outcome. Data from 670 of7399 microarray features were significantly associatedwith a good or a bad outcome in the preliminarygroup (P

MOLECULAR PROFILING OF SURVIVAL IN DIFFUSE LARGE-B-CELL LYMPHOMAAProbabilityof SurvivalNO. AT RISKAll Patients1.00.8IPI score, 0–10.60.4IPI score, 2–30.2IPI score, 4–50.0P

The New England Journal of Medicinesignatures were found to predict survival in the preliminarygroup, in the validation group, and in thegroup of all patients (Table 2).Individual genes that were not in these four signaturesbut that predicted the likelihood of survival in aunivariate analysis of the preliminary group (P

MOLECULAR PROFILING OF SURVIVAL IN DIFFUSE LARGE-B-CELL LYMPHOMAARelative Level ofExpression (log 2 )2¡2Germinal-center B-cell–likeGerminal-CenterB-Cell Signature0Type 3Activated B-cell–like20¡2Germinal-center B-cell–likeMHC Class IISignatureType 3Activated B-cell–like20¡2Germinal-center B-cell–likeLymph-NodeSignatureType 3Activated B-cell–like20¡2Germinal-center B-cell–likeProliferationSignatureType 3Activated B-cell–like20¡2Germinal-center B-cell–likeBMP6Type 3Activated B-cell–like20¡2Germinal-center B-cell–likeOutcome-PredictorScoreType 3Activated B-cell–likeBSubgroup of Diffuse Large-B-Cell LymphomaGerminal-center B-cell–like Type 3 Activated B-cell–likeGerminal-center B-cellsignatureMHC class II signatureLymph-node signatureProliferation signatureBMP6Outcome-predictorscore0.25 0.50 1.00 2.00 4.00Relative Level of Expression (¬ median value)CProbabilityof SurvivalNO. AT RISKQuartiles 1 and 2Quartiles 3 and 4Germinal-Center B-Cell–like1.00.8Quartiles 1 and 20.60.4Quartiles 3 and 40.2P

