Biomarkers of potential prognostic significance in diffuse large B-cell lymphoma

Biomarkers of Potential Prognostic Significance inDiffuse Large B-Cell Lymphoma

Grace Wu, M.D.

Armand Keating, M.D.

Department of Medical Oncology and Hematology,Princess Margaret Hospital/Ontario Cancer Insti-tute, University of Toronto, Toronto, Ontario, Can-ada.

Address for reprints: Grace Wu, M.D., Departmentof Medical Oncology and Hematology, PrincessMargaret Hospital, 5-211, 610 University Ave.,Toronto, Ontario M5G 2M9, Canada; Fax: (416)946-4530; E-mail: [email protected]

Received June 14, 2005; accepted July 22, 2005.

Diffuse large B-cell lymphoma (DLBCL) is a biologically heterogeneous disease for

which the current approach to treatment is only successful for 50% of patients. The

prognostic value of various clinical and biological factors in predicting treatment

outcome is discussed in this paper. A review of the English literature was per-

formed including original articles and relevant reviews from MEDLINE that ad-

dressed the topics of DLBCL biology and potential prognostic factors. The Inter-

national Prognostic Index is, to date, the most successful clinical model for

predicting outcome. In addition, a rapidly expanding list of molecules has been

identified by conventional and newer diagnostic methods that may be of signifi-

cant prognostic value. Gene expression profiling has led to the discovery of new

biological subtypes of DLBCL based on patterns of gene expression, and a host of

new genes that may play important roles in this disease. Various derangements in

apoptosis, cell-cycle regulation, differentiation, and signal transduction have been

noted, while the host environment and immune response also appear to modify

clinical outcome. Although to our knowledge, the fundamental abnormalities

underlying DLBCL remain elusive, progress is being continuously made to further

the understanding of the biological heterogeneity of this disease and the use of

various clinical and biological variables to predict treatment outcome. The goal is

to be able to identify subgroups of patients at high risk of treatment failure and

develop more effective treatment based on specific biological defects that may

represent new rational therapeutic targets. Cancer 2006;106:247–57.

© 2005 American Cancer Society.

KEYWORDS: diffuse large B-cell lymphoma, biomarkers, prognosis, internationalprognostic index, gene expression profiling.

D iffuse large B-cell lymphoma (DLBCL), the most common subtypeof non-Hodgkin lymphoma (NHL) in adults,1 is a potentially

curable disease. Nonetheless, with currently available treatment,long-term remission can only be achieved in about 50% of patients.2,3

The ability to predict, at diagnosis, which patients will respond tostandard therapy, and who will likely experience relapse, can provideinvaluable information for stratifying patients according to risk andguiding research efforts to identify the best treatment for differentsubgroups of patients. Patients with poor prognosis disease, for ex-ample, may benefit from participating in studies investigating moreaggressive regimens or other experimental therapies. Herein, we dis-cuss the prognostic value of various clinical and biologic factors forpatients with DLBCL based on a review of the available literature.

Clinical ParametersClinical parameters that measure disease burden and patient charac-teristics have been shown in various studies to be significantly asso-

247

© 2005 American Cancer SocietyDOI 10.1002/cncr.21586Published online 8 December 2005 in Wiley InterScience (www.interscience.wiley.com).

ciated with outcome.4 In the early 1990s, a new prog-nostic model was developed based on five clinicalparameters that were independent predictors of out-come.4 Known as the International Prognostic Index(IPI), this index remains the best-validated and mostsuccessful tool in predicting survival to date. Com-posed of five clinical variables of age, stage of disease,extranodal sites, performance status, and serum lac-tate dehydrogenase (LDH) level, newly diagnosed pa-tients with low-risk IPI scores were found to have a5-year survival estimate of 73%, compared with 26%for the high-risk group.4 For relapsed patients � 60years who are eligible for high-dose therapy with stemcell support, a modified version of the IPI—the age-adjusted IPI based on disease stage, performance sta-tus, and serum LDH level—retains its usefulness as aprognostic tool.5,6 However, further efforts to intensifytreatment based on poor risk as predicted by the indexhave met with inconsistent results.7 One explanationmay be that the IPI, constructed according to theability to predict clinical outcome, may not, and is notmeant to, reflect the biologic heterogeneity which un-derlies this diagnostic entity. Inherent biological dif-ferences may prove to be stronger determinants ofresponse to specific types of treatment. Until the bio-logic differences within DLBCL are better understood,and demonstrated to affect treatment outcome, theIPI remains an important and valuable tool for pre-dicting outcome in patients with DLBCL.

