transcript profiling in peripheral t-cell lymphoma, not otherwise specified… · ptcl-nos...

Transcript profiling in peripheral T-cell lymphoma, nototherwise specified, and diffuse large B-cell lymphomaidentifies distinct tumor profile signatures

Daruka Mahadevan,1 Catherine Spier,2

Kimiko Della Croce,1 Susan Miller,4

Benjamin George,1 Chris Riley,1 Stephen Warner,3

Thomas M. Grogan,2 and Thomas P. Miller1

Departments of 1Medicine, 2Pathology, and 3Pharmacology and4Arizona Research Labs, Arizona Cancer Center, University ofArizona, Tucson, Arizona

AbstractTo glean biological differences and similarities of periph-eral T-cell lymphoma–not otherwise specified [PTCL-NOS]to diffuse large B-cell lymphoma (DLBCL), a transcripto-some analysis was done on five PTCL-NOS and fourDLBCL patients and validated by quantitative real-timereverse transcription-PCR on 10 selected genes. Normalperipheral blood T cells, peripheral blood B cells, andlymph node were used as controls. The resultant geneexpression profile delineated distinct ‘‘tumor profilesignatures’’ for PTCL-NOS and DLBCL. Several highlyoverexpressed genes in both PTCL-NOS and DLBCLinvolve the immune network, stroma, angiogenesