Normal lymphocyte differentiation is,in some sense,adisaster waiting t - PDF document

Normal lymphocyte differentiation is,in some sense,adisaster waiting to happen.B cells put their genomicintegrity in danger during the formation and revisionoftheir antigen receptors.A second potentially dan-gerous event is the response to antigen.When thisresponse functions normally,the clonal expansion ofB cells is regulated tightly by homeostatic controls.However,chronic infections can wreak havoc on lym-phocyte homeostasis,as can abnormal responses toself-antigens,and both ofthese mechanisms might and discuss how the relationship between a lymphomaand its normal B-cell counterpart might be exploitedto understand and treat these cancers.We discuss alsohow oncogenic alterations in these cancers subverthomeostatic regulation oflymphocyte responses.The perils of normal B-cell differentiationThe first dangerous hurdle in B-cell differentiation isrearrangement ofthe immunoglobulin genes ofB-cellprecursors in the bone marrow to form a B-cell receptor(BCR).This molecular process,V(D)J RECOMBINATION A.L.Shaffer,Andreas Rosenwald and Louis M.StaudtWhen the regulation of B-cell differentiation and activation is disrupted, lymphomas andleukaemias can occur. The processes that normally create immunoglobulin diversity might bemisdirected, resulting in oncogenic chromosomal translocations that block differentiation,prevent apoptosis and/or promote proliferation. Prolonged or unregulated antigenic stimulation NATURE REVIEWS  VOLUME 2 Metabolism Branch,Center for Cancer Research,National Cancer Institute,National Institutes of V(D)J RECOMBINATIONThe somatic rearrangement ofvariable (V),diversity (D) andjoining (J) regions ofantigen-receptor genes,which leads tothe repertoire diversity ofboth  T- and B-cell receptors. GERMINAL CENTREThe structure that is formed bythe clonal expansion ofantigen-activated B-cell blasts that havemigrated to the follicles oflymph nodes.The B cells in thesestructures proliferate and theirimmunoglobulin genes undergosomatic hypermutation,beforethe cells leave as plasma cells or VOLUME 2    ,although they sometimes involve DNAregions with altered structure.The IgH breaks oft(11;14) in mantle-cell lymphoma seem to occur beforeheavy-chain diversity and joining segment (Drearrangement,which indicates that this translocationoccurs early in B-cell differentiation.As RAGs are notexpressed after the immature B-cell stage,t(14;18)might occur in a pre-GERMINAL CENTRE(GC) B cell as well. (BOX 1),and t(11;14),which involves the geneencoding cyclin D1 and the IgH locus in mantle-celllymphoma (BOX 1).The structures ofthe recombinationbreakpoints in these translocations are consistent withRAG-mediated cleavage ofthe IgH locus,guided byrecombination signal sequences (RSSs).However,therecombination sites in the partner genes lack clear RSSsand do not have cryptic sequences that could function Mature B-cell malignancies Follicular lymphomaAn often indolent B-cell lymphoma with a follicular growth pattern.Most are characterized by the overexpression ofBCL-2,owing to t(14;18).They comprise ~22% ofnon-Hodgkin lymphomas (NHLs).They cannot be cured byconventional chemotherapy and the survival rate is 73% at 10 years.Mantle-cell lymphoma A B-cell lymphoma that localizes to the mantle region ofsecondary follicles.Mantle-cell lymphoma (MCL) is associatedwith t(11;14),which results in the overexpression ofcyclin D1.MCLs comprise 6% ofall NHLs,have a malepredominance and occur at a median age of60.With current chemotherapy regimens,patients with MCL can achievecomplete remission,but long-term remission is rare and median survival is 3Ð5 years.Burkitt lymphomaAn aggressive B-cell lymphoma ofchildren and young adults that is associated invariably with translocations ofc-MYCThe endemic form involves EpsteinÐBarr virus (EBV) infection ofmalignant cells,whereas the sporadic form is EBVindependent.These lymphomas can be cured in more than 80% ofcases.Multiple myelomaAn incurable malignancy ofplasma cells with a median survival ofthree years.Multiple myeloma constitutes ~10% ofall haematological malignancies,with a median age at diagnosis of~65.Neoplastic cells are located in the bonemarrow,and osteolytic bone lesions are characteristic.Reciprocal chromosomal translocations between one oftheimmunoglobulin loci and various other genes,including those that encode cyclin D1,cyclin D3,MMSET(multiple myeloma SET-domain protein) or fibroblast growth factor receptor 3 (),are considered to be primaryoncogenic events.Diffuse large B-cell lymphoma Diffuse large B-cell lymphoma (DLBCL) is the most common type ofNHL (30Ð40% ofcases).Up to one third ofcases have abnormalities of,and ~20% ofcases have translocations of.DLBCLs are clinically,morphologically and molecularly heterogeneous.40% ofpatients with DLBCL can be cured by conventionalchemotherapy.Hodgkin lymphomaThis type oflymphoma accounts for ~10% ofall lymphoid malignancies,and it usually arises in the lymph nodes ofyoung adults.It can be subdivided into a classical subtype and a less common nodular lymphocyte predominantsubtype.Cure rates ofmore than 80% can be achieved with present therapies.Lymphoplasmacytic lymphomaThis is a rare form ofNHL that comprises ~1.5% ofnodal lymphomas.It is usually indolent and frequently involvesbone marrow,lymph nodes and spleen.Most patients have monoclonal immunoglobulin M in their serum,and thetumour cells have a plasmacytic morphology.A subset oflymphoplasmacytic lymphomas is characterized by recurrentt(9;14),which involves the PAX5(paired box gene 5) and immunoglobulin heavy-chain loci.Marginal-zone lymphomaThis extranodal lymphoma occurs in organs that normally lack organized lymphoid tissue (such as the stomach,salivary glands,lungs and thyroid glands),and it comprises 7Ð8% ofall B-cell lymphomas.In many cases,chronicinflammation or an autoimmune process precedes development ofthe lymphoma.Gastric mucosal-associatedlymphoid tissue (MALT) lymphoma,the most common type,is associated with Helicobacter pyloriinfection,and 70%ofpatients at early stages have complete remission after eradication ofthis bacterium.At later stages,the acquisition ofgenetic abnormalities might lead to H.pylori-independent growth ofthe tumour cells or to transformation to anaggressive DLBCL.Chronic lymphocytic leukaemia The most common type ofleukaemia,chronic lymphocytic leukaemia (CLL),is often an indolent disease with a median age ofonset of65.CLL is molecularly and clinically related to a nodal lymphoma known as smalllymphocytic lymphoma.Current therapy can reduce symptoms,but it is not curative and does not prolongsurvival. NATURE REVIEWS  VOLUME 2 presentation will influence its clinical behaviour andresponsiveness to therapy.Several possible mechanisms could account for theapparent developmental arrest in many lymphoidmalignancies.First,oncogenic alterations could inter-fere with regulatory networks that control lymphocytedifferentiation.As discussed later,translocation ofmight cause lymphomas,in part,by blocking plasma-cytic differentiation.Second,the malignant lymphocytemight lose responsiveness to external cues,such as anti-gen or other immune cells that regulate normal differ-entiation.Third,it is conceivable that an oncogenicevent might activate pathways that mimic a particularstage ofnormal differentiation.This possibility seemsless likely in some lymphoid malignancies,as describedlater,that share extensive gene-expression profiles andbiological functions with particular stages ofB-cell differentiation.The analysis ofsomatic mutations in the rearrangedimmunoglobulin loci oflymphoid malignancies showsthat there are clear differences between the diagnosticcategories (TABLE 1).Most types ofnon-Hodgkin lym-phoma have highly mutated immunoglobulin genesthat bear the hallmarks ofSHM.A prominent exceptionto this rule might be mantle-cell lymphoma,whichindicates that this lymphoma might be pre-GC in ori-gin.Ofcourse,the mere presence ofimmunoglobulinmutations in a lymphoid malignancy only indicates thatthe cell that gave rise to the tumour had passed througha stage ofB-cell differentiation during which SHMoccurs.In some lymphomas,however,individualtumour