MorphologyThe heterogeneity of DLBCL is readily apparent in thediverse morphologic features found within this diag-nostic entity, which has historically formed the basisof its subclassification.8 The latest classification, as setout by the World Health Organization (WHO), recog-nizes a number of morphologic subtypes includingcentroblastic, immunoblastic, anaplastic, and moreuncommon variants of DLBCL.1 Although severalstudies demonstrated a significant association be-tween the centroblastic subtype and a favorable out-come,9,10 other studies were unable to detect any sur-vival difference.11,12 There is also concern regardingthe intra- and interobserver reproducibility in estab-lishing morphologic subtypes.1 Therefore, at the cur-rent time, there are insufficient data to support the useof morphologic features as prognostic factors.

Early BiomarkersSurface immunophenotypeThe advent of immunophenotyping techniques hasgreatly facilitated the diagnosis of lymphoproliferativedisorders. For patients with DLBCL, immunopheno-

typing may also uncover new subsets of disease basedon the presence of specific surface markers.

In addition to markers of B-cell lineage (e.g.,CD19, CD20, and CD79a), 25–50% of de novo DLBCL(not arising from antecedent follicular lymphoma) arealso found to express CD10.1,13 A membrane metallo-proteinase expressed primarily in early lymphoid pro-genitors and B lymphocytes in the germinal center(GC) of lymphoid follicles,14 the prognostic signifi-cance of CD10 is presently unclear, with conflictingreports of superior,15,16 neutral,17–19 and inferior out-come.20,21 Its role, if any, in DLBCL is also uncertain.However, CD10 is a marker of cells originating fromlymphoid germinal centers and, as such, has beenstrongly associated with the GC-like subtype of DL-BCL16 –18 as defined by gene expression studies.22,23

Gene expression profiling studies have demon-strated two distinct subsets of DLBCL based on pat-terns of gene expression.22,23 One subset’s pattern ofexpression resembles that of lymphocytes found inlymph node GCs, termed GC-like. The other subset,with a gene expression pattern corresponding to thatof activated peripheral blood B cells, is known as ac-tivated B cell-like (ABC). CD10 is expressed primarilyin the GC subset of DLBCL, a subset that is associatedwith a significantly better outcome compared to theABC subtype.22,23 CD10 may, in fact, define a furthersubgroup of DLBCL within the GC group that is asso-ciated with the t(14;18) translocation,23 which juxtaposesBCL-2 with the immunoglobulin heavy chain.17,24 Theresultant overproduction of bcl-2, an antiapoptotic pro-tein, may confer an adverse disease phenotype and neg-atively affect the outcome of cases with this transloca-tion. With uncertain predictive value, CD10 shouldprimarily be used as one of the markers of the GC sub-type of DLBCL, rather than a prognostic marker.

CD5, a T-cell marker also found in mantle celllymphoma and chronic lymphocytic leukemia, maydefine a unique subset of DLBCL. Accounting for up to10% of de novo DLBCL, CD5-positive cases have beenassociated with an inferior survival18,19,25 and, inter-estingly, a distinct gene expression profile in a smallstudy.26 The molecular abnormalities or exact role ofCD5 in DLBCL are currently unclear.

CD40, present in 67–76% of DLBCL, is a memberof the tumor necrosis factor (TNF) receptor family thatcan be found in all stages of B-cell development.18 Itsexpression appears to be critical for lymphocyte mat-uration in GCs, and has been associated with pro-longed survival in one study.18 Of interest are thespeculations regarding its mechanisms of action, in-cluding enhancement of the immune response viacross-linking of CD40 and its ligand on activated Tlymphocytes, which corroborates the importance of a

248 CANCER January 15, 2006 / Volume 106 / Number 2

competent immune response in disease control. An-other proposed mechanism is the modulation of ap-optosis via up-regulation of a proapoptotic protein(bax).18 The prognostic significance of CD40 awaitsconfirmation by other groups.

