TCL1A expression delineates biological andclinical variability in B-cell lymphoma

Mohit Aggarwal14 Raquel Villuendas1 Gonzalo Gomez2 Socorro M Rodriguez-Pinilla1Margarita Sanchez-Beato1 David Alvarez1 Nerea Martinez1 Antonia Rodriguez1Maria E Castillo1 Francisca I Camacho1 Santiago Montes-Moreno1 Jose A Garcia-Marco3Eva Kimby4 David G Pisano2 and Miguel A Piris1

1Molecular Pathology Programme Spanish National Cancer Research Centre (CNIO) Madrid Spain2Bioinformatics Unit Structural Biology and Biocomputing Programme CNIO Madrid Spain 3Departmentof Haematology Hospital Universitario Puerta de Hierro Madrid Spain and 4Division of HematologyDepartment of Internal Medicine at Huddinge Karolinska Institutet Stockholm Sweden

The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkinrsquoslymphoma has identified increased T-cell leukemialymphoma 1A (TCL1) expression in multiple lymphomatypes and cases and has enabled the investigation of the functional and clinical importance of TCL1expression Specifically Burkittrsquos lymphoma cases show a homogeneously strong expression of TCL1whereas diffuse large B-cell lymphoma follicular lymphoma mantle cell lymphoma chronic lymphocyticleukemia nodal marginal zone lymphoma and splenic marginal zone lymphoma display a striking variability inthe intensity of TCL1 staining This was validated in two independent series A Gene-Set Enrichment Analysis ofthe genes correlated with TCL1A expression found that variation in the level of expression of TCL1A wassignificantly associated with some of the most important gene signatures recognizing B-cell lymphomapathogenesis and heterogeneity such as germinal center B-cell receptor NF-jB (and its target genes) deathMAP kinases TNFR1 TOLL and IL1R Additionally TCL1 expression was correlated with shorter time totreatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle celllymphoma series thus indicating the clinical and biological significance of TCL1 expression and suggestingTCL1A as a potential therapeutic targetModern Pathology (2009) 22 206ndash215 doi101038modpathol2008148 published online 26 September 2008

Keywords TCL1 B-cell lymphoma gene expression clinical variability

Molecular pathogenesis of non-Hodgkinrsquos lymphomais determined by an interaction between the tumorcell and its microenvironment1 This interactioncontrols the activation of B-cell receptor NF-kB andother signaling pathways and eventually regulatesapoptosis and cell cycle23 Some primary andsecondary cytogenetic alterations have been identi-fied that involve oncogenes such as c-MYCin Burkittrsquos lymphoma BCL-2 translocation (t1418)in follicular lymphoma CCND1 translocation (t1114)in mantle cell lymphoma TCL1A deregulation inchronic lymphocytic leukemia4 and BCL-6 deregu-lation in diffuse large B-cell lymphoma56 Howeverthere is an urgent need to identify additional accurate

molecular markers and to apply our accumulatedknowledge about lymphoma pathogenesis to targetedtherapy Although the effort expended on recognizingdisease-specific molecular markers has led to theidentification of many individual markers such asCCND1 BCL6 ZAP70 and CD30 the molecularpathogenesis of the different types of non-Hodgkinrsquoslymphoma seems to be characterized by the activationof some major B-cell lymphoma pathways that arecommonly deregulated in B-cell non-Hodgkinrsquos lymp-homa types To date however little has been done toidentify nonlymphoma-type-specific alterations thatcould consistently determine lymphoma pathogenesis

Here we have focused on the biological andclinical relevance of TCL1A expression and itsrelation with B-cell receptor activation The involve-ment of B-cell receptor signaling7 and the TCL1Aoncogene8 in pathogenesis of B-cell non-Hodgkinrsquoslymphoma has already been demonstrated Dys-regulation of TCL1A expression in T cells bychromosome rearrangements or in B cells possibly

Received 18 July 2008 revised and accepted 14 August 2008published online 26 September 2008

Correspondence Dr MA Piris MD PhD Centro Nacional deInvestigaciones Oncologicas CMelchor Fernandez Almagro no 3Madrid E-28029 SpainE-mail mapiriscnioes

Modern Pathology (2009) 22 206ndash215amp 2009 USCAP Inc All rights reserved 0893-395209 $3200

wwwmodernpathologyorg

by EpsteinndashBarr virus infection or microRNA target-ing enhances cell proliferation and survival andleads to cell transformation following prolongedlatency49ndash11 Dysregulation of TCL1A secondary tomiR-29 and miR-181 loss is a causal event in thepathogenesis of the aggressive form of chroniclymphocytic leukemia and probably of splenicmarginal zone lymphoma912 Aberrant TCL1Aexpression also promotes malignant transformationof germinal center B cells13 A role of TCL1Aoncogene in the pathogenesis of other B-cell non-Hodgkinrsquos lymphoma malignancies has alreadybeen proposed4 but the exact mechanisms bywhich TCL1A regulates tumor development innon-Hodgkinrsquos lymphoma is not yet fully under-stood14 Here we analyze gene-expression profilingdata obtained from the analysis of the various B-celllymphoma types to investigate the functionalsignificance of TCL1A in B-cell non-Hodgkinrsquoslymphoma and the clinical relevance of increasedTCL1A expression in mantle cell lymphoma andchronic lymphocytic leukemia

Materials and methods

Patients and Controls

The microarray data (GSE9327) from 187 cases ofB-cell non-Hodgkinrsquos lymphoma available in theGEO public repository under MIAME regulations(httpwww ncbinlmnihgovgeoqueryacccgitokenfrac14 ftenzyyykicqqfiampaccfrac14GSE9327) were usedin this study The microarray data have beenpreviously published by our group and includethose for Burkittrsquos lymphoma (9 cases) diffuse largeB-cell lymphoma (36 cases)15 mantle cell lymp-homa (38 cases)16 chronic lymphocytic leukemia(38 cases)17 follicular lymphoma (33 cases)18 nodalmarginal zone lymphoma (6) and splenic marginalzone lymphoma (27 cases)19 Reactive lymph nodesfrom eight patients and spleen from three patientswere also analyzed The cases were selected on thebasis of the standard WHO Classification crite-ria2021 All samples were fresh frozen diagnosticsamples taken previous to treatment and wereprovided by the hospitals and the CNIO TumorBank under the supervision of the Local EthicCommittees