cells in the malignant clone have distinctimmunoglobulin sequences,which indicates that thetumour is frozen at a stage ofdifferentiation at which(TABLE 1)The presence ofimmunoglobulin mutations in lym-phoid malignancies is usually taken as evidence that thecell oforigin ofthe tumour passed through the GCmicroenvironment.Although most SHM takes place in,recent work indicates that it can occur also out-side ofclassical GC structures.Signalling through is required to initiate and maintain the GC reactionand studies ofhyper-IgM patients with genetic deficien-) have shown that someSHM can take place in the absence ofCD40 signallingIn particular,a CD27IgMIgDsomatically mutated memory B cells is retained in theperipheral blood ofthese patients,whereas other mem-ory B-cell subpopulations are absent.These studiesindicate that SHM can occur outside ofthe GC,andthey are reminiscent ofearlier work in lymphotoxin-deficient mice,which lack GCs but can initiate SHMafter several immunizations.A direct observation ofSHM outside ofGCs was reported recently using a trans-genic mouse engineered to synthesize anti-IgG antibod-ies (rheumatoid factors).Clonal expansion ofanti-IgG-specific B cells was observed in the T-zoneÐred-pulpborder ofthe spleen,and the B cells in these proliferativefoci were shown to have ongoing SHM at a rate similarto that seen in GC B cells.Given the possibility ofextra-GC SHM,the presence ofimmunoglobulin mutations This possibility is interesting because follicular lym-phomas seem to be arrested at the GC stage ofdifferen-tiation (see later),which indicates that a naive B cellthat has acquired a translocation can neverthelessparticipate in an antigen-driven GC response.After antigen encounter,naive B cells follow one ofthree pathways:they can enter the GC microenviron-ment,where they interact with T cells,;they can differentiate into short-lived PLASMABLASTSoutside ofthe GC;or they can enteran unresponsive state known as anergy (FIG.1a).In theGC,two molecular processes remodel DNA Ñimmunoglobulin CLASS-SWITCH RECOMBINATIONimmunoglobulin SOMATIC HYPERMUTATIONBoth CSR and SHM generate DNA breaksare,therefore,dangerous mechanisms that might pre-dispose to chromosomal translocations.The DNAbreaks that are induced by CSR and SHM coincidewith the sites ofchromosomal translocations thatinvolve the IgH locus in certain lymphoid malignan-cies.SHM is probably involved in t(8;14) in endemicBurkitt lymphomas (BOX 1)c-MYCoften joined to the IgH locus in a rearranged andsomatically mutated IgH variable (V) region.SHMcan also target non-immunoglobulin loci,such as(REFS 15Ð17),and the involvement ofthese genesin translocations is probably a byproduct ofthisprocess.CSR is the culprit in many ofthe transloca-tions that occur in multiple myeloma (BOX 1)radic Burkitt lymphoma,because the translocationbreakpoints occur in IgH switch regionsCell of originHistorically,the relationship between normal B-cellsubpopulations and types oflymphoma has beenassessed by a combination ofmicroscopic appearanceand immunophenotype.By these criteria,mostmature B-cell malignancies seem to be ÔtrappedÕat par-ticular stages ofnormal B-cell development.Follicularlymphomas,for example,have growth patterns thatresemble those ofnormal GC B cells,and they areinfiltrated with follicular dendritic cells and T cells.The tumour cells also express the membrane metallo-,which is a hallmark ofhumanGC B cells,leaving little doubt that follicular lym-phoma is a disease ofGC B cells.However,in somelymphomas,the tumour cells show a spectrum ofmorphological differentiation,ranging from GC-likecells to plasmacytic cells,which indicates that the blockin differentiation is not complete.When we speak ofcell oforigin we are,by necessity,referring to the relationship between the phenotype ofthe tumour on clinical presentation and a normalstage ofB-cell differentiation.We cannot observehuman lymphoid tumours during their natural evolu-tion from a normal B cell.Therefore,as mentionedearlier,oncogenic translocations might occur at anearly stage ofB-cell differentiation,after which thetransformed B cell might differentiate further andarrest at a later stage ofdifferentiation.The importantpoint is that the phenotype ofthe tumour at clinicalmemory B cells. (FDCs).Cells with a dendriticmorphology that are present inlymph nodes,where they presentintact antigens held in immunecomplexes to B cells.A dividing B cell that iscommitted to plasma-celldifferentiation.CLASS-SWITCHRECOMBINATIONDNA rearrangement ofthe VDJregion from immunoglobulin Mto any ofthe IgG,IgA or IgEconstant genes at the heavy-chain locus.Recombinationoccurs in repetitive sequences ofDNA that are located upstreamofeach constant gene.SOMATIC HYPERMUTATIONÔuntemplatedÕnucleotides orsmall deletions targeted to arearranged VDJ or VJ segment,which occurs only in B cells.The mutations are foundbetween the promoter andenhancer ofthe rearranged gene(including non-coding regions),but they are found at the highestfrequency in ÔhotspotsÕ(RGYW) that are located in thecomplementarity-determiningregions. VOLUME 2    and BCL-6) and many new genesofunknown function that were identified by high-throughput sequencing ofcomplementary DNA librariesfrom normal GC B cells.Expression ofthe GCB-cellsignature genes is maintained in some lymphoma cell,which indicates that this signature is a stablechange in gene expression and does not require the cell-ular interactions that are present in the GC microenvir-onment to be maintained.So,the GC B cell is at a discretestage ofB-cell differentiation and is not just a specializedtype ofactivated lymphocyte. in a lymphoid malignancycannot be taken as definitiveevidence for a GC or post-GC cell oforigin.Recently,the relationship ofB-cell malignancies tonormal stages ofB-cell differentiation and activation hasbeen clarified using genomic-scale gene-expression pro-filing.A unique gene-expression signature distinguishesGC B cells from other stages ofB-cell differentiation,including resting naive and memory blood B cells andmitogenically activated blood B cells(FIG.1b).The GCB-cell signature contains several hundred genes,includ-ing well-known GC markers (such as the genes encoding signaturesignatureGC reaction Figure 1| Mature B-cell lymphomas: cell of origin.aface three cell fates: clonal expansion and selection in a germinal centre (GC), clonal expansion and differentiation at extra-sites, or anergy. Eventually, B cells either die or differentiate to memory B cells or antibody-secreting plasma cells. expression profiling shows a relationship between stages of B-cell differentiation and several types of mature B-cell lymphoma.Each column represents the results of gene-expression profiling from a single messenger RNA sample of normal or malignantB cells. Each row represents the expression of a single gene. Genes were chosen on the basis of their ability to distinguishbetween diffuse large B-cell lymphomas (DLBCLs) of GC and non-GC phenotype. Samples are compared with a commonreference RNA pool, and relative gene expression is shown using a colour scale in which shades of red indicate genes that areexpressed at a higher than median level, shades of green indicate genes that are expressed at a lower than median level, andblack indicates genes that are expressed at the median level across all samples. A 16-fold range of gene expression is shown.Germinal-centre B-cell signature genes Ñ for example, those that encode CD10, , BCL-6 and CD77 synthase Ñ relatenormal GC B cells to some lymphomas (follicular lymphomas, Burkitt lymphomas and the GC B-cell-like DLBCL subgroup).Genes that are expressed at a higher level inmitogenically activated peripheral-blood B cells than in GC B cells Ñ for example,(forkhead box P1), CD44, cyclin D2 and IRF4 (interferon-regulatory factor 4) Ñ uniquely identify theactivated B-cell-like DLBCL subgroup. Potential cell types of origin for these lymphomas are indicated in pink (activated B celand blue (GC B cell) in part NATURE REVIEWS  VOLUME 2 unpublished observations).Together,these observationspoint to a GC B cell as the cell oforigin for GCBDLBCLs,and they show that these tumours are trappedat this stage ofdifferentiation.Another