CD138, also known as syndecan-1, is a heparansulfate proteoglycan expressed in normal and malig-nant plasma cells, as well as lymphomas exhibitingplasmacytic differentiation.27 Identified in only 2% ofDLBCL in one study,27 but much more commonly inhuman immunodeficiency virus (HIV)-associatednon-Hodgkin lymphoma,28 CD138 is primarily used asa marker of DLBCL arising from a post-GC stage ofdevelopment rather than an independent prognosticfactor.27

Cytogenetic abnormalities in DLBCLRecurrent cytogenetic abnormalities found in DLBCLmay pinpoint candidate genes that are potentially re-sponsible for the early steps of lymphomagenesis. Themajority of genes identified to date appear to affectthe apoptotic and/or cellular proliferation pathways.For example, two of the most common translocationsfound in DLBCL, t(14;18) and t(3;14), respectively, jux-tapose BCL-2, which encodes an antiapoptotic proteinon 18q21,29,30 and BCL-6, which encodes a prolifera-tive transcription factor on 3q27,31 with the immuno-globulin heavy chain on 14q32, leading to their up-regulation. Other recurrent chromosomal gains, suchas 8q32–34 and 2p,32,35 are correlated with up-regula-tion of c-myc, a proliferative protein, and rel, whichplays a role in preventing apoptosis. Loss of 17p,31,33,35

conversely, correlates with deletion of the tumor sup-pressor gene encoding for the p53 protein. The cellularpathways affected by alteration of the above genes, aswell as other key molecules implicated in DLBCL, willbe discussed in the following sections.

Derangement of Apoptosis: BCL-2, NF-�BMolecular abnormalities in DLBCLApoptosis, or programmed cell death, is mediated byboth an intrinsic, mitochondrial pathway, as well as anextrinsic, death receptor-mediated pathway.36 The ex-trinsic pathway leads to eventual cell death via a seriesof caspases that are counteracted by a number ofmolecules found upregulated by gene expression pro-filing studies in DLBCL (as explained below).36 –38 Theintrinsic apoptotic pathway is regulated by the bal-ance of pro- versus antiapoptotic proteins,36,38 ofwhich bcl-2 is the most extensively studied in relationto DLBCL.

BCL-2Normally suppressed in the GC stage of B-cell devel-opment,39,40 bcl-2 protein is overexpressed in 30 –50%of DLBCL,1 affecting both the GC and ABC subsets asdefined by gene expression profiling studies.22 It isbelieved to confer a survival advantage by opposingapoptosis, and mediates resistance to conventionalchemotherapy.41 Consequently, BCL-2 expression isgenerally associated with an inferior outcome in DL-BCL.17,35,41– 48

In addition to gene amplification,31,32,34,35,49 over-expression of bcl-2 may result from juxtaposition withthe promoter region of the immunoglobulin heavychain29,30 or activation by the nuclear factor kappa B(NF-�B) antiapoptotic pathway.38,50 The t(14;18)translocation, leading to deregulated transcription ofBCl-2, appears to be found primarily within DLBCL ofthe GC subtype.23,24 It may define a subset with apotentially distinct pattern of gene expression com-pared with other GC cases.51 In one study, cases pos-itive for t(14;18), found in 20 –30% of DLBCL, appearedto overexpress genes associated with apoptosis, genetranscription, and cell-adhesion, whereas t(14;18)-negative cases seemed to involve cell-cycle regulatorygenes.51 To our knowledge, there is currently no con-sensus regarding the predictive value of t(14;18), withstudies demonstrating no difference24,41,44,51,52 in out-come, or inferior survival.53

NF-�B Antiapoptotic PathwayThe NF-�B family of transcription factors promotescell survival by the induction of other antiapoptoticproteins, including inhibitors of apoptosis (IAP), c-flip,and antiapoptotic mitochondrial proteins (e.g., bcl-2).37 c-Rel, a member of the NF-�B family, was foundprimarily within the GC subtype of DLBCL.23 Its prog-nostic significance is not established.54 Survivin is anIAP which blocks apoptotic caspase activity.38 It re-sults in increased bcl-2 expression,55 and may play anadditional antiapoptotic role in the transition from G2

to M-(mitosis) phase in the cell cycle.56 Its associationwith an unfavorable outcome is demonstrated by aretrospective analysis of data from a prospective ther-apeutic trial.56 c-Flip, which blocks the activation ofproapoptotic caspase 8,36,37 is associated with the ABCsubgroup,22 although its prognostic value, in isolation,is unclear at this time.