In addition an already published series of 98cases of chronic lymphocytic leukemia hybridizedon a chronic lymphocytic leukemia-specific 19KOligonucleotide Microarray (Agilent Technology)was used for TCL1A analysis22 Main clinicalcharacteristics of the patients22 are summarized inTable 1a

Additional 64 patients diagnosed as mantle celllymphoma23 were collected by the CNIO TumorBank Network under the supervision of the LocalEthic Committees All cases were histologicallyreviewed by a team of pathologists (SMRP andMAP) in accordance with the WHO criteria2021

Table 1 Main clinical findings from (a) CLL (98 patients) and (b)MCL (64 patients)

Clinical variables Total

(a) CLLSex

Male 60Female 38

Age at diagnosis (year)a

o60 3460ndash69 30X70 32

RAI stage0 5240 46

Binet stageA 76B+C 22

CD38 expressiona

o20 65X20 23

First-line treatment (at 60 months of follow-up)Yes 43No 55

(b) MCLSex

Male 45Female 19

Agea

o60 years 21X60 years 30

Stagesa

I 0II 4III 7IV 48

Extranodal involvementa

o2 39X2 25

Serum LDHa

pNormal 384Normal 12

ECOG performance statusa

o2 42X2 11

International prognostic indexa

Lowlow-intermediate 27Highhigh-intermediate 25

Overall survival (at 60 months)Alive 36Dead 28

CLL chronic lymphocytic leukemia ECOG Eastern CooperativeOncology Group LDH lactate dehydrogenase MCL mantle celllymphomaaData are not available for all the cases

TCL1A expression in B-cell lymphomaM Aggarwal et al

207

Modern Pathology (2009) 22 206ndash215

These samples were used to do a tissue microarray23

(see below) Tumoral diagnostic samples wereobtained from untreated patients Patient recordsand clinical data were reviewed retrospectively Thepatient data for analyzable cases are summarized inTable 1b

Gene-Set Enrichment Analysis and RegulatoryNetwork Analysis

We conducted Gene-Set Enrichment Analysis tounderstand the functional pathways that are cor-egulated with the expression of TCL1A The gene setdatabase included Biocarta pathways24 clusters offunctionally related coregulated genes identified inunsupervised clustering by us and molecularsignatures defined for lymphoma in the Staudtmolecular signature database (httplymphochipnihgovsignaturedb)25 The gene sets that werecoregulated with TCL1A expression were identifiedusing Pearsonrsquos correlation with a minimum of 15genes in a gene set required to qualify for furtheranalysis

ARACNE was used to generate direct regulatoryinteractions with TCL1A26 ARACNE is an algorithmthat reverse engineers a gene regulatory networkfrom microarray gene-expression data The algo-rithm uses mutual information an informationtheoretical measure to compute the nonlinearcorrelation between pairs of genes and infer a best-fit network of probable interactions The resultsshow those best candidate genes (Po005) that sharea direct regulatory relationship with a particulargene target

Tissue Microarray and Immunohistochemistry

A tissue microarray23 with 64 cases of mantle celllymphoma was stained with TCL1A (clone 27D620MBL International MA) and cyclin D1 antibodies(NeoMarkers-LabVision Thermo Fisher ScientificInc Fremont CA) The diagnosis of the cases wascorroborated by positive cyclin D1 staining

The expression of the studied markers wasdetermined by the ARIOL semiautomated compu-terized training system (httpwwwaicorpcomproducts02pathhtm)23 The system was trained bya team of technicians and pathologists to quantifyTCL1A and CCND1 expression The operation issimilar to flow cytometry but on tissue sectionsinvolving quantitative measurement (number ofcells in a core of the patientrsquos sample) andqualitative assessment (intensity of staining)

Statistical Analysis

Survival curves were produced by the KaplanndashMeiermethod All deaths were attributed to the tumor

Figure 1 TCL1 gene expression in B-cell non-Hodgkinrsquos lympho-ma types using cases hybridized on OncoChip The expressionfor the cases was normalized with respect to the expression ofreactive lymph nodes whereas gene expression for splenicmarginal zone lymphoma was normalized against normal spleen

Figure 2 Oncomine analysis of TCL1A in non-Hodgkinrsquos lym-phoma (a) Differential expression of TCL1A in lymphoma typesBurkittrsquos lymphoma (HIVthorn ) diffuse large B-cell lymphoma(centroblastic) diffuse large B-cell lymphoma (immunoblastic)diffuse large B-cell lymphoma hairy-cell Leukemia Burkittrsquoslymphoma follicular lymphoma B-cells mantle cell lymphomaand chronic lymphocytic leukemia respectively (wwwoncomineorg) Data link source httpwwwncbinlmnihgovprojectsgeoqueryacccgiaccfrac14GSE235026 (b) TCL1A expression indiffuse large B-cell lymphoma follicular lymphoma and chroniclymphocytic leukemia Data link sources httpllmppnihgovlymphomaanalysisshtml httpwwwncbinlmnihgovgeoqueryacccgiaccfrac14GSE6035

TCL1A expression in B-cell lymphomaM Aggarwal et al

208

Modern Pathology (2009) 22 206ndash215

Statistical significance of associations betweenindividual variables and survival was determinedusing the log-rank test Pearsonrsquos correlation was

applied to TCL1A and CCND1 expression analysisAll statistical analyses were performed using SPSSversion 130 (SPSS Inc Chicago IL)

Figure 3 Heat map showing genes coregulated with variable expression of TCL1A in the B-cell non-Hodgkinrsquos lymphoma series

Table 2 Pathways coregulated with TCL1A expression in B-cell NHL GSEA reporting on functionally relevant pathways that arestatistically significant (Po005 FDRo25) and report on positive Pearsonrsquos correlation with high level of expression of TCL1A