subgroup ofDLBCLs,representing ~30%ofcases,are known as activated B-cell-like DLBCLs(ABC DLBCLs),because these lymphomas resemblemitogenically activated peripheral B cells,and not GC B cells,in their gene-expression profile(FIG.1).Animportant feature ofABC DLBCLs is the high level ofexpression ofnuclear factor-B (NF-B) target genes,including those that encode BCL-2,interferon regula-tory factor 4 ((FLICE-like inhibitoryprotein) and cyclin D2(see below).These lymphomashave a high level ofimmunoglobulin somatic muta-tions,but they do not have ongoing SHM.Nearly allABC DLBCLs express a high level ofIgM(A.R.andL.M.S.,unpublished observations),which indicates thatthey have not undergone immunoglobulin class-switchrecombination,a finding that is unexplained so far.The cell oforigin for ABC DLBCLs is less clear thanfor GCB DLBCLs,although the absence ofthe GC B-cell gene-expression signature and the lack ofongoingSHM do not indicate a GC B-cell origin.ABC DLBCLsresemble pre-plasma cells in terms ofgene expression inthat they have higher levels ofexpression ofimmuno-globulin,X-box binding protein 1 (),IRF4 andother plasma-cell genes than GCB DLBCLs,and a lowerlevel ofexpression ofBCL-6 (REF.29;A.R.and L.M.S.,unpublished observations).GCs contain a subpopulation Several types ofB-cell lymphoma express GC B-cellsignature genes,including follicular lymphomas,Burkittlymphomas and a subgroup ofdiffuse large B-cell lym-BOX 1FIG.1).This findingestablishes that these malignancies are derived from aGC B cell and not from a post-GC somaticallymutated B cell.Although these malignancies retainexpression ofmost ofthe GC B-cell signature genes,thelymphoma from an individual patient might have lostexpression ofany one GC B-cell marker.Furthermore,expression ofa single gene is usually insufficient to estab-lish the relationship between a malignancy and its nor-mal counterpart,because many ofthe gene-expressiondifferences between stages ofdifferentiation are quanti-tative,not qualitative,in nature.Therefore,a ÔdiagnosisÕofa GC B-cell origin must be based on the expression ofseveral GC B-cell signature genes to be accurate.By con-trast,other types oflymphoid malignancy fail to expressthese GC B-cell genes,and they have their own gene-expression signatures that relate them to other stages ofB-cell differentiation (TABLE 1)About halfofall DLBCLs fall into a gene-expressionsubgroup known as GC B-cell-like DLBCLs (GCBDLBCLs),which have a gene-expression profile thatclosely resembles that ofnormal GC B cellsFIG.1BOX 2).Furthermore,these lymphomas have highlymutated immunoglobulin genes and SHM is ongoingin malignant clones.Gene-expression profiling indi-cates also that most GCB DLBCLs have undergoneimmunoglobulin class switching(A.R.and L.M.S., Table 1 |Characteristics of mature B-cell malignanciesMalignancySHMOngoing SHMGC B-cellPutative cell of origin*expressionprofileMantle-cell lymphomaNo (exceptNoNoPre-GC B cellpercentageChronic lymphocyticYes and noNoNoAntigen-experiencedleukaemia (CLL)B cell (pre- or post-GC)Burkitt lymphomaYesNoYesGC B cellFollicular lymphomaYesYesYesGC B cellMarginal-zone lymphomaYes (except forYes (prevalentNoGC B cell or post-GCÑ nodal, extranodal (MALT)some splenicin MALTB celland splenicvariants)lymphomasGC B-cell-like DLBCLYesYesYesGC B cellActivated B-cell-like DLBCLYesNoNoGC B-cell subset orLymphoplasmacyticYesYesNoMultiple myelomaYesNoNoPost-GC B cellHodgkin lymphomaYesNoNoGC or post-GC B cell(classical type)Hodgkin lymphomaYesYesYesGC B cell(nodular lymphocyte pre-dominant type)*Based on the presence or absence of somatic hypermutation (SHM) and the gene-expression profile. CLL consists of two clinicallydistinct subtypes, one with SHM and one without. In some CLLs, subclones can accumulate additional mutations through SHM oranother mutational process. The gene-expression profile of marginal-zone lymphomas (MZLs) has yet to be determined, but MZL B cellslack germinal-centre (GC) markers (such as CD10 and BCL-6) and express marginal-zone markers (such as CD21 and CD35)gene-expression profile has yet to be determined, but LPL has characteristics that relate it to post-GC plasma cells (such as c. DLBCL, diffuse large B-cell lymphoma; MALT, mucosal-associated lymphoid tissue. VOLUME 2    metabolism (for example,glycolytic enzymes),and theyare turned on as lymphocytes ÔblastÕin response to mito-genic stimuli.Many ofthese genes are targets ofthetranscription factor c-MYC.Although GC B cellsexpress c-MYC,they express lower levels ofc-MYCmessenger RNA than other dividing cells.Given thedrive for positive and negative selection in the GC,itmight be appropriate that cells tip the balance in favourofproliferation rather than cell growth to expand thepool ofselectable B cells as rapidly as possible.The expression ofc-MYC is altered by transloca-tions,mutations and/or overexpression in many GC B-cell-derived lymphomas.All Burkitt lymphomas havetranslocations ofc-MYCto immunoglobulin loci,andthese translocations occur also in some DLBCLsBurkitt lymphomas and DLBCLs can also accumulatesomatic mutations ofc-MYCthat might alter its func-tion as a transcription factor.These oncogeniclesions ofc-MYC could increase cell growth and pro-mote tumour-cell proliferationGC B cells seem to be poised forapoptosis unless they are rescued by positive selectionMany anti-apoptotic proteins,such as BCL-2,A1 andBCL-X,are expressed at low levels in most GC B cells.The NF-B signalling pathway transcrip-tionally activates several ofthese genes and delivers apotent anti-apoptotic stimulus to cells.NF-B tran-scription factors are kept in an inactive state in the cyto-plasm by interactions with inhibitor ofNF-B proteinsBs).Signalling through various cell-surface receptorsactivates IB kinase (IKK),which phosphorylates I ofBCL-6cells,which are a possiblenormal counterpart ofABC DLBCLs.Alternatively,asplasma-cell differentiation and SHM can occur outside of,it is possible that ABC DLBCLs are derived fromB cells that have never entered a GC.Oncogenic lesions in lymphomasThe various chromosomal translocations,amplifica-tions,mutations and deletions that occur in B-cell lym-phomas disrupt normal B-cell homeostasis in threeways:by driving the cells through the cell cycle,by pre-venting the normal induction ofcell death and byblocking terminal differentiation.Analysis ofnormalGC B-cell gene expression and function indicates howthese oncogenic events might perturb mature B-cell dif-ferentiation to give a selective advantage to the trans-formed cells (FIG.2)Enhancing cell growth and proliferation.GC B cells are some ofthe most rapidly proliferating cells in thebody,doubling in number every seven hours,and gene-expression profiling has shown that they have a corre-spondingly high level ofexpression ofcell-cycle progres-sion genes that function in the G2/M phase ofthe cellcycle Ñ for example,the genes that encode CDC2(cell-division cycle 2),(polo-like kinase) and BUB1ding uninhibited by benzimidazoles 1 homologue)Interestingly,however,genes that control cell growth(increase in cell size) are expressed at low levels in GC B cells.These genes encode components ofthe protein-translation machinery (for example,ribosomal proteinsand translation-initiation factors) and intermediary Diffuse large B-cell lymphoma: many diseases in one diagnostic category On the basis ofmorphological and clinical criteria,the diagnostic framework that is used at present places diffuse largeB-cell lymphomas (DLBCLs) in a single category and,consequently,all patients receive the same therapy.However,patients with DLBCL are markedly heterogeneous in their response to multi-agent chemotherapy in that ~40% can becured,whereas the remainder succumb to the diseaseRecent gene-expression profiling ofDLBCL has shown that this single diagnostic category includes more than onemolecularly and clinically distinct disease.DLBCL consists ofat least three gene-expression subgroups,known asgerminal-centre B-cell-like (GCB),activated B-cell-like (ABC) and type 3 .The gene-expression subgroupsdiffer by the expression ofmore than 1,000 genes,which makes them as distinct as acute lymphoblastic and acutemyelogenous leukaemias.As discussed in detail in this