Constitutive activation of the NF-�B pathway ap-pears to be a prominent feature of DLBCL of the ABCsubtype, as shown by an elegant study using ABC-likecell lines.57 In that study, inhibition of NF-�B activitywas shown to produce cell-cycle arrest and apoptosisin cell lines of the ABC but not the GC subtype of

DLBCL Prognostic Markers/Wu and Keating 249

DLBCL.57 Activation of NF-�B leads to overexpressionof BCL-2, c-FLIP, CCND2, and MUM1/IRF-4. Cyclin D2promotes cell-cycle progression from the G1 to S-(DNA synthesis) phase. MUM1/IRF-4, implicated inlymphocyte activation and terminal B-cell differentia-tion,58 is a marker of DLBCL of the ABC subtype. Itappears to be associated with inferior survival.16,42

Activation of NF-�B appears to be sustained byincreased I�B (inhibitor of NF-�B) kinase activity andenhanced degradation of NF-�B inhibitors (I�B).57

This provides prosurvival and proliferation signals tomalignant cells, potentially forming the basis of infe-rior clinical outcome encountered in the ABC subtypeof DLBCL.57 This unopposed activation may be antag-onized by a proteasome inhibitor such as bortezomib(Velcade; Millennium Pharmaceuticals, Cambridge,MA), which inhibits the degradation of I�B (NF-�Binhibitor) in the ubiquitation/proteosome pathway,among other mechanisms of action.59

Derangement of Cell-Cycle Progression: BCL-6, p53, c-MYC, and OthersIn addition to escaping normal apoptotic mecha-nisms, lymphoma cells can acquire further growthadvantage via breakdown of cell-cycle control, leadingto unchecked cellular proliferation.38 The critical junc-tion between G1 (gap 1) and S-phase appears to be amajor site of derangement in DLBCL.38 Cell-cycle pro-gression to S-phase is promoted by a complex com-posed of regulatory D cyclins (e.g., cyclin D1, D2, andD3) and cyclin-dependent kinases (CDK). Expressionof cyclin D2 in DLBCL, upregulated by bcl-6 and theNF-�B pathway, has been associated with a poor prog-nosis.16,42

The pro-proliferative D cyclin/CDK complex is in-hibited by a number of CDK inhibitors, includingp21CIP1, p27KIP1, p16INK4a, and p19ARF.38 These CDKinhibitors are, in turn, regulated by other transcriptionfactors (e.g., c-myc) and tumor suppressors (e.g., p53)that appear to be variably deranged in DLBCL.

p53Inhibitory p21CIP1 is induced by p53, a tumor suppres-sor gene sometimes viewed as “guardian of the ge-nome.” When genomic damage is detected, p53 ini-tiates cell-cycle arrest in G1 to allow time for repair, or,if damage is too extensive, directs the cell towardsapoptosis.38 Mutated or deleted in many solid tumorsand hematologic malignancies,38 p53 abnormalitieshave been found in at least 15–20% of DLBCL.17,35

Associated with p53 derangement are drug resis-tance60 and decreased survival in a number of stud-ies,60 – 62 but not others.17,43,47,48

p19ARF supports p53 by binding to, and thereby

inhibiting, mdm2, a molecule that promotes p53 deg-radation.38 p16INK4a, which directly inhibits the D cy-clin/CDK complex, appears to be inactivated by ge-netic and epigenetic mechanisms in a number ofDLBCL cases,38,63 and has been shown to be an ad-verse prognostic marker.63 A fourth inhibitor, p27KIP1,is a target of repression by the growth-promoting tran-scription factors bcl-6 and c-myc.38

c-MYCc-MYC, deregulated in Burkitt lymphoma, is also over-expressed in up to 15% of DLBCL as a result of the t(8,14)translocation or gene amplification (8q24).31,35,38,49 It ac-tivates genes that promote cell-cycle progression (e.g.,cyclin D1, D2, and CDK4) and suppresses inhibitoryproteins like p21CIP1 and p27KIP1.38 Paradoxically, c-mycalso promotes apoptosis via the p19ARF/p53 apoptoticpathway.38 c-Myc appears to be more commonly foundin DLBCL with extranodal involvement (especially in-volving the gastrointestinal tract) and HIV-positive pa-tients,64–66 and is generally, but not consistently65,67 as-sociated with a more aggressive clinical course.49,64,68

BCL-6Bcl-6 promotes cell proliferation and blocks differen-tiation13,38,69,70 by acting as a transcriptional repressorof a number of genes, including inhibitory p27KIP1,cyclin D2 (CCND2), and BLIMP-1 (a repressor of c-myc), as well as other molecules involved in B-cellactivation and inflammatory response.31,38

Expressed in over 50% of DLBCL, bcl-6 is primar-ily found in B lymphocytes in the GC stage of devel-opment.69,70 It may be up-regulated as a result of genemutation,71 amplification,32 or chromosomal translo-cation.31 In translocation, it most frequently associateswith the immunoglobulin heavy chain (IgH), found in20 –30% of DLBCL cases, but also associates with non-IgH partners.72 Two recent studies reported that trans-locations involving the IgH were associated with in-creased bcl-6 expression and a better prognosis,whereas translocations with non-IgH partners re-sulted in lower bcl-6 expression and an inferior out-come.73,74