Name of Gene-Set from GSEA Size ES NES NOM p FDR q Pathways

CD40 signaling during GC dev 91 053 194 000 003 GCGC B cell 90 039 152 006 012 GCGC T-helper cell 166 041 160 002 009 GCBlood pan-B cell 27 077 224 000 000 BCSMZ B cell 25 064 178 004 005 BCBCR signal pathway 23 051 168 004 006 BCToll pathway 17 065 184 000 005 BCNF-kB pathway 16 069 173 000 005 NF-kBNF-kB target genes 18 052 131 020 021 NF-kBTNFR2 pathway 15 064 169 004 006 NF-kBDeath pathway 23 043 133 013 019 NF-kBIL1R pathway 19 052 148 008 014 NF-kBIL2 pathway 18 049 146 013 015 NF-kBIL2RB pathway 27 043 137 010 017 NF-kBTCR pathway 30 049 167 002 006 TCCD8 T-cell differentiation 47 044 154 004 012 TCCD4 T-cell differentiation 78 040 153 000 012 TCB-cell and T-cell calcium signaling 76 043 144 005 015 CalciumBlood NK cell 18 049 145 010 014 TCResting dendritic cell 30 045 140 010 016 DCPlasmacytoid dendritic cell 104 043 174 000 006 DCCREB pathway 18 043 129 019 021 CalciumMAPK pathway 59 037 148 004 014 NF-kB calcium BC TCBLIMP-1 targets 88 048 166 007 005 GC

GC Germinal center NF-kB nuclear factor-kB pathway BC B cells TC T cells DC dendritic cells calcium calcium-signaling pathways ESenrichment score NES normalized enrichment score NOM p nominal P-value SMZ splenic marginal zone lymphoma BCR B-cell receptorTNFR tumor-necrosis factor receptor IL interleukin TCR transcription-coupled repair NK cells natural killer cells CREB cAMP responseelement-binding protein MAPK mitogen-activated protein kinase BLIMP-1 B lymphocyte-induced maturation protein-1 GSEA Gene-SetEnrichment Analysis

TCL1A expression in B-cell lymphomaM Aggarwal et al

209

Modern Pathology (2009) 22 206ndash215

Results

TCL1A Expression in B-Cell Non-HodgkinrsquosLymphoma

The assembly of a collection of gene-expressionsignatures of the major types of B-cell non-Hodg-kinrsquos lymphoma enabled the functional and clinicalrelevance of genes presumably involved in thepathogenesis of specific tumor types to be investi-gated An unexpected finding in our microarrayseries was the increased expression of TCL1A indifferent lymphoma types and cases (Figure 1) inspite of this gene expression being restricted to asubset of mantle zone cells in normal lymph nodesThus Burkittrsquos lymphoma cases had a consistentlystrong expression of TCL1A whereas diffuse largeB-cell lymphoma follicular lymphoma mantle celllymphoma chronic lymphocytic leukemia nodalmarginal zone lymphoma and splenic marginalzone lymphoma displayed striking variability inthe intensity of TCL1A staining This increasedexpression of TCL1A in different non-Hodgkinrsquoslymphoma types and cases was validated by anOncomine (wwwoncomineorg) search from alreadypublished microarray data where great variability inthe intensity of TCL1A expression was noticed inmost B-cell lymphoma types (Figure 2) To under-stand better the functional relevance of theincreased expression of TCL1A we performed anunsupervised hierarchical clustering of genes in theOncoChipt that showed the TCL1A oncogene to becoregulated in a cluster including B-cell receptor-pathway genes such as BTK and LYN and genesinvolved in lymphoma pathogenesis (eg POU2AF1and BCL11A Supplementary Figure 1) thus con-firming our previous observations linking B-cellreceptor genes with TCL1A expression in chronic

lymphocytic leukemia which were made using adifferent platform and series of cases17 Hence wehave used gene-expression data to generate testablehypotheses about B-cell lymphoma pathogenesis

Pathways coregulated with TCL1A across non-Hodgkinrsquos lymphoma typesPearsonrsquos correlation identified the genes coregu-lated with the expression of TCL1A (Figure 3) It isof particular note that although increased micro-array expression was seen in all the B-cell lympho-ma series it specifically appeared to establishdistinct heterogeneity in chronic lymphocytic leu-kemia mantle cell lymphoma diffuse large B-celllymphoma follicular lymphoma and splenic mar-ginal zone lymphoma cases whereby the groupsfeatured either high or low levels of expression ofTCL1A that were associated with changes in theexpression of a large set of genes To explore this ingreater depth we performed comparative Gene-SetEnrichment Analysis of the genes whose expressionwas correlated with TCL1A expression and foundthat variation in the level of expression of TCL1Awas significantly associated (Po005) with some ofthe most important pathways controlling B-celllymphoma pathogenesis and heterogeneity(Table 2) These pathways included those of thegerminal center B-cell receptor NF-kB (and itstarget genes) death MAP kinases TNFR1 TOLLand IL1R The association of TCL1A with theexpression of all these pathways implies that itmay have a central role in B-cell lymphomagenesisTo explore further the genes that share a directregulatory relationship with TCL1A we appliedARACNE Reverse engineering for TCL1A regulatorynetwork confirmed most of the results obtained byGene-Set Enrichment Analysis including the genesLYN BCL2 TNFRSF7 and TCL6 (Figure 4)

The functional relevance of TCL1A expressionwas validated by analyzing a previously publishedindependent series of 98 chronic lymphocyticleukemia22 patients with Gene-Set Enrichment Ana-lysis Cases that strongly expressed TCL1A morestrongly expressed genes clustered in the gene setsidentified as BLIMP-1 targets and the CREBP path-way including important B-cell receptor signalinggenes such as SYK BTK and AKT-1 A low level ofexpression of TCL1A was associated with NF-kBand its associated pathways such as the CD40signaling during GC development and IL1R pathway(Figure 5) TCL1A and ZAP70 expression weresignificantly correlated in this series (Pfrac14 0007)

Clinical significance of TCL1A expression in chroniclymphocytic leukemiaB-cell receptors in TCL1A transgenic mice showvery low level of somatic hypermutation resemblingthose of aggressive human chronic lymphocyticleukemia with shorter time to treatment and pooreroverall survival10 Additionally the expression ofTCL1A in human chronic lymphocytic leukemia has

Figure 4 Network analysis for TCL1A in B-cell non-Hodgkinrsquoslymphoma using ARACNE (Algorithm for the Reconstruction ofAccurate Cellular Networks) The figure shows those bestcandidate genes (Po005) that share a direct regulatory relation-ship with TCL1A