review,these subgroups seem to be derived from different stagesofnormal B-cell differentiation.The DLBCL gene-expression subgroups have distinct mechanisms ofmalignant transformation,which shows thatthey are pathogenetically distinct diseases.The t(14;18),which involves ,is seen exclusively in GCB DLBCLs and ispresent in ~20% ofthese cases.Similarly,amplification ofthe locus on chromosome 2p occurs only in GCB.By contrast,activation ofthe anti-apoptotic nuclear factor-B (NF-B) pathway is a feature ofABC DLBCL,but not GCB DLBCLThe molecular distinctions between subgroups ofDLBCL are important because the subgroups differ in their abilityto be cured by the multi-agent chemotherapy that is used at present.Patients with GCB DLBCL have the mostfavourable cure rate (60% five-year survival),whereas patients with ABC and type 3 DLBCL have five-year survivalrates ofonly 36% and 39%,respectively.These clinical differences might be owing,in part,to the ability ofthe NF-pathway to block many forms ofcell death,including that induced by chemotherapyFurther gene-expression differences between DLBCLs can affect the success ofchemotherapy.The expression ofgenes that are associated with proliferation predicts poor outcome.By contrast,expression ofMHC class II genes bylymphoma cells predicts a favourable outcome,which indicates that DLBCLs might downregulate expression ofthesegenes to evade an immune response.Some patients mount a reactive lymph-node response to DLBCL cells thatinvolves macrophages,natural killer cells and stromal cells,and this innate immune response is associated with survivalafter chemotherapy NATURE REVIEWS  VOLUME 2 to the nucleus.Gastric mucosal-associated lymphoidtissue (MALT) lymphomas frequently acquire a t(11;18)that leads to the overexpression ofMALT1(REF.53),whichis an activator ofNF-.Less commonly,these lym-phomas have translocations ofthe ,whichencodes a MALT1-interacting proteinthat is requiredfor the activation ofNF-B downstream ofantigen-receptor signalling.In approximately 50% ofHodgkinlymphomas,the malignant cells are infected withEpsteinÐBarr virus (EBV) and they express latent mem-brane protein 1 (),a viral protein that mimicsCD40 signalling and activates NF-Constitutive activation ofthe IKK complex byunknown mechanisms leads to the nuclear localizationofNF-B in ABC DLBCLsand in some Hodgkin lym-.Two cell-line models ofABC DLBCL haveconstitutive IKK activity,rapid degradation ofIconstitutive nuclear localization ofNF-.One possi-ble explanation for this IKK activity is that ABCDLBCLs are arrested at a stage ofB-cell differentiationthat involves the upregulation ofNF-Alternatively,unknown oncogenic alterations in ABCDLBCLs might activate the NF-B pathway.Interferencewith NF-B signalling results in the death ofABCDLBCL cells,but it has no effect on GCB DLBCL cellstherefore,drugs that inhibit the NF-B pathway areattractive candidates for the treatment ofpatients withAmong the DLBCLs,the gene,which encodesan NF-B subunit,is amplified and overexpressed exclu-sively in a subset ofGCB DLBCLs.The selective advan-tage that is conferred by this oncogenic event is unclear,because the GCB DLBCLs that have an amplification ofthe gene do not have increased expression ofNF-(A.R.and L.M.S.,unpublishedobservations).One explanation could be that lym-phoma cells with amplification ofreceiveenhanced anti-apoptotic and/or proliferative signalswhen they are triggered through receptors that activateIKK.In this scenario,amplification might have acrucial role in the early stages ofthe malignant processwhen the lymphoma cell still receives stimulation fromantigen and/or CD40L-bearing activated T cells in theGC.On clinical presentation,these DLBCLs might notbe receiving these signals any longer,so that c-RELwould remain complexed with IB in the cytoplasmand be unable to activate NF-B target genes.Blocking differentiation.In addition to driving prolifera-tion or preventing cell death,the genetic lesions in somelymphomas seem to arrest differentiation In the case ofGC B cells,developmental arrest at this stage is particularly dangerous as these cells areprogrammed to divide extremely rapidly.The mostcommon genetic abnormalities in non-Hodgkin lym-phomas are translocations and mutations ofthe gene,which encodes a transcriptional regulator ofGCB-cell differentiation and proliferation.The gene is the target ofpromiscuous translocations involv-ing many partner chromosomes,and it might be fur-ther dysregulated by SHM ofthe regulatory regions in promoting its degradation by the proteasome.This leadsto the nuclear accumulation ofNF-B and the upregula-tion ofexpression ofa characteristic set ofNF-.In contrast to blood B cells that are stimu-lated through the BCR or CD40,GC B cells have a lowlevel ofexpression ofmany NF-B target genes.Thereduced activity ofthe NF-B pathway in most GC B cells might contribute to apoptosis and negative selec-tion during the GC reaction.However,CD40 and BCRsignalling in some GC B cells (centrocytes) might acti-vate the NF-B pathway and upregulate expression ofBCL-X,A1 and/or BCL-2 ,leading to thesurvival and positive selection ofthose cells.GC B-cell-derived lymphomas use several strategiesto overcome this propensity for apoptosis.Most follicu-lar lymphomas (~90%) and some DLBCLs have ato the IgH locus.Also,the genomic locus can be amplified in DLBCLs,andis transcriptionally upregulated in ABC.In addition,B-cell lymphomas activate theNF-B pathway by various means.Some DLBCLs havetranslocations and truncations ofthe which encodes a subunit ofthe dimeric NF-factors.In Hodgkin lymphomas(BOX 1),gene can be inactivated by deletions or pointmutations,which releases NF-B for translocation Plasma cellGC B cellPlasmablast/plasma cellBCL-6mechanismBBBBBBBBBBApoptosis Figure 2| Mechanisms of malignant transformation of germinal-centre B cells.germinal centre (GC) is a microenvironment in which B cells undergo rapid clonal expansion in thepresence of T cells, follicular dendritic cells (FDCs) and antigen. In a process of positive selection,GC B cells mutate their immunoglobulin genes, and those that acquire mutations that maintain orimprove the affinity of the B-cell receptor (BCR) for antigen are rescued from programmed celldeath and can differentiate further. Most GC B cells express BCL-6 but lack expression ofinterferon-regulatory factor 4 (IRF4) and B-lymphocyte-induced maturation protein 1 (BLIMP1).Some GC B cells have a plasmablastic (PB) phenotype, have turned off expression of BCL-6 andhave turned on expression of IRF4 and BLIMP1. These cells are likely to become plasma cellsc-MYC, which is not expressed highly by normal GC B cells, is often expressed as a result oftranslocation by Burkitt lymphoma cells, which promotes clonal expansion. Apoptosis is blockedby oncogenic activation of the nuclear factor-B) pathway, which occurs in diffuse largevirus-related lymphomas. Apoptosis is also abrogated by translocation, amplification orgene in follicular lymphomas and DLBCLs. Translocation differentiation and promotes the proliferation of GC B cells. Translocation of PAX5(paired box gene 5) in lymphoplasmacytic lymphomas is also likely to block plasmacytic differentiation. VOLUME 2    During normal plasmacytic differentiation,BLIMP1and BCL-6 are involved in a double-negative regulatory(FIG.3),as the expression ofBLIMP1 decreases theexpression ofBCL-6 (REF.71).In the GC,most B cells areBCL-6,but a minority are BCL-6,and these cells are probably in the process ofplasmacytic differentiation and exit from the GC.TheBCL-6GC B cells also express IRF4,which is a target ofNF-B signalling.This is interesting given that twoactivators ofthe NF-B pathway,BCR and CD40 sig-nalling,cause mRNA levels ofBCL-6 to drop markedlyand BCR signalling causes BCL-6 protein degradationThese observations might indicate a model in whichstrong signals through the BCR or CD40 cause theexpression ofBCL-6 to be reduced,which allows thelevel ofBLIMP1 to rise and plasmacytic differentiationto ensue.In this model,translocations make thegene less responsive to these physiological regula-tory influences,thereby blocking the expression ofBLIMP1 and plasmacytic differentiation.In addition to blocking terminal