The New England Journal of Medicineports our view that the subgroups of diffuse large-B-cell lymphoma represent distinct disease entities. 3Further support for this idea is supplied by the findingthat the germinal-center B-cell–like subgroup had asignificantly greater likelihood of survival after chemotherapythan did the activated B-cell–like and type 3subgroups.In the second approach, we used clinical and geneexpressiondata to identify individual genes that predictedthe outcome and then combined these variablesinto a multivariate model. This model incorporateddifferences in the levels of gene expression among thesubgroups of diffuse large-B-cell lymphoma that influencedthe outcome, as well as other differences ingene expression that were associated with the likelihoodof survival.The predictive genes fell within four biologic groupsdefined on the basis of gene-expression signatures. Theproliferation signature was the best predictor of anadverse outcome — a finding that is consistent withthose of previous analyses of tumor-cell proliferationin diffuse large-B-cell lymphoma. 12,13 Two of the geneexpressionsignatures that were associated with a goodoutcome suggest that the immune response to thetumor cells may be a crucial determinant of survivalafter chemotherapy. The MHC class II gene-expressionsignature correlated with a good outcome, suggestingthat antigen presentation to the immune systemhas a role in therapeutic responses. 14 The genesin the lymph-node signature that were associated witha good outcome encode components of the extracellularmatrix and connective-tissue growth factor,a mediator of fibrosis that promotes the synthesis ofthe matrix. 15 Sclerotic reactions occur in some casesof diffuse large-B-cell lymphoma, and the lymph-nodesignature may reflect these reactions. Other genes inthe lymph-node signature are characteristically expressedin macrophages and natural killer cells, againsuggesting that an antitumor immune response improvessurvival after chemotherapy.The prognostically favorable germinal-center B-cellgene-expression signature is notable because cell linesderived from germinal-center B-cell–like diffuse large-B-cell lymphomas have decreased activity of the nuclearfactor kB signaling pathway. 16 This protectivepathway interferes with the apoptotic effect of chemotherapy.17 In contrast, in activated B-cell–like diffuselarge-B-cell lymphomas, there is constitutive activationof this pathway, 16 which, in principle, could block theapoptosis induced by chemotherapy and thus accountfor the relatively poor outcome in this subgroup.Our outcome predictor may help identify patientswith diffuse large-B-cell lymphoma who are unlikelyto be cured by conventional therapy. On the basis oftheir predictor scores, one quarter of the 240 patientsin this study could be assigned to a risk group with afive-year survival rate of 15 percent. Among patientsat intermediate risk according to the internationalprognostic-indexscore, use of the outcome predictorindicated that 55 percent of these patients had a fiveyearsurvival rate of 18 percent, whereas this groupof patients had a five-year survival rate of 41 percentoverall. Even among patients with a low risk accordingto the international-prognostic-index score, use of theoutcome predictor showed that 16 percent of the patientsin this group had a five-year survival rate of only28 percent (data not shown).One of the features of our analysis is that the outcomepredictor involves a small number of genes andthus multiplexed quantitative reverse-transcriptase–polymerase-chain-reaction assays or customized DNAmicroarrays could easily be developed for clinical application.Regardless of the eventual choice of technique,our study highlights the need to use moleculardiagnosis in patients with diffuse large-B-cell lymphoma,since these patients have molecularly distinct diseasesthat may require individualized and molecularlytargeted therapies.Supported by a Director’s Challenge grant (UO1-CA84967) from theNational Cancer Institute, by the Center for Cancer Research of the NationalCancer Institute, and by the Cancer Genome Anatomy Project. Dr.Rosenwald was supported in part by the Deutsche Krebshilfe, Bonn, Germany.Ms. Averett was a Howard Hughes Medical Institute ResearchScholar at the National Institutes of Health during the study.We are indebted to Bob Strausberg, Narajan Bhat, and ButchHopkins for preparing the messenger RNA reference pool and forhelpful discussions, and to Ken Buetow and Jenny Kelly for verifyingthe sequence of Lymphochip clones.REFERENCES1. Coiffier B. Diffuse large cell lymphoma. Curr Opin Oncol 2001;13:325-34.2. Fisher RI, Gaynor ER, Dahlberg S, et al. Comparison of a standard regimen(CHOP) with three intensive chemotherapy regimens for advancednon-Hodgkin’s lymphoma. N Engl J Med 1993;328:1002-6.3. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse largeB-cell lymphoma identified by gene expression profiling. Nature 2000;403:503-11.4. The International Non-Hodgkin’s Lymphoma Prognostic FactorsProject. 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MOLECULAR PROFILING OF SURVIVAL IN DIFFUSE LARGE-B-CELL LYMPHOMA11. Huang JZ, Sanger WG, Greiner TC, et al. The t(14;18) defines aunique subset of diffuse large B-cell lymphoma with a germinal centerB-cell gene expression profile. Blood 2002;99:2285-90.12. Grogan TM, Lippman SM, Spier CM, et al. Independent prognosticsignificance of a nuclear proliferation antigen in diffuse large cell lymphomasas determined by the monoclonal antibody Ki-67. Blood 1988;71:1157-60.13. Silvestrini R, Costa A, Boracchi P, Giardini R, Rilke F. Cell proliferationas a long-term prognostic factor in diffuse large-cell lymphomas. Int JCancer 1993;54:231-6.14. Miller TP, Lippman SM, Spier CM, Slymen DJ, Grogan TM. HLA-DR (Ia) immune phenotype predicts outcome for patients with diffuselarge cell lymphoma. J Clin Invest 1988;82:370-2.15. Grotendorst GR. Connective tissue growth factor: a mediator of TGFbetaaction on fibroblasts. Cytokine Growth Factor Rev 1997;8:171-9.16. Davis RE, Brown KD, Siebenlist U, Staudt LM. Constitutive nuclearfactor kappaB activity is required for survival of activated B cell-like diffuselarge B cell lymphoma cells. J Exp Med 2001;194:1861-74.17. Baldwin AS. Control of oncogenesis and cancer therapy resistance bythe transcription factor NF-kappaB. J Clin Invest 2001;107:241-6.Copyright © 2002 Massachusetts Medical Society.N Engl J Med, Vol. 346, No. 25 · June 20, 2002 · www.nejm.org · 1947The New England Journal of MedicineDownloaded from nejm.org on February 11, 2013. For personal use only. No other uses without permission.Copyright © 2002 Massachusetts Medical Society. All rights reserved.