TCL1A expression in B-cell lymphomaM Aggarwal et al

210

Modern Pathology (2009) 22 206ndash215

Results

TCL1A Expression in B-Cell Non-HodgkinrsquosLymphoma

The assembly of a collection of gene-expressionsignatures of the major types of B-cell non-Hodg-kinrsquos lymphoma enabled the functional and clinicalrelevance of genes presumably involved in thepathogenesis of specific tumor types to be investi-gated An unexpected finding in our microarrayseries was the increased expression of TCL1A indifferent lymphoma types and cases (Figure 1) inspite of this gene expression being restricted to asubset of mantle zone cells in normal lymph nodesThus Burkittrsquos lymphoma cases had a consistentlystrong expression of TCL1A whereas diffuse largeB-cell lymphoma follicular lymphoma mantle celllymphoma chronic lymphocytic leukemia nodalmarginal zone lymphoma and splenic marginalzone lymphoma displayed striking variability inthe intensity of TCL1A staining This increasedexpression of TCL1A in different non-Hodgkinrsquoslymphoma types and cases was validated by anOncomine (wwwoncomineorg) search from alreadypublished microarray data where great variability inthe intensity of TCL1A expression was noticed inmost B-cell lymphoma types (Figure 2) To under-stand better the functional relevance of theincreased expression of TCL1A we performed anunsupervised hierarchical clustering of genes in theOncoChipt that showed the TCL1A oncogene to becoregulated in a cluster including B-cell receptor-pathway genes such as BTK and LYN and genesinvolved in lymphoma pathogenesis (eg POU2AF1and BCL11A Supplementary Figure 1) thus con-firming our previous observations linking B-cellreceptor genes with TCL1A expression in chronic

lymphocytic leukemia which were made using adifferent platform and series of cases17 Hence wehave used gene-expression data to generate testablehypotheses about B-cell lymphoma pathogenesis

Pathways coregulated with TCL1A across non-Hodgkinrsquos lymphoma typesPearsonrsquos correlation identified the genes coregu-lated with the expression of TCL1A (Figure 3) It isof particular note that although increased micro-array expression was seen in all the B-cell lympho-ma series it specifically appeared to establishdistinct heterogeneity in chronic lymphocytic leu-kemia mantle cell lymphoma diffuse large B-celllymphoma follicular lymphoma and splenic mar-ginal zone lymphoma cases whereby the groupsfeatured either high or low levels of expression ofTCL1A that were associated with changes in theexpression of a large set of genes To explore this ingreater depth we performed comparative Gene-SetEnrichment Analysis of the genes whose expressionwas correlated with TCL1A expression and foundthat variation in the level of expression of TCL1Awas significantly associated (Po005) with some ofthe most important pathways controlling B-celllymphoma pathogenesis and heterogeneity(Table 2) These pathways included those of thegerminal center B-cell receptor NF-kB (and itstarget genes) death MAP kinases TNFR1 TOLLand IL1R The association of TCL1A with theexpression of all these pathways implies that itmay have a central role in B-cell lymphomagenesisTo explore further the genes that share a directregulatory relationship with TCL1A we appliedARACNE Reverse engineering for TCL1A regulatorynetwork confirmed most of the results obtained byGene-Set Enrichment Analysis including the genesLYN BCL2 TNFRSF7 and TCL6 (Figure 4)

The functional relevance of TCL1A expressionwas validated by analyzing a previously publishedindependent series of 98 chronic lymphocyticleukemia22 patients with Gene-Set Enrichment Ana-lysis Cases that strongly expressed TCL1A morestrongly expressed genes clustered in the gene setsidentified as BLIMP-1 targets and the CREBP path-way including important B-cell receptor signalinggenes such as SYK BTK and AKT-1 A low level ofexpression of TCL1A was associated with NF-kBand its associated pathways such as the CD40signaling during GC development and IL1R pathway(Figure 5) TCL1A and ZAP70 expression weresignificantly correlated in this series (Pfrac14 0007)

Clinical significance of TCL1A expression in chroniclymphocytic leukemiaB-cell receptors in TCL1A transgenic mice showvery low level of somatic hypermutation resemblingthose of aggressive human chronic lymphocyticleukemia with shorter time to treatment and pooreroverall survival10 Additionally the expression ofTCL1A in human chronic lymphocytic leukemia has

Figure 4 Network analysis for TCL1A in B-cell non-Hodgkinrsquoslymphoma using ARACNE (Algorithm for the Reconstruction ofAccurate Cellular Networks) The figure shows those bestcandidate genes (Po005) that share a direct regulatory relation-ship with TCL1A

TCL1A expression in B-cell lymphomaM Aggarwal et al

210

Modern Pathology (2009) 22 206ndash215

been associated with the presence of unmutatedIgVH genes11 but to the authorsrsquo knowledge nodirect proof of the clinical prognostic value ofTCL1A expression in chronic lymphocytic leukemiahas previously been published

The analysis of the data generated by gene-expression profiling of an independent set of 98chronic lymphocytic leukemia patients22 with aspecific microarray platform shows that overexpres-sion of TCL1A is associated with shorter time to

treatment in chronic lymphocytic leukemia patients(Po0001 Figure 6) In this analysis the series wasdivided into four quartiles according to TCL1Aexpression and cases in the lower quartile werecompared with those in the other three quartiles

Clinical significance of TCL1A expression in mantlecell lymphomaA new series of 64 mantle cell lymphoma23 caseswas analyzed in a tissue microarray to establish

CA

MK

2DS

OS

1P

RK

CB

1R

AC

1M

AP

K3

AK

T1

PR

KA

BIA

GR

B2

CR

EB

1M

AP

K1

INP

P5

DLC

KC

SK

BL

NK

TR

AF

5P

CN

AB

TK

CD

37

YW

HA

QF

CE

R2

CD

22

CR

2D

AX

XS

YK

IL1

BB

CL

6C

D4

4IE

R3

TN

FS

F9

MA

P3

K8

CF

LA

RN

FK

B2

BC

L2A

1N

FK

BIE

GA

DD

45B

ICA

M1

IRA

K2

MA

PK

14IL

1A

TR

AF

6M

AP

2K

3N

FK

B1

IL1

R1

IL6

MA

PK

8

BLIMP-1 TARGETSCREBP PATHWAYCD40 SIGNALLING PATHWAYIL1R PATHWAY

Figure 5 Heat map showing genes coregulated with variable expression of TCL1A oncogene in chronic lymphocytic leukemia (98 cases)Cases with stronger TCL1A expression show increased expression of coregulated pathways such as B-cell receptor genes Cases withweaker TCL1A expression have an NF-kB target-gene signature