differentiation,BCL-6 might also promote the proliferation ofGC B cells.Another gene that is repressed by BCL-6 is which encodes an inhibitor ofcyclin-dependent kinasesBy inhibiting the expression ofp27KIP1,BCL-6 mightfacilitate the many rounds ofcell division that occur dur-ing a normal GC reaction.In this regard,it is intriguingthat BCL-6 was selected from a genetic screen offibro-blasts for inhibitors ofcellular senescence.Senescence isa programmed cellular response that blocks the cell cycleafter many rounds ofcell division.In fibroblasts,cyclinD1 was found to be required for the inhibition ofsenes-cence by BCL-6,but normal GC B cells have low levels ofcyclin D1.As p27KIP1 can induce senescence,it is possi-ble that the repression ofcould contribute to theability ofBCL-6 to inhibit senescence.In summary,this model proposes that translo-cations trap B cells at the GC stage by simultaneouslyblocking differentiation and promoting unlimited celldivision.Secondary oncogenic changes,possibly medi-ated by errors ofSHM or CSR,might then lead to clini-cally evident lymphomas.DLBCLs,for example,canaccumulate mutations in c-MYCPAX5(paired box gene 5) and other genes as a byproduct of.Therefore,prolonged residence ofa B cell at theGC stage ofdifferentiation might allow cells that acquireoncogenic mutations to be selected,leading to malignantprogression.Escape from terminal B-cell differentiation is also afeature ofanother mature B-cell malignancy,lympho-plasmacytic lymphoma (LPL) (BOX 1),which is a diseaseofpost-GC,immunoglobulin-secreting cells.In 50% ofLPL cases,a translocation juxtaposes the IgH and PAX5.PAX5 is a transcription factor that activates variousB-cell-specific genes,and it is required for the commit-ment ofbone-marrow progenitors to the B-cell lineagePAX5 acts as a master regulator ofB-cell identity byblocking differentiation to other haematopoietic.PAX5 participates in the complicated regu-latory network that guides plasmacytic differentiation byrepressing (REF.81)(FIG.3),another transcription end.In all cases,the coding region remainsintact and so the lymphomas presumably co-opt thenormal function ofBCL-6 in the GC.As the expressionofBCL-6 is lost during terminal plasmacytic differentia-tion,it is probable that translocations cause lym-phomas by prolonging the expression ofBCL-6 beyondits normal developmental limit.To appreciate how thismight be oncogenic,it is important to understand thenormal function ofBCL-6.BCL-6 is a transcriptional repressor that is expressedat the highest level in GC B cells.Mice that are defi-cient in BCL-6 fail to form GCs during T-cell-dependentimmune responses,and they succumb to a fatal inflam-matory disease mediated by T helper 2 (T2) cellsAn insight into this complex phenotype was providedby the demonstration that BCL-6 represses genes thatare involved in B-cell activation,inflammation andterminal differentiation.One important target ofBCL-6 is B-lymphocyte-induced maturation protein 1,which is a crucial regulator ofplasma-cell differentiation.BLIMP1 is a transcriptional repres-sor that extinguishes the entire mature B-cell gene-expression programme,thereby blocking BCR signalling,CSR,SHM and other mature B-cell functions.BLIMP1causes this profound change in gene expression bydirectly repressing the expression oftranscription factorsthat themselves regulate several downstream targets.BLIMP1 also represses c-MYC,which contributes to thecell-cycle arrest that is characteristic ofplasma cellsSo,BLIMP1 causes a global change in gene expression,which results in plasma cells that have only minimalresemblance to mature B cells. GC B cellPlasma cellBLIMP1PAX5 Figure 3| The relationship between B-cell differentiation and lymphomagenesis.transcription factors BCL-6, BLIMP1 (B-lymphocyte-induced maturation protein 1), PAX5 (pairedbox gene 5) and XBP1 (X-box protein 1) form a regulatory circuit that controls the progression ofgerminal-centre (GC) B cells to fully differentiated, immunoglobulin-secreting plasma cells. TheGC B-cell repressor, BCL-6, blocks expression of BLIMP1, which is a master regulator of plasma-cell differentiation. BCL-6 also promotes proliferation by blocking expression of the cell-cycle(REF.67). BLIMP1, when expressed, reciprocally inhibits the expression of BCL-6, extinguishes the B-cell gene-expression programme and inhibits the expression ofproliferation-inducing genes. BLIMP1 also represses PAX5, which induces the expression of B-cell genes and itself represses is required for plasma-cell differentiation. XBP1 probably upregulates the expression of genesthat are essential for plasma-cell functions, such as immunoglobulin secretion. This modelproposes that translocations of PAX5initiate lymphomas by dysregulating thisnetwork, thereby blocking plasmacytic differentiation and promoting proliferation. DLBCL, diffuse large B-cell lymphoma; LPL, lymphoplasmacytic lymphoma. NATURE REVIEWS  VOLUME 2 V-region sequences from these leukaemias Ñ one sub-type has somatically mutated immunoglobulin genes(immunoglobulin-mutated CLL) and the other subtypehas immunoglobulin genes that are close or identical togerm-line sequences (immunoglobulin-unmutated.These subtypes can be distinguished easily bygene-expression profiling.Patients with immuno-globulin-mutated and -unmutated CLL have markedlydifferent clinical courses:immunoglobulin-unmutatedCLL is a progressive disease that often requires earlytreatment,whereas most immunoglobulin-mutatedCLLs are indolent and require late or no treatmentIn keeping with the generally benign course ofimmuno-globulin-mutated CLLs,a significant proportion ofasymptomatic elderly individuals have clonal popula-tions ofB cells that have a CLL surface phenotype andmutated immunoglobulin genesSeveral lines ofevidence point to a role for antigen in(FIG.4).CLL B cells use a biased Vrepertoire andhave non-random combinations ofV,D and J segmentsthat are not characteristic ofnormal blood B cellsFurthermore,certain Vgenes are used differentially byimmunoglobulin-unmutated and -mutated forms ofCLL.For example,the gene is associated almostexclusively with immunoglobulin-unmutated CLL,whereas other Vgenes,such as ,are over-represented in immunoglobulin-mutated CLLAlthough Vbiases point to a role for antigen in thenatural history ofCLL,it is unclear still whether con-tinuous antigenic stimulation is required or whetherantigen is involved only during early clonal expansion.An ongoing role for antigen is favoured by a recentanalysis ofa set ofimmunoglobulin-mutated CLLswith rearrangements.Unlike patients withother types ofimmunoglobulin-mutated CLL,thesepatients have an aggressive disease,with median sur-vival times that are indistinguishable from those ofpatients with immunoglobulin-unmutated CLL.Thecomplementarity-determining regions 3 (CDR3s) ofsegments are restricted in length and sequenceand they are associated preferentially in these patientswith a single immunoglobulin light-chain V region,V(REF.105).The association ofa particular V region with acharacteristic clinical behaviour strongly indicates a rolefor ongoing antigenic stimulation.The nature ofthis antigenic stimulus is unknown,but for some CLL B cells it is probably an autoantigen.Most immunoglobulins ofCLLs react with self-antigens,such as single- and double-stranded DNA,and IgG,andmany are polyreactive.Indeed,two ofthe commonCLL V genes,,are used frequently toproduce rheumatoid factor in other diseases.Theubiquitous presence ofthese self-antigens in the bodywould allow them to stimulate the malignant clonechronically,even as it accumulates to a large cell number.In some patients with CLL,the leukaemic cells havefeatures in common with anergic B cells.Anergic B cells have markedly decreased levels ofsurface IgMwhich is also characteristic ofCLL.The low level ofexpression ofsurface IgM by anergic B cells is owing to factor that is required for plasmacytic differentiationBLIMP1 represses PAX5directly,thereby relieving therepression of(REF.71).The sustained expression ofPAX5 in LPLs,owing to chromosomal translocation,islikely to disrupt this regulation,allowing LPL cells toavoid terminal differentiation.The role of antigen in lymphoid malignanciesDirect and