With few exceptions,17,75 cases of DLBCL with in-creased BCL-6 expression have a favorable progno-sis,17,42,74,76,77 although the exact mechanism(s) thatmay confer a better outcome are unclear. One expla-nation may be the strong association of bcl-6 with theGC subtype of DLBCL, which is, in turn, correlatedwith a superior outcome.22,23 However, bcl-6 expres-sion can also be found in 31% of the ABC subtype ofDLBCL (vs. 91% of GC subtype).22 Further researchinto the molecular processes that lead to or resultfrom BCL-6 expression may provide clues to the


role(s) and prognostic significance of BCL-6 in theheterogeneous disease known as DLBCL.

Gene Expression ProfilingMicroarray technology provides an exciting and pow-erful new tool in the study of lymphoma biology. Us-ing DNA or oligonucleotide microarrays, the activity ofa whole host of genes and molecular pathways can beassessed simultaneously. This provides an unprece-dented opportunity to study the heterogeneity of DL-BCL, identify genes and molecular pathways that mayplay critical roles in this disease, and generate newclassification systems based on biological characteris-tics.22,23,75

Cell of OriginGene expression profiling studies have sought to sub-classify DLBCL based on similarities in gene expres-sion22,23 as well as prespecified outcomes like “cured”vs. “fatal/refractory.”75 Using hierarchical clustering,which groups cases according to degrees of similarityin gene expression, Alizadeh et al.22 successfully sep-arated DLBCL into two groups with gene expressionpatterns that resembled, respectively, those of germi-nal centers (GC) B-cells and activated peripheralblood B cells (ABC). Thus classified based on putativecells of origin (GC vs. postgerminal center/peripheralblood), it was found that DLBCL of the GC B cell-likesubtype was associated with a significantly superior5-year survival compared with the ABC subtype (76%vs. 16%; P � 0.01).22

In the study, genes strongly identified with the GCsubtype of DLBCL were CD10, BCL-6, and LMO2, amolecule with potential roles in angiogenesis42 andinhibition of B-cell differentiation.22 In a differentstudy, BCL-6 and LMO2 were found to be strong pre-dictors of survival.42 Representative genes in the ABCsubtype include IRF4/MUM1, FLIP, and BCL-2.22 IRF4(interferon regulatory factor 4), also known as MUM1(multiple myeloma oncogene 1), is a transcription fac-tor involved in the final stage of B-cell differentiationin the GC toward plasma cells.16,27 It is expressed in50% of DLBCL, a marker of cells postgerminal-centerstage of development.16 Flip, or FLICE-like inhibitoryprotein, blocks the apoptotic activity of caspase 8, asdiscussed previously.25 Two other genes overex-pressed in the ABC phenotype, CCND2 (cyclin D2) andSCYA3, were, along with BCL-2, found to be three ofthe strongest predictors of a poor outcome in anotherstudy.42 Incidentally, cyclin D2 and SCYA3 are down-stream targets of repression by bcl-6, a marker of theGC subtype of DLBCL and a strongly favorable prog-nostic factor.42

A confirmatory study was performed on a larger

dataset of 240 cases of DLBC,23 which identified athird subgroup, provisionally termed “type 3.” Thisnew subgroup demonstrated minimal expression ofgenes characteristic of either the GC or ABC subtypes.Five-year survival of type 3 DLBCL was found to be39%, similar to the 35% of the ABC subgroup, andsignificantly inferior to the 60% survival enjoyed bythe GC subgroup.23 This third group appears to beheterogeneous, and likely encompasses multiple sub-types. In fact, a subsequent study using a differentapproach to analyze the gene expression data (Bayes-ian method of analysis) was able to reassign a signif-icant proportion of “type 3” DLBCL to either the GC orABC subgroup.52

Cured versus Fatal/Refractory PhenotypeA different approach to study DLBCL using gene ex-pression profiling techniques is supervised learning,which classifies genes based on their ability to predictparticular outcomes. In one study, genes highly ex-pressed in curative cases were grouped together toform a “cured” pattern of expression, as opposed tocases that were fatal (or refractory to treatment),which formed the “fatal/refractory” pattern.75 Thir-teen genes with the greatest outcome predictive value(cured vs. fatal) were selected to form a predictivemodel that was used to separate DLBCL cases into twogroups. The 2 groups differed significantly in survival(5-year survival of 70% vs. 12%, P � 0.00004).75