TCL1A expression in B-cell lymphomaM Aggarwal et al

211

Modern Pathology (2009) 22 206ndash215

Figure 6 Prognostic significance of TCL1A oncogene in chroniclymphocytic leukemia KaplanndashMeier analysis shows TCL1Amicroarray data in 98 cases of chronic lymphocytic leukemia(log-rank test P-valueo0001) The cases were divided intoquartiles of TCL1A expression and the lowest was compared tothe top three quartiles TFS Treatment-free survival

Figure 7 Expression of TCL1 in mantle cell lymphoma cases (a and b) Expression of TCL1A in mantle zone cells in a reactive tonsil (c) amantle cell lymphoma case showing strong TCL1A expression (d) an example of a mantle cell lymphoma case with TCL1A-negativeexpression

Figure 8 Prognostic value of TCL1A protein expression in mantlecell lymphoma The KaplanndashMeier curve indicates better lym-phoma-specific survival (LSS) for cases with weaker TCL1Aexpression in mantle cell lymphoma (Pfrac14002) The cases weredivided into quartiles of TCL1A expression and the lowest wascompared to the top three quartiles

TCL1A expression in B-cell lymphomaM Aggarwal et al

212

Modern Pathology (2009) 22 206ndash215

the clinical and biological significance of TCL1Aprotein expression (Figure 7) In this case strongerTCL1A expression was associated with shortersurvival probability (Pfrac14 002 Figure 8) For thisanalysis the mantle cell lymphoma cases in thelowest quartile of TCL1A expression which repre-sents the 25 of cases with the lowest TCL1Aintensity were compared with those of the otherthree quartiles

The correlation between TCL1A and cyclin D1protein expression was also investigated TCL1A andcyclin D1 expression were significantly correlated(Pearsonrsquos Rfrac14 0283 Pfrac14 0024) This same associationwas seen in the mRNA-expression array data of theLymphochip (httpllmppnihgov data not shown)

Discussion

Gene-expression profiling analysis of lymphomashas allowed collecting data from a large series ofcases including all the major lymphoma types Oneof the surprising findings of this study was theincreased expression of TCL1A gene in multiple B-cell non-Hodgkinrsquos lymphoma types which con-firms and extends previously published findingsconcerning precise lymphoma types27

Physiological expression of TCL1A is largelylimited to precursorimmature T and B lymphocytesand a subset of mantle zone B cells1127 Howeverlymphoma analysis shows that TCL1A expression isincreased in specific cases or tumor types presum-ably deriving from TCL1A-negative cells subsetsAdditionally all tumor types with the notableexception of Burkittrsquos lymphoma show strikingvariability in the intensity of TCL1A expressionAlthough this had been previously described inspecific lymphoma types our observations indicatethat increased TCL1A expression is a generalphenomenon occurring to some extent in all majorB-cell lymphoma types122728 Interestingly the onlylymphoma type in which TCL1A expression wasconsistently high was Burkittrsquos lymphoma a tumorwhose normal counterpart is TCL1A-negative germ-inal center cells Indeed TCL1A expression has beenproposed as being a useful ancillary test of use in thediagnosis of Burkittrsquos lymphoma2930

The oncogenic capacity of TCL1A has beendemonstrated by several groups Thus overexpres-sion of the TCL1A gene in murine B-cells leads to aCD5(thorn ) B-cell lymphoproliferative disorder withmany of the features seen in the aggressive variant ofhuman B-cell chronic lymphocytic leukemiaincluding a very low level of Ig somatic hypermuta-tion410 Hoyer and co-worker have also shown thatdysregulated TCL1 promotes multiple classes ofmature B-cell lymphoma27

In this study we have demonstrated an asso-ciation between TCL1A expression and essentialpathways for B-cell survival including B-cellreceptor CD-40 signaling NF-kB TOLL and

calcium-signaling pathways These findings seemto place TCL1A at the center of a crossroads thatis essential for malignant B-cell survival whereB-cell apoptosis induced by antigen receptor cross-linking is blocked by a T-cell signal through CD4031

This is consistent with the interpretation thatthe increased expression of TCL1A observed inmultiple B-cell lymphoma types is the consequenceof the escape from GC mechanisms of TCL1Arepression13

ARACNE generates a putative transcriptional net-work to predict broader functional relationships andcould help in the understanding the biological roleof TCL1A One of the important findings of networkanalysis was probable regulatory interaction ofTCL1A and CD27 It is known that TNFRSF7CD27binds to CD70 and plays a key role in regulatingB-cell activation and immunoglobulin synthesis Ittransduces signals that lead to the activation of NF-kB and MAPK8JNK Adaptor proteins TRAF2 andTRAF5 have been shown to mediate the signalingprocess and are important in the apoptosis inducedby TNFRSF7 TRAF1 and BCL2 are frequently foundoverexpressed in human chronic lymphocytic leu-kemia and this cooperation has been proposed tocontribute to the development of chronic lympho-cytic leukemia32 Interestingly the ARACNE studysuggests a potential mechanism where TCL1Aoncogene may provide a common link of functionalregulation between B-cell receptor component(LYN) NF-kB pathway (pro-apoptotic signal viaTNFRSF7) and survival pathway (BCL2) in B-cellnon-Hodgkinrsquos lymphoma

We have also demonstrated the prognostic sig-nificance of TCL1A expression in chronic lympho-cytic leukemia and mantle cell lymphoma inpatients using RNA microarray and protein studiesrespectively This together with the data showingthat TCL1A expression is correlated with an adverseclinical outcome in diffuse large B-cell lymphoma33

is consistent with the proposed oncogenic roleplayed by TCL1A expression Gene-silencing stu-dies may reveal how B-cell receptor and othercoregulated pathways interact with the TCL1Aoncogene at the molecular level

Thus these data contribute to the identification oflymphoma as the result of multiple oncogeniclesions and dysregulated pathways some of thekey drivers being B-cell receptor signaling andTCL1A expression acting via the calcium-signalingpathway A potential therapeutic implication ofthese findings has been shown by Zanesi et al34

where rapamycin inhibition of the Tcl1AktmTORpathway in Em-TCL1 transgenic mice prolonged thelife of all treated animals

Acknowledgements

This study was supported by grants from theMinisterio de Sanidad y Consumo (G03179