indirect evidence indicates that antigenicstimulation has a role in the pathogenesis ofmany typesoflymphoid malignancy.Despite the frequent occur-rence ofoncogenic translocations involving the IgHlocus,most B-cell malignancies express surface immuno-globulin.Indeed,most IgH translocations involve non-productively rearranged alleles,which indicates that,atleast early in the history ofthe malignant clone,the trans-formed B cell relied on expression ofa functional BCR forsurvival.In most types oflymphoid malignancy,how-ever,it is unclear whether ongoing stimulation ofthetumour cells by antigen has a role in the disease.Direct evidence ofa pathogenic role for antigencomes from a study ofa type ofmarginal-zone lym-(BOX 1)known as gastric MALT lymphomaPatients with this type oflymphoma often have gastritisand/or peptic ulcers on presentation,and many ofthemare infected with Helicobacter pylori.Strikingly,70% ofthese patients can be cured oftheir MALT lymphomasby antibiotic treatment targeted at the H.pylorition.Even some patients with primary DLBCL ofthestomach who are infected with H.pylorican be cured byantibiotic treatmentH.pylori-specific T cells fromthese patients can stimulate the proliferation oftheirMALT lymphoma cells in culture.The BCRs that areexpressed by the lymphoma cells have biased usage ofcertain V-family members and they frequently showintraclonal diversification,but it is not clear which anti-gens they recognizeInfection with hepatitis C virus has been associatedwith lymphoid malignancies in some epidemiological,but not others.Some patients with a type ofmarginal-zone lymphoma known as splenic lymphomawith villous lymphocytes are infected with hepatitis Cvirus,and treatment ofthis infection with interferon-) eradicates the lymphoma in most ofthese.IFN-treatment had no effect on the lym-phomas ofother patients with this disease who werenot infected with hepatitis C virus.Chronic hepatitis Cvirus infection might,therefore,have a pathogenic rolein some lymphomas,and this might involve directstimulation ofthe lymphoma by viral antigensA role for antigen in CLL?Chronic lymphocytic(BOX 1),the most common type ofhuman leukaemia,involves the clonal expansion ofmature B cells that express and have a low level ofexpression ofsurface immunoglobulin.Leukaemic cellsfrom patients with CLL share expression ofa characteris-tic set ofgenes that distinguishes CLL from other lym-phoid malignancies,and so CLL should be viewed as asingle disease.Nevertheless,two subtypes ofCLL wererevealed by the analysis ofrearranged immunoglobulin VOLUME 2    autoimmunity,without an acquired oncogenic abnor-mality in the leukaemic cells.Concluding remarksIt is clearly useful to think like an immunologist whentrying to understand lymphoid malignancies.Thisreview has emphasized that many lymphoid malignan-cies ÔinheritÕa gene-expression programme and,there-fore,part oftheir biology from a stage ofnormal B-celldifferentiation.However,lymphoid malignanciesdiverge from their normal counterparts as a result ofoncogenic alterations that subvert the homeostatic con-trol ofB-cell proliferation,apoptosis and differentiation.Recent molecular insights into these malignanciesprovide several potential targets for therapy.Many lym-phomas engage the NF-B pathway Ñ including ABCDLBCLs,gastric MALT lymphomas,EBV-associatedlymphomas and Hodgkin lymphomas Ñ which makesit an attractive therapeutic target.Velcade/PS-341 is aproteasome inhibitor that inhibits the NF-B pathwaysby stabilizing IB proteins.In Phase II clinical trials,velcade has been shown to induce remission in patientswith multiple myeloma,and it will be an interestingagent to evaluate in patients with lymphoma.BCL-2 isalso an attractive therapeutic target given its frequent a block in transport from the endoplasmic reticulum tothe medial golgi,and this is a feature ofsome CLLsBCR signalling is ineffective in anergic B cells and ischaracterized by diminished calcium oscillations,as is the.Finally,anergic B cells are charac-terized by the constitutive nuclear localization ofNFATP(pre-existing component ofnuclear factor ofactivated T cells) without nuclear translocation ofNF-.CLLcells can also have constitutive nuclear localization ofNFATP,and their gene-expression profiles show noevidence ofNF-B activitypresents a model ofCLL pathogenesis thatemphasizes the potential role ofantigen.In this model,the biological and clinical behaviours ofthe leukaemiadepend on which immunoglobulin V regions wererearranged in the progenitor ofthe malignant clone.Immunoglobulin-unmutated CLL is derived presum-ably from a pre-GC B cell that is nevertheless Ôantigen-experiencedÕ.Immunoglobulin-mutated CLL involvespresumably a B cell that has participated in a GC reac-tion,and that might or might not receive ongoing anti-genic stimulation.One sixth ofall patients with CLLhave leukaemic cells at diagnosis that have no clonalchromosomal abnormalities.So,CLL might begin asa defect in normal B-cell homeostasis similar to Oncogenic hit?Oncogenic hit?Oncogenic hit? Figure 4| Chronic lymphocytic leukaemia: a disease of antigen-experienced B cells.The two subtypes of chronic immunoglobulin-mutated and immunoglobulin-unmutated CLL are distinguished by the presenceor absence of immunoglobulin variable (V)-region mutations, by differences in gene expression and by their clinical courses. Thmodel emphasizes the potential role of antigenic stimulation in the progression of this disease. Immunoglobulin-unmutated and -mutated forms have a different repertoire of heavy-chain V-region (V) gene rearrangements, which indicates that the type of CLL thatresults is dictated by the specificity of the B-cell receptor (BCR). Many studies have implicated an antigen or autoantigen indirectly inthe pathogenesis of CLL. Antigenic stimulation might occur before and/or after the B cell acquires a genetic change (oncogenic unmutated CLL most probably originates from a pre-germinal centre (pre-GC) B cell. Immunoglobulin-mutated CLL might originatefrom a post-GC B cell. Alternatively, immunoglobulin-mutated CLL might originate from a pre-GC B cell that is nevertheless drivantigen through a GC reaction. The clinical manifestations of CLL might be related to whether antigen drives continued clonal expansion or induces an anergic state. Disease progression might be influenced by the accumulation of additional oncogenic hits NATURE REVIEWS  VOLUME 2 1. Fugmann, S. D., Lee, A. I., Shockett, P. E., Villey, I. J. &Schatz, D. G. The RAG proteins and V(D)J recombination:Annu. Rev. Immunol.2. Marculescu, R., Le, T., Simon, P., Jaeger, U. & Nadel, B.V(D)J-mediated translocations in lymphoid neoplasms: afunctional assessment of genomic instability by cryptic sites.3. Boehm, T. . Alternating purinepromote chromosomal translocations seen in a variety of4. Welzel, N. . Templated nucleotide addition andimmunoglobulin JH-gene utilization in t(11;14) junctions:implications for the mechanism of translocation and theorigin of mantle-cell lymphoma. 5. Yu, W. . Continued RAG expression in late stages of B-cell development and no apparent re-induction after6. Gartner, F., Alt, F. W., Monroe, R. J. & Seidl, K. J. Antigen-independent appearance of recombination activating gene(RAG)-positive bone-marrow B cells in the spleens of7. MacLennan, I. C. Germinal centers. Annu. Rev. Immunol.8. Liu, Y. J., Zhang, J., Lane, P. J., Chan, E. Y. & MacLennan, I. C. Sites of specific B-cell activation in primary andsecondary responses to T-cell-dependent and T-cell-Eur. J. Immunol.9. Chua, K. F., Alt, F. W. & Manis, J. P. The function of AID in somatic mutation and class-switch recombination:upstream or downstream of DNA breaks. 10. Honjo, T., Kinoshita, K. & Muramatsu, M. Molecularmechanism of class-switch recombination: linkage withAnnu. Rev. Immunol.11. Papavasiliou, F. N. & Schatz, D. G. Somatic hypermutationof immunoglobulin genes: merging mechanisms for geneticdiversity. 