Surprisingly, when the group attempted to reas-sign their DLBCL cases according to genes belongingto the GC versus ABC phenotype, no significant differ-ence in outcome could be found.75 This issue wasaddressed in a later study by the Lymphoma/Leuke-mia Molecular Profiling Project (LLMPP) group usinga different and presumably more discriminating set ofgenes on the same dataset. The LLMPP group con-firmed that a significant difference in survival did existbetween the two groups (62% vs. 26%, P � 0.005).52

Host Response: The Immune System andMicroenvironmentThe potential importance of the host response in DL-BCL is illustrated by earlier studies of immune re-sponse in DLBCL, as well as gene expression profilingstudies that isolated patterns of gene expression asso-ciated with components of the immune system andmicroenvironment.23

It has been shown, for example, that host immuneresponse, as evidenced by T-cell infiltration, may leadto improved tumor control and survival.78 Although anumber of studies investigated the importance ofCD8-positive (cytotoxic) T-cells in DLBCL, anotherstudy found that the presence of CD4-positive (helper)


human leukocyte antigen (HLA)-DR-positive T-cellsalso resulted in improved survival.78 The LLMPPgroup confirmed a positive survival effect of HLA-DRexpression.79 These findings suggest that the host im-mune response, from antigen presentation (HLA-DR)to helper (CD4-positive) T-cells that facilitate the ac-tivation of cytotoxic (CD8-positive) T-cells, may play arole in containing lymphoma cells.

In addition, two of four distinct gene expressionsignatures generated by hierarchical clustering in agene expression profiling study were associated withthe presence of infiltrating reactive cells and antigenpresentation to the immune system. Known as the“lymph node” and “MHC Class II” signatures, bothpatterns were associated with a favorable outcome.23

Immunohistochemical TechniquesGene expression profiling technology, while immenselypowerful, is not readily available in the clinical setting.Fortunately, a number of studies demonstrated the fea-sibility of employing immunohistochemical techniquesand a select group of genes to classify DLBCL into GC orABC subgroups.16,17 CD10, bcl-6, MUM1, and sometimesCD138 are the markers commonly used to distinguishbetween the two subtypes.16,17

In one study that compared the classification ofcases based on gene expression versus immunohisto-chemical detection of proteins (i.e., gene products),the GC phenotype was defined as bcl-6�, MUM1-,and either CD10 positive or negative, while all othercombinations were classified as non-GC.16 Thus sep-arated, a significant difference in survival was foundbetween the two groups (76% vs. 34%; P � 0.001),similar to the results from gene expression profilingstudies. More interestingly, case assignment by immu-nohistochemical techniques demonstrated better cor-relation with clinical outcome than classificationbased on DNA microarrays. For example, eight casesclassified as GC phenotype by immunohistochemistrybut ABC subtype by DNA microarray were found tohave a superior 5-year survival of 76%. Likewise, 22cases classified as ABC in the study but GC by DNAmicroarray were associated with a suboptimal 34%survival.16

Gene expression profiling is a powerful newmethod that has uncovered new subtypes of DLBCL,as well as genes and molecular pathways that mayplay important roles in DLBCL. Data from such stud-ies can help guide further research into the pathologicmechanisms of DLBCL and the development of ratio-nally designed targeted therapeutic agents.

DISCUSSIONDiffuse large B-cell lymphoma is a heterogeneous dis-ease characterized by diverse morphologic and immu-nophenotypic features, cytogenetic abnormalities,and patterns of gene expression. Scrutiny into the roleand prognostic significance of various clinical andbiologic variables has resulted in the construction of aprognostic index, the IPI, based on select clinical pa-rameters, in addition to the discovery of an expandingnumber of genes and molecular pathways that arepotentially involved in the pathogenesis of DLBCL.Some of the molecules implicated appear to contrib-ute to derangements in apoptosis (e.g., bcl-2, NF-�B),cell-cycle regulation (e.g., bcl-6, c-myc, p53), cellulardifferentiation, and signal transduction. The putativeorigin of the lymphoma cells, host environment, andimmune response may also play a significant role.Table 1 summarizes the prognostic value of the moreextensively studied molecules discussed here.Granted, the majority of studies were retrospective innature and were limited by the availability of appro-priate pathologic specimens. Selection bias could notbe ruled out. Nonetheless, given that only half of thepatients newly diagnosed with DLBCL will be curedwith current treatment, there is a sense of urgency toimprove our ability in identifying patients who are lesslikely to respond to standard treatment, and to de-velop more effective options for those with “high risk”disease based on clinical parameters and/or biologiccharacteristics.