TCL1A expression in B-cell lymphomaM Aggarwal et al

213

Modern Pathology (2009) 22 206ndash215

Figure 6 Prognostic significance of TCL1A oncogene in chroniclymphocytic leukemia KaplanndashMeier analysis shows TCL1Amicroarray data in 98 cases of chronic lymphocytic leukemia(log-rank test P-valueo0001) The cases were divided intoquartiles of TCL1A expression and the lowest was compared tothe top three quartiles TFS Treatment-free survival

Figure 7 Expression of TCL1 in mantle cell lymphoma cases (a and b) Expression of TCL1A in mantle zone cells in a reactive tonsil (c) amantle cell lymphoma case showing strong TCL1A expression (d) an example of a mantle cell lymphoma case with TCL1A-negativeexpression

Figure 8 Prognostic value of TCL1A protein expression in mantlecell lymphoma The KaplanndashMeier curve indicates better lym-phoma-specific survival (LSS) for cases with weaker TCL1Aexpression in mantle cell lymphoma (Pfrac14002) The cases weredivided into quartiles of TCL1A expression and the lowest wascompared to the top three quartiles

TCL1A expression in B-cell lymphomaM Aggarwal et al

212

Modern Pathology (2009) 22 206ndash215

the clinical and biological significance of TCL1Aprotein expression (Figure 7) In this case strongerTCL1A expression was associated with shortersurvival probability (Pfrac14 002 Figure 8) For thisanalysis the mantle cell lymphoma cases in thelowest quartile of TCL1A expression which repre-sents the 25 of cases with the lowest TCL1Aintensity were compared with those of the otherthree quartiles

The correlation between TCL1A and cyclin D1protein expression was also investigated TCL1A andcyclin D1 expression were significantly correlated(Pearsonrsquos Rfrac14 0283 Pfrac14 0024) This same associationwas seen in the mRNA-expression array data of theLymphochip (httpllmppnihgov data not shown)

Discussion

Gene-expression profiling analysis of lymphomashas allowed collecting data from a large series ofcases including all the major lymphoma types Oneof the surprising findings of this study was theincreased expression of TCL1A gene in multiple B-cell non-Hodgkinrsquos lymphoma types which con-firms and extends previously published findingsconcerning precise lymphoma types27

Physiological expression of TCL1A is largelylimited to precursorimmature T and B lymphocytesand a subset of mantle zone B cells1127 Howeverlymphoma analysis shows that TCL1A expression isincreased in specific cases or tumor types presum-ably deriving from TCL1A-negative cells subsetsAdditionally all tumor types with the notableexception of Burkittrsquos lymphoma show strikingvariability in the intensity of TCL1A expressionAlthough this had been previously described inspecific lymphoma types our observations indicatethat increased TCL1A expression is a generalphenomenon occurring to some extent in all majorB-cell lymphoma types122728 Interestingly the onlylymphoma type in which TCL1A expression wasconsistently high was Burkittrsquos lymphoma a tumorwhose normal counterpart is TCL1A-negative germ-inal center cells Indeed TCL1A expression has beenproposed as being a useful ancillary test of use in thediagnosis of Burkittrsquos lymphoma2930

The oncogenic capacity of TCL1A has beendemonstrated by several groups Thus overexpres-sion of the TCL1A gene in murine B-cells leads to aCD5(thorn ) B-cell lymphoproliferative disorder withmany of the features seen in the aggressive variant ofhuman B-cell chronic lymphocytic leukemiaincluding a very low level of Ig somatic hypermuta-tion410 Hoyer and co-worker have also shown thatdysregulated TCL1 promotes multiple classes ofmature B-cell lymphoma27

In this study we have demonstrated an asso-ciation between TCL1A expression and essentialpathways for B-cell survival including B-cellreceptor CD-40 signaling NF-kB TOLL and

calcium-signaling pathways These findings seemto place TCL1A at the center of a crossroads thatis essential for malignant B-cell survival whereB-cell apoptosis induced by antigen receptor cross-linking is blocked by a T-cell signal through CD4031

This is consistent with the interpretation thatthe increased expression of TCL1A observed inmultiple B-cell lymphoma types is the consequenceof the escape from GC mechanisms of TCL1Arepression13

ARACNE generates a putative transcriptional net-work to predict broader functional relationships andcould help in the understanding the biological roleof TCL1A One of the important findings of networkanalysis was probable regulatory interaction ofTCL1A and CD27 It is known that TNFRSF7CD27binds to CD70 and plays a key role in regulatingB-cell activation and immunoglobulin synthesis Ittransduces signals that lead to the activation of NF-kB and MAPK8JNK Adaptor proteins TRAF2 andTRAF5 have been shown to mediate the signalingprocess and are important in the apoptosis inducedby TNFRSF7 TRAF1 and BCL2 are frequently foundoverexpressed in human chronic lymphocytic leu-kemia and this cooperation has been proposed tocontribute to the development of chronic lympho-cytic leukemia32 Interestingly the ARACNE studysuggests a potential mechanism where TCL1Aoncogene may provide a common link of functionalregulation between B-cell receptor component(LYN) NF-kB pathway (pro-apoptotic signal viaTNFRSF7) and survival pathway (BCL2) in B-cellnon-Hodgkinrsquos lymphoma

We have also demonstrated the prognostic sig-nificance of TCL1A expression in chronic lympho-cytic leukemia and mantle cell lymphoma inpatients using RNA microarray and protein studiesrespectively This together with the data showingthat TCL1A expression is correlated with an adverseclinical outcome in diffuse large B-cell lymphoma33

is consistent with the proposed oncogenic roleplayed by TCL1A expression Gene-silencing stu-dies may reveal how B-cell receptor and othercoregulated pathways interact with the TCL1Aoncogene at the molecular level

Thus these data contribute to the identification oflymphoma as the result of multiple oncogeniclesions and dysregulated pathways some of thekey drivers being B-cell receptor signaling andTCL1A expression acting via the calcium-signalingpathway A potential therapeutic implication ofthese findings has been shown by Zanesi et al34

where rapamycin inhibition of the Tcl1AktmTORpathway in Em-TCL1 transgenic mice prolonged thelife of all treated animals

Acknowledgements

This study was supported by grants from theMinisterio de Sanidad y Consumo (G03179