12. Pelicci, P. G., Knowles, D. M. 2nd, Magrath, I. & Dalla-Favera, R. Chromosomal breakpoints and structuralalterations of the c-myc locus differ in endemic and sporadicProc. Natl Acad. Sci. USA13. Rabbitts, T. H., Hamlyn, P. H. & Baer, R. Altered nucleotidesequences of a translocated c-myc gene in Burkitt14. Goossens, T., Klein, U. & Kuppers, R. Frequent occurrenceof deletions and duplications during somatic hypermutation:implications for oncogene translocations and heavy-chainProc. Natl Acad. Sci. USA15. Pasqualucci, L. . BCL-6 mutations in normal germinal-center B cells: evidence of somatic hypermutation actingoutside Ig loci. Proc. Natl Acad. Sci. USA16. Shen, H. M., Peters, A., Baron, B., Zhu, X. & Storb, U.gene in normal B cells by the process of somatic hypermutation of Ig genes. 17. Pasqualucci, L. . Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. This study shows that diffuse large B-cell lymphomas(DLBCLs) can accumulate mutations in non-immunoglobulin genes as a result of somatic18. Neri, A., Barriga, F., Knowles, D. M., Magrath, I. T. & Dalla-Favera, R. Different regions of the immunoglobulin heavy-chain locus are involved in chromosomal translocations inProc. Natl19. Bergsagel, P. L. . Promiscuous translocations intoimmunoglobulin heavy-chain switch regions in multipleProc. Natl Acad. Sci. USA20. Jacob, J., Kelsoe, G., Rajewsky, K. & Weiss, U. Intraclonalgeneration of antibody mutants in germinal centers. 21. Han, S. . Cellular interaction in germinal centers. Rolesof CD40 ligand and B7-2 in established germinal centers. 22. Kawabe, T. . The immune responses in CD40-deficientmice: impaired immunoglobulin class switching andgerminal center formation. 23. Weller, S. CD40L-independent Ig genehypermutation suggests a second B-cell diversificationProc. Natl Acad. Sci. USA24. Matsumoto, M. . Affinity maturation without germinalcentres in lymphotoxin--deficient mice. 25. William, J., Euler, C., Christensen, S. & Shlomchik, M. J.Evolution of autoantibody responses via somatichypermutation outside of germinal centers. References 23Ð25 provide evidence for SHM in theabsence of recognizable germinal centres (GCs).26. Alizadeh, A. A. . Distinct types of diffuse large B-celllymphoma identified by gene-expression profiling. 27. Shaffer, A. L. . Signatures of the immune response.28. Alizadeh, A. . The Lymphochip: a specialized cDNAmicroarray for the genomic-scale analysis of geneexpression in normal and malignant lymphocytes. 29. Rosenwald, A. . The use of molecular profiling to predictsurvival after chemotherapy for diffuse large-B-cellN. Engl. J. Med.References 26 and 29 show the utility of gene-expression profiling for the analysis of human B-celllymphomas, and show that a single diagnosticdistinct diseases with different clinical outcomes.30. Lossos, I. S. mutation in germinal center B-cell-like, but not in activated B-cell-like, diffuse large-cell lymphomas. Proc. Natl Acad.31. Davis, R. E., Brown, K. D., Siebenlist, U. & Staudt, L. M.Constitutive nuclear factor-B activity is required for survivalof activated B-cell-like diffuse large B-cell lymphoma cells. constitutively active in activated B-cell-like DLBCLs,and that it is a potential therapeutic target.32. Angelin-Duclos, C., Cattoretti, G., Lin, K. I. & Calame, K.Commitment of B lymphocytes to a plasma-cell fate isassociated with Blimp-1 expression in vivo33. Falini, B. expression of the MUM1/IRF4 protein in a subset ofgerminal-center B cells, plasma cells and activated T cells.34. Levens, D. Disentangling the MYC web. 35. Martinez-Valdez, H. express the apoptosis-inducing genes , but not the survival gene 36. Cutrona, G. et al. The propensity to apoptosis of centrocytesand centroblasts correlates with elevated levels of intracellularmyc protein. Eur. J. Immunol.238 (1997).37. Dalla-Favera, R., Martinotti, S., Gallo, R. C., Erikson, J. &Croce, C. M. Translocation and rearrangements of the c-myc oncogene locus in human undifferentiated B-cell38. Akasaka, T. . Molecular and clinical features of non-s, diffuse large-cell lymphoma of B-cell typeassociated with the c-MYC/immunoglobulin heavy-chainJ. Clin. Oncol.39. Hoang, A. T. . A link between increased transformingactivity of lymphoma-derived MYC mutant alleles, theirdefective regulation by p107 and altered phosphorylation of40. Liu, Y. J. . Germinal-center cells express bcl-2 proteinafter activation by signals which prevent their entry intoEur. J. Immunol.41. Pezzella, F. . Expression of the bcl-2 oncogene protein isnot specific for the 14;18 chromosomal translocation. 42. Craxton, A., Chuang, P. I., Shu, G., Harlan, J. M. & Clark, E. A. The CD40-inducible Bcl-2 family member A1 protectsB cells from antigen receptor-mediated apoptosis. 43. Karin, M. & Lin, A. NF-B at the crossroads of life and death.44. Hinz, M. B maintains high expressionof a characteristic gene network, including CD40, CD86 anda set of antiapoptotic genes in Hodgkin/Reed-Sternberg45. Pahl, H. L. Activators and target genes of Rel/NF-46. Tuscano, J. M. . Bcl-X rather than Bcl-2 mediatesCD40-dependent centrocyte survival in the germinal center.Does BCR signalling contribute to the proliferation ofmalignant lymphocytes,and should this be targeted ther-apeutically? Lymphomas that occur in the context ofchronic stimulation by H.pylorior hepatitis C virus pro-vide precedents for these possibilities.Lymphomas mightbe stimulated also by autoantigens,as might be the casefor CLL.In this regard,it is interesting that patients withrheumatoid arthritis,and possibly other autoimmunediseases,have an increased risk oflymphoma in cohort.It is conceivable,therefore,that polymorphicalleles ofkey immunoregulatory genes could be associ-ated with an increased risk ofboth autoimmune diseaseand lymphoid malignancies.Although many oftheseconsiderations are speculative,recent progress has pro-vided hope that further insights into the pathogenesis oflymphoid malignancies will come from our knowledge ofnormal immune homeostasis.overexpression in lymphomas by various mechanisms.Recent studies have indicated that small-moleculeinhibitors that target the BCL-2 might beeffective as they could allow pro-apoptotic BH3-onlyproteins to interact with (BCL-2-associated Xprotein) and/or BAK(BCL-2 antagonist/killer) and initiate apoptosisMany questions remain.In most lymphomas andleukaemias,we have no idea which signalling pathwaysdrive proliferation.The importance ofidentifying thesepathways is highlighted by the fact that one ofthe mostimportant factors predicting poor survival afterchemotherapy for DLBCL is proliferation.Do onco-genic alterations in the genomes oflymphoma cells driveproliferation? Do B-cell lymphomas depend on T-cellinteractions during some phase oftheir generation and,ifso,would inhibition ofCD40 signalling be beneficial? VOLUME 2    47. Bakhshi, A. . Cloning the chromosomal breakpoint oft(14;18) human lymphomas: clustering around JH onchromosome 14 and near a transcriptional unit on 18. 48. Tsujimoto, Y., Cossman, J., Jaffe, E. & Croce, C. M.Involvement of the bcl-2 gene in human follicular lymphoma.49. Monni, O. . BCL2 overexpression associated withchromosomal amplification in diffuse large B-cell lymphoma.50. Neri, A. . Molecular analysis of cutaneous B- and T-cell51. Cabannes, E., Khan, G., Aillet, F., Jarrett, R. F. & Hay, R. T.Mutations in the gene in Hodgkintumour suppressor role for I52. Jungnickel, B. . Clonal deleterious mutations in the Igene in the malignant cells in HodgkinJ. Exp.53. Akagi, T. . A novel gene, MALT1 at 18q21, is involved int(11;18) (q21;q21) found in low-grade B-cell lymphoma of54. Lucas, P. C. . Bcl10 and MALT1, independent targets ofchromosomal translocation in MALT lymphoma, cooperatein a novel NF-B signaling pathway. 55. Uren, A. G. . Identification of paracaspases andmetacaspases: two ancient families of caspase-likeproteins, one of which plays a key role in MALT lymphoma.56. Zhang, Q. . Inactivating mutations and overexpressionof BCL10, a caspase recruitment domain-containing gene,in MALT lymphoma with t(1;14)(p22;q32). 57. Ruland, J. . Bcl10 is a positive regulator of antigenreceptor-induced activation of NF-closure. 