The IPI incorporates a number of well-studiedclinical parameters that likely reflect the extent ofdisease and host characteristics. Even though it re-mains the most validated and successful prognostictool for patients with DLBCL, more recent researchsuggests that biological differences, which may not befully accounted for by the index, do substantially mod-ify the clinical outcome as predicted by the IPIalone.22,53,75 Whereas intensifying treatment for pa-tients with clinically defined high-risk disease has notresulted in significant success,7 the corollary thattreatment aimed at putative biologic abnormalitiesmay lead to improved outcome awaits confirmationby future studies. The first step is to define the criticalpathways that underlie the pathogenesis of DLBCL,which have remained somewhat elusive to date.

Morphologic differences have not proven to be ofconsistent prognostic value, and additionally sufferfrom limited intra- and interobserver reproducibility.1

Conversely, immunophenotypic, cytogenetic, and im-munohistochemical data, readily available in the clin-ical setting, can provide valuable information regard-ing potential biologic subtypes and likely prognosis


with current treatment, even though the roles andmechanisms of action of many molecules/biomarkersremain incompletely understood. Gene expressionprofiling has provided a more comprehensive view ofthe genes and molecular pathways that may be active

in DLBCL, in addition to generating robust new DL-BCL subtypes22,52,75 and a whole host of new genes,like HGAL,80 which appear to be prognostically signif-icant and differentially expressed within DLBCL sub-types.

TABLE 1Biomarkers of Potential Prognostic Significance in DLBCL

Biomarker Role Prognostic Significance

Morphologic subtypesCentroblastic N/A Favorable.9,10

No difference.11,12

Immunoblastic N/A Unfavorable.9,10

No difference.11,12

Immunophenotypic markersCD5 T-cell marker, role unclear. Distinct gene expression pattern.24 10% of DLBCL. Inferior survival.18,19,25,26

No difference.83

CD10 Marker of germinal center (GC) B-cells and DLBCL of GC subtype. Associated witht(14,18). 50% of de novo DLBCL.

Favorable prognosis.15,16

No difference.17,18,19

Inferior outcome. 20,21

CD40 Cell surface receptor of the TNF family. Potential involvement in apoptosis and T-cellresponse. 76% of DLBCL.25

Favorable.18

CD138 Marker of plasma cells and lymphoma of post-GC, plasmacytic differentiation. N/ACytogenetic abnormalities

t(14,18)(q32,q21) BCL-2/immunoglobulin (Ig) heavy chain translocation. Dysregulated bcl-2 expression,primarily in subset of DLBCL with GC phenotype. 20-30% of DLBCL.

No difference41,44,45,51

Inferior outcome.53,84

t(3,14)(q27,q32) Immunoglobulin (Ig) heavy chain partner. Increased bcl-6 expression.40,41 20-30%. Favorable.73,74

3q23/non-Ig gene partners Decreased bcl-6 expression.40,41 Unfavorable.73,74

Genes involved in apoptosisBcl-2 Anti-apoptotic mitochondrial protein, downstream target of NF-�B. Chemotherapy

resistance. 30-50% of DLBCL.Unfavorable.17,35,41,43-48

Insignificant.82

Rel Transcription factor of the rel/NF-�B family, anti-apoptotic. Associated with GCsubtype of DLBCL.17

Significance unclear.54

Survivin Inhibitor of apoptotic proteins. Inhibits caspases. Unfavorable.56

No difference.55

FLIP Negative mimic of caspase 8. Inhibits extrinsic apoptotic pathway. Overexpressed inABC subtype.16

Prognostic significance unclear.

Transcription factorsBCL-6 Transcriptional repressor affecting cellular proliferation, differentiation, and immune

response. Marker of GC phenotype. � 50% of DLBCL.Favorable.16,22,23,42,76,77

Neutral.65,85,86

Unfavorable.87

Myc Transcriptional factor promoting cell growth, apoptosis, differentiation, andadhesion.38 Up to 15% of DLBCL.

More aggressive disease and inferioroutcome.23,42,49,64,68

LMO2 Transcriptional factor overexpressed in GC phenotype.16 Good outcome.42

IRF4/MUM1 Transcriptional factor involved in differentiation from GC to plasma cells. Marker ofABC phenotype.16

Inferior survival.16,42

Genes involved in cell-cycleprogression

p53 Tumor suppressor gene – cell cycle arrest for DNA repair and/or apoptosis. Mutated/deleted in 15-20% of DLBCL.

No difference17,43,48

Poor outcome.35,42,61

Cyclin D2 (CCND2) Promotes cell-cycle progression from G1 to S-phase. Downstream target of BCL-6.Part of non-GC phenotype.