TCL1A expression in B-cell lymphomaM Aggarwal et al

213

Modern Pathology (2009) 22 206ndash215

the clinical and biological significance of TCL1Aprotein expression (Figure 7) In this case strongerTCL1A expression was associated with shortersurvival probability (Pfrac14 002 Figure 8) For thisanalysis the mantle cell lymphoma cases in thelowest quartile of TCL1A expression which repre-sents the 25 of cases with the lowest TCL1Aintensity were compared with those of the otherthree quartiles

The correlation between TCL1A and cyclin D1protein expression was also investigated TCL1A andcyclin D1 expression were significantly correlated(Pearsonrsquos Rfrac14 0283 Pfrac14 0024) This same associationwas seen in the mRNA-expression array data of theLymphochip (httpllmppnihgov data not shown)

Discussion

Gene-expression profiling analysis of lymphomashas allowed collecting data from a large series ofcases including all the major lymphoma types Oneof the surprising findings of this study was theincreased expression of TCL1A gene in multiple B-cell non-Hodgkinrsquos lymphoma types which con-firms and extends previously published findingsconcerning precise lymphoma types27

Physiological expression of TCL1A is largelylimited to precursorimmature T and B lymphocytesand a subset of mantle zone B cells1127 Howeverlymphoma analysis shows that TCL1A expression isincreased in specific cases or tumor types presum-ably deriving from TCL1A-negative cells subsetsAdditionally all tumor types with the notableexception of Burkittrsquos lymphoma show strikingvariability in the intensity of TCL1A expressionAlthough this had been previously described inspecific lymphoma types our observations indicatethat increased TCL1A expression is a generalphenomenon occurring to some extent in all majorB-cell lymphoma types122728 Interestingly the onlylymphoma type in which TCL1A expression wasconsistently high was Burkittrsquos lymphoma a tumorwhose normal counterpart is TCL1A-negative germ-inal center cells Indeed TCL1A expression has beenproposed as being a useful ancillary test of use in thediagnosis of Burkittrsquos lymphoma2930

The oncogenic capacity of TCL1A has beendemonstrated by several groups Thus overexpres-sion of the TCL1A gene in murine B-cells leads to aCD5(thorn ) B-cell lymphoproliferative disorder withmany of the features seen in the aggressive variant ofhuman B-cell chronic lymphocytic leukemiaincluding a very low level of Ig somatic hypermuta-tion410 Hoyer and co-worker have also shown thatdysregulated TCL1 promotes multiple classes ofmature B-cell lymphoma27

In this study we have demonstrated an asso-ciation between TCL1A expression and essentialpathways for B-cell survival including B-cellreceptor CD-40 signaling NF-kB TOLL and

calcium-signaling pathways These findings seemto place TCL1A at the center of a crossroads thatis essential for malignant B-cell survival whereB-cell apoptosis induced by antigen receptor cross-linking is blocked by a T-cell signal through CD4031

This is consistent with the interpretation thatthe increased expression of TCL1A observed inmultiple B-cell lymphoma types is the consequenceof the escape from GC mechanisms of TCL1Arepression13

ARACNE generates a putative transcriptional net-work to predict broader functional relationships andcould help in the understanding the biological roleof TCL1A One of the important findings of networkanalysis was probable regulatory interaction ofTCL1A and CD27 It is known that TNFRSF7CD27binds to CD70 and plays a key role in regulatingB-cell activation and immunoglobulin synthesis Ittransduces signals that lead to the activation of NF-kB and MAPK8JNK Adaptor proteins TRAF2 andTRAF5 have been shown to mediate the signalingprocess and are important in the apoptosis inducedby TNFRSF7 TRAF1 and BCL2 are frequently foundoverexpressed in human chronic lymphocytic leu-kemia and this cooperation has been proposed tocontribute to the development of chronic lympho-cytic leukemia32 Interestingly the ARACNE studysuggests a potential mechanism where TCL1Aoncogene may provide a common link of functionalregulation between B-cell receptor component(LYN) NF-kB pathway (pro-apoptotic signal viaTNFRSF7) and survival pathway (BCL2) in B-cellnon-Hodgkinrsquos lymphoma

We have also demonstrated the prognostic sig-nificance of TCL1A expression in chronic lympho-cytic leukemia and mantle cell lymphoma inpatients using RNA microarray and protein studiesrespectively This together with the data showingthat TCL1A expression is correlated with an adverseclinical outcome in diffuse large B-cell lymphoma33

is consistent with the proposed oncogenic roleplayed by TCL1A expression Gene-silencing stu-dies may reveal how B-cell receptor and othercoregulated pathways interact with the TCL1Aoncogene at the molecular level

Thus these data contribute to the identification oflymphoma as the result of multiple oncogeniclesions and dysregulated pathways some of thekey drivers being B-cell receptor signaling andTCL1A expression acting via the calcium-signalingpathway A potential therapeutic implication ofthese findings has been shown by Zanesi et al34

where rapamycin inhibition of the Tcl1AktmTORpathway in Em-TCL1 transgenic mice prolonged thelife of all treated animals

Acknowledgements

This study was supported by grants from theMinisterio de Sanidad y Consumo (G03179

TCL1A expression in B-cell lymphomaM Aggarwal et al

213

Modern Pathology (2009) 22 206ndash215

PI051623 PI052800 CIBER-ER) the Ministerio deCiencia y Tecnologıa (SAF2005-00221 SAF2004-04286 BIO 2005-01078) the Fundacion la CaixaSpain and the National Institute of Bioinformatics(wwwinaborg) a platform of Genoma Spain Thisstudy was also supported by a Marie Curie PhDEarly Stage Research Training fellowship Weappreciate the grammatical and linguistic reviewprovided by Dr Philip Mason

Disclosureconflict of interest

The authors declare no conflicts of interest

References

1 Herreros B Sanchez-Aguilera A Piris MA Lymphomamicroenvironment culprit or innocent Leukemia20082249ndash58

2 Sanchez-Beato M Sanchez-Aguilera A Piris MA Cellcycle deregulation in B-cell lymphomas Blood20031011220ndash1235

3 Staudt LM Dave S The biology of human lymphoidmalignancies revealed by gene expression profilingAdv Immunol 200587163ndash208

4 Bichi R Shinton SA Martin ES et al Human chroniclymphocytic leukemia modeled in mouse by targetedTCL1 expression Proc Natl Acad Sci USA2002996955ndash6960

5 Cattoretti G Pasqualucci L Ballon G et al DeregulatedBCL6 expression recapitulates the pathogenesis ofhuman diffuse large B cell lymphomas in mice CancerCell 20057445ndash455