58. Sylla, B. S. Barr virus-transforming proteinlatent infection membrane protein 1 activates transcriptionfactor NF-B through a pathway that includes the NF-inducing kinase and the INatl Acad. Sci. USA59. Krappmann, D. . Molecular mechanisms of constitutiveB/Rel activation in Hodgkin/Reed-Sternberg cells.60. Staudt, L. M., Dent, A. L., Shaffer, A. L. & Yu, X. Regulationof lymphocyte cell-fate decisions and lymphomagenesis byInt. Rev. Immunol.61. Dalla-Favera, R. . Molecular pathogenesis of B-cellmalignancy: the role of BCL-6. Curr. Top. Microbiol.62. Allman, D. . BCL-6 expression during B-cell activation.63. Cattoretti, G. . Bcl-6 protein is expressed in germinal-center B cells. 64. Dent, A. L., Shaffer, A. L., Yu, X., Allman, D. & Staudt, L. M.Control of inflammation, cytokine expression and germinal-center formation by BCL-6. 65. Ye, B. H. . The BCL-6 proto-oncogene controlsgerminal-centre formation and T66. Fukuda, T. . Disruption of the gene results in animpaired germinal-center formation. References 64Ð66 use Bcl-6-deficient mice to showthat Bcl-6 is required for GC formation and that itregulates inflammation.67. Shaffer, A. L. . BCL-6 represses genes that function inlymphocyte differentiation, inflammation and cell-cyclecontrol. 68. Reljic, R., Wagner, S. D., Peakman, L. J. & Fearon, D. T.Suppression of signal transducer and activator ofdifferentiation by BCL-6. 69. Vasanwala, F. H., Kusam, S., Toney, L. M. & Dent, A. L.Repression of AP-1 function: a mechanism for the regulationof Blimp-1 expression and B-lymphocyte differentiation bythe B-cell lymphoma-6 protooncogene. References 67Ð69 identify genes that are inhibited byBCL-6 in B cells, including genes that control the cell) and differentiation (70. Turner, C. A. Jr, Mack, D. H. & Davis, M. M. Blimp-1, a novelzinc-finger-containing protein that can drive the maturationof B lymphocytes into immunoglobulin-secreting cells. 71. Shaffer, A. L. . Blimp-1 orchestrates plasma-celldifferentiation by extinguishing the mature B-cell gene-expression program. This study shows that BLIMP1 acts as the masterregulator of plasma-cell differentiation byextinguishing gene-expression programmes thatdirect proliferation and GC B-cell functions, such asclass-switch recombination. BLIMP1 mediates thesebroad effects by regulating the expression of othertranscription factors directly and by reciprocallyrepressing 72. Lin, Y., Wong, K. & Calame, K. Repression of c-myctranscription by Blimp-1, an inducer of terminal B-celldifferentiation. 73. Niu, H., Ye, B. H. & Dalla-Favera, R. Antigen-receptorand degradation of the BCL-6 transcription factor. Dev.74. Shvarts, A. . A senescence rescue screen identifiesBCL6 as an inhibitor of anti-proliferative p19(ARF)Genes Dev.75. Alexander, K. & Hinds, P. W. Requirement for p27(KIP1) inretinoblastoma protein-mediated senescence. 76. Iida, S. et al. The t(9;14)(p13;q32) chromosomal translocationassociated with lymphoplasmacytoid lymphoma involves thePAX-5 gene. 77. Urbanek, P., Wang, Z. Q., Fetka, I., Wagner, E. F. &Busslinger, M. Complete block of early B-cell differentiationand altered patterning of the posterior midbrain in micelacking Pax5/BSAP. 78. Nutt, S. L., Heavey, B., Rolink, A. G. & Busslinger, M.79. Mikkola, I., Heavey, B., Horcher, M. & Busslinger, M.expression. 80. Horcher, M., Souabni, A. & Busslinger, M. Pax5/BSAPmaintains the identity of B cells in late B lymphopoiesis.References 77Ð80 delineate the essential role of PAX5in committing cells to and maintaining them in the 81. Reimold, A. M. . Transcription factor B-cell lineage-specific activator protein regulates the gene for human X-box binding protein 1. 82. Reimold, A. M. . Plasma-cell differentiation requires the83. Lin, K. I., Angelin-Duclos, C., Kuo, T. C. & Calame, K. Blimp-1-dependent repression of Pax-5 is required fordifferentiation of B cells to immunoglobulin-M-secreting84. Vaandrager, J. W. . DNA fiber fluorescence in situhybridization analysis of immunoglobulin class switching inB-cell neoplasia: aberrant CH gene rearrangements in folliclecenter-cell lymphoma. 85. Lam, K. P., Kuhn, R. & Rajewsky, K. surface immunoglobulin on mature B cells by inducible genetargeting results in rapid cell death. 86. Cavalli, F., Isaacson, P. G., Gascoyne, R. D. & Zucca, E.MALT lymphomas. Hematology (Am. Soc. Hematol. Educ.87. Wotherspoon, A. C. . Regression of primary low-grade88. Bayerdorffer, E. . Regression of primary gastriclymphoma of mucosa-associated lymphoid tissue type aftercure of infection. MALT LymphomaStudy Group. 89. Morgner, A. . Complete remission of primary high-gradeB-cell gastric lymphoma after cure of J. Clin. Oncol.90. Hussell, T., Isaacson, P. G., Crabtree, J. E. & Spencer, J. Theresponse of cells from low-grade B-cell gastric lymphomas91. Qin, Y. associated lymphoid tissue-type B-cell lymphoma. 92. Du, M. . Ongoing mutation in MALT lymphomaplays a role in the clonal expansion. 93. Zuckerman, E. . Hepatitis C virus infection in patients94. Rabkin, C. S. . Prospective study of hepatitis C viralinfection as a risk factor for subsequent B-cell neoplasia.95. Hermine, O. . Regression of splenic lymphoma withvillous lymphocytes after treatment of hepatitis C virusN. Engl. J. Med.96. Quinn, E. R. . The B-cell receptor of a hepatitis C virus(HCV)-associated non-Hodgkin lymphoma binds the viral E2envelope protein, implicating HCV in lymphomagenesis.97. Jaffe, E. S., Harris, N. L., Stein, H., Vardiman, J. W. Tumoursof Haematopoietic and Lymphoid Tissues(IARC, Lyon,98. Klein, U. . Gene-expression profiling of B-cell chroniclymphocytic leukemia reveals a homogeneous phenotyperelated to memory B cells. 99. Rosenwald, A. . Relation of gene expression phenotypeto immunoglobulin mutation genotype in B-cell chroniclymphocytic leukemia. References 98 and 99 use gene-expression profilingto show that chronic lymphocytic leukaemia (CLL) is asingle disease with at least two variants.100. Damle, R. N. . Ig V gene mutation status and CD38expression as novel prognostic indicators in chroniclymphocytic leukemia. 101. Fais, F. . Chronic lymphocytic leukemia B cells expressrestricted sets of mutated and unmutated antigen receptors.102. Hamblin, T. J., Davis, Z., Gardiner, A., Oscier, D. 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Preclinical and clinical evaluation of proteasomeinhibitor PS-341 for the treatment of cancer. Curr. Opin.Chem. Biol.122. Letai, A. activate mitochondrial apoptosis, serving as prototypecancer therapeutics. Cancer Cell123. Mellemkjaer, L. . Rheumatoid arthritis and cancer risk.Eur. J. Cancer124. Coiffier, B. Diffuse large-cell lymphoma. Curr. Opin. Oncol.125. Huang, J. Z. diffuse large B-cell lymphoma with a germinal center B-cellgene-expression profile. 126. Baldwin, A. S. Control of oncogenesis and cancer-therapyresistance by the transcription factor NF-J. Clin. Invest.127. Shipp, M. A. et al. Diffuse large B-cell lymphomaoutcome prediction by gene-expression profiling andsupervised machine learning. 128. Hagman, J. . Pax-5/BSAP: regulator of specific geneexpression and differentiation in B lymphocytes. Curr. Top.129. Usui, T. . Overexpression of B-cell-specific activatorprotein (BSAP/Pax-5) in a late B cell is sufficient to suppressdifferentiation to an Igproducer cell with plasma-cell130. Rosenwald, A. & Staudt, L. M. Clinical translation of gene-expression profiling in lymphomas and leukemias. 131. Thorselius, M. . Somatic hypermutation and V(H) geneusage in mantle-cell lymphoma. Eur. J. Haematol.132. Algara, P. . Analysis of the IgV(H) somatic mutations insplenic marginal-zone lymphoma defines a group ofunmutated cases with frequent 7q deletion and adverseclinical course. 133. Thiede, C. . Ongoing somatic mutations and clonalexpansions after cure of gastric mucosa-associated lymphoid tissue B-cellJ. Clin. Oncol.134. Gurrieri, C. . Chronic lymphocytic leukemia B cells can undergo somatic hypermutation and intraclonalWe wish to thank the members of the Staudt lab and theLymphoma/Leukemia Molecular Profiling Project for discussions The following terms in this article are linked online to:Cancer.gov:http://www.cancer.gov/search/Entrez:http://www.ncbi.nlm.nih.gov/Entrez/EBV | LMP1http://www.ncbi.nlm.nih.gov/LocusLink/CD5 | CD10 | CD40 | CD40L | CD44 | CD77 synthase | CDC2 | | cyclin D1 | cyclin D2 | cyclin D3 | FGFR3 || IRF4 | JAW1 | lymphotoxin-MALT1 | NFATP | | PAX5 | PIM1 | PLK | RAG1 |Swiss-Prot:http://ca.expasy.org/sprot/sprot-top.htmlLymphoma/Leukemia Molecular Profiling Project:Access to this interactive links box is free online.