Unfavorable.16,42

Gene expression profilingGerminal center (GC) subtype Similar expression pattern to GC B-cells, associated with BCL-6, CD10, t(14,18). Improved survival.16,22,23,52

Activated B cell-like (ABC)subtype

Similar to mitogen activated B-cells. Associated with BCL-2, MUM1, FLIP,16

constitutive activation of NF-�B.48Poor survival.16,22,23,52

Cured phenotype N/A Good outcome.75

Fatal/refractory N/A Poor outcome.75

DLBCL: diffuse large B-cell lymphoma; NA: not applicable; GC: germinal center; TNF: tumor necrosis factor; Ig: immunoglobulin; NF-�B: nuclear factor kappa B; ABC: activated B-cell.


How, then, can we make use of the wealth ofdiagnostic data to understand the disease of individ-ual patients, and attempt to stratify them according tospecific biologic subtypes and/or risk categories? Toour knowledge, there is currently no single prognosticmodel that is entirely satisfactory. Combining all avail-able pointers may be the most rational approach, tak-ing into account the interconnectedness among anumber of biological variables. For example, CD10and bcl-6 denote a group of DLBCL of GC origin,which, according to gene expression profiling studies,is associated with a significantly superior outcomecompared with DLBCL beyond the GC stage of devel-opment (marked by expression of MUM1 and/orCD138).16,22,23,52 The prognostic significance of CD10,which may define a unique subset within the GCgroup of DLBCL, may very well hinge on its closeassociation with the t(14;18) translocation, and theup-regulation of bcl-2 that ensues.23,24 Multifunc-tional bcl-6, conversely, is generally associated with afavorable outcome with standard anthracycline-basedtherapy.17,42,74,76,77 Further attempts to improve theclinical outcome of patients with bcl-6� DLBCL of theGC subtype, however, require a better understandingof the exact role of bcl-6 in DLBCL, the defining ab-normality(ies) of the GC subtype, and what featuresmake this subtype of disease more responsive to treat-ment. Alternatively, it may be the exclusion of otherfeatures in the GC subtype, like overexpression ofbcl-2, one of the strongest negative predictive factorsin DLBCL,42 or constitutive activation of the antiapop-totic NF-�B pathway prominent in the ABC subtype,57

that offers a better outcome in comparison.Bcl-2 is an antiapoptotic protein that confers che-

motherapy resistance and survival advantage to lym-phoma cells, rendering it an attractive target of treat-ment. Particularly exciting is the apparent mitigationof the adverse effect of bcl-2 by the anti-CD20 anti-body, rituximab (Rituxan; Genentech, San Francisco,CA), as demonstrated in a retrospective analysis ofrandomized clinical trial data.81 Other attempts to tar-get bcl-2 include the use of the antisense oligonucle-otide, oblimersen (Genasense; Genta, BerkeleyHeights, NJ), which is currently under clinical trial.82 Itmay be of interest to determine which subgroup(s) ofbcl-2-positive DLBCL patients appear to derive themost benefit from anti-bcl-2 treatment. With bcl-2-positive disease associated with NF-�B activation aspart of the ABC phenotype, an alternate agent thattargets the NF-�B pathway, a dominant defect in thissubtype, may prove to be of greater therapeutic effect.

Other biologic markers, such as CD5, p53, andc-myc, as well as newer molecules uncovered by geneexpression profiling, have been found to be of prog-

nostic significance, and are appealing as potentialtreatment targets. In addition to unraveling the mo-lecular basis of the various subtypes of DLBCL, thenext step is to devise further studies to assess the mostappropriate treatment for individual patients accord-ing to their lymphoma’s unique pathology and riskprofile. Patients with disease of the ABC subtype whoalso score as high-risk according to the IPI, for exam-ple, are highly unlikely to respond to standard ther-apy. This may be a group that benefits from the addi-tion of an agent that antagonizes the NF-�B pathway,or consolidation with autologous stem cell transplan-tation during first remission.

DLBCL is a heterogeneous disease that will likelybenefit from further refinement of treatment based onspecific clinical and biological risk factors, rather thanthe current uniform approach that is failing half of allnewly diagnosed patients. New technology has madeit possible to better understand the molecular path-ways involved in different subtypes of DLBCL. Furtherresearch is required to develop new therapeutic agentsand treatment approaches that will improve the clin-ical outcome of all patients with DLBCL.

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Biomarkers of potential prognostic significance in diffuse large B-cell lymphoma