6 Polo JM Juszczynski P Monti S et al Transcriptionalsignature with differential expression of BCL6 targetgenes accurately identifies BCL6-dependent diffuselarge B cell lymphomas Proc Natl Acad Sci USA20071043207ndash3212

7 Kuppers R Mechanisms of B-cell lymphoma patho-genesis Nat Rev Cancer 20055251ndash262

8 Teitell MA The TCL1 family of oncoproteinsco-activators of transformation Nat Rev Cancer20055640ndash648

9 Pekarsky Y Santanam U Cimmino A et al Tcl1expression in chronic lymphocytic leukemia is regu-lated by miR-29 and miR-181 Cancer Res20066611590ndash11593

10 Yan XJ Albesiano E Zanesi N et al B cell receptorsin TCL1 transgenic mice resemble those of aggressivetreatment-resistant human chronic lymphocyticleukemia Proc Natl Acad Sci USA 200610311713ndash11718

11 Herling M Patel KA Khalili J et al TCL1shows a regulated expression pattern in chroniclymphocytic leukemia that correlates with molecularsubtypes and proliferative state Leukemia 200620280ndash285

12 Ruiz-Ballesteros E Mollejo M Mateo M et al Micro-RNA losses in the frequently deleted region of 7q inSMZL Leukemia 2007212547ndash2549

13 Kuraishy AI French SW Sherman M et al TORC2regulates germinal center repression of the TCL1oncoprotein to promote B cell development and

inhibit transformation Proc Natl Acad Sci USA200710410175ndash10180

14 Johnson AJ Lucas DM Muthusamy N et alCharacterization of the TCL-1 transgenic mouse as apreclinical drug development tool for humanchronic lymphocytic leukemia Blood 20061081334ndash1338

15 Tracey L Perez-Rosado A Artiga MJ et al Expressionof the NF-kappaB targets BCL2 and BIRC5Survivincharacterizes small B-cell and aggressive B-celllymphomas respectively J Pathol 2005206123ndash134

16 Martinez N Camacho FI Algara P et al The molecularsignature of mantle cell lymphoma reveals multiplesignals favoring cell survival Cancer Res2003638226ndash8232

17 Rodriguez A Martinez N Camacho FI et al Variabilityin the degree of expression of phosphorylated Ikappa-Balpha in chronic lymphocytic leukemia cases withnodal involvement Clin Cancer Res 2004106796ndash6806

18 Ruiz-Vela A Aggarwal M de la Cueva P et alLentiviral (HIV)-based RNA interference screen inhuman B-cell receptor regulatory networks revealsMCL1-induced oncogenic pathways Blood20081111665ndash1676

19 Ruiz-Ballesteros E Mollejo M Rodriguez A et alSplenic marginal zone lymphoma proposal of newdiagnostic and prognostic markers identified aftertissue and cDNA microarray analysis Blood20051061831ndash1838

20 Harris NL Jaffe ES Stein H et al A revised European-American classification of lymphoid neoplasms aproposal from the International Lymphoma StudyGroup Blood 1994841361ndash1392

21 Jaffe ES World Health Organization Pathology andGenetics of Tumours of Haematopoietic and LymphoidTissues IARC Press Oxford University Press (distri-butor) Lyon Oxford 2001

22 Rodriguez A Villuendas R Yanez L et al Molecularheterogeneity in chronic lymphocytic leukemia isdependent on BCR signaling clinical correlationLeukemia 2007211984ndash1991

23 Tracey L Aggarwal M Garcia-Cosio M et al Somatichypermutation signature in B-cell low-grade lympho-mas Haematologica 2008931186ndash1194

24 Wei G Twomey D Lamb J et al Gene expression-based chemical genomics identifies rapamycin as amodulator of MCL1 and glucocorticoid resistanceCancer Cell 200610331ndash342

25 Shaffer AL Wright G Yang L et al A library of geneexpression signatures to illuminate normal and patho-logical lymphoid biology Immunol Rev 200621067ndash85

26 Basso K Margolin AA Stolovitzky G et al Reverseengineering of regulatory networks in human B cellsNat Genet 200537382ndash390

27 Said JW Hoyer KK French SW et al TCL1 oncogeneexpression in B cell subsets from lymphoid hyperpla-sia and distinct classes of B cell lymphoma Lab Invest200181555ndash564

28 Narducci MG Pescarmona E Lazzeri C et al Regu-lation of TCL1 expression in B- and T-cell lymphomasand reactive lymphoid tissues Cancer Res2000602095ndash2100

29 Harris NL Horning SJ Burkittrsquos lymphomamdashthemessage from microarrays N Engl J Med 20063542495ndash2498

TCL1A expression in B-cell lymphomaM Aggarwal et al

214

Modern Pathology (2009) 22 206ndash215

30 Rodig SJ Vergilio JA Shahsafaei A et al Characteristicexpression patterns of TCL1 CD38 and CD44 identifyaggressive lymphomas harboring a MYC translocationAm J Surg Pathol 200832113ndash122

31 Tsubata T Wu J Honjo T B-cell apoptosis induced byantigen receptor crosslinking is blocked by a T-cellsignal through CD40 Nature 1993364645ndash648

32 Zapata JM Krajewska M Morse III HC et al TNFreceptor-associated factor (TRAF) domain and Bcl-2cooperate to induce small B cell lymphomachroniclymphocytic leukemia in transgenic mice Proc NatlAcad Sci USA 200410116600ndash16605

33 Ramuz O Bouabdallah R Devilard E et al Identifica-tion of TCL1A as an immunohistochemical marker ofadverse outcome in diffuse large B-cell lymphomas IntJ Oncol 200526151ndash157

34 Zanesi N Aqeilan R Drusco A et al Effect ofrapamycin on mouse chronic lymphocytic leukemiaand the development of nonhematopoietic malignan-cies in Emu-TCL1 transgenic mice Cancer Res200666915ndash920

35 Alizadeh AA Eisen MB Davis RE et al Distinct typesof diffuse large B-cell lymphoma identified by geneexpression profiling Nature 2000403503ndash511

Supplementary Information accompanies the paper on Modern Pathology website (httpwwwnaturecommodpathol)

TCL1A expression in B-cell lymphomaM Aggarwal et al

215

Modern Pathology (2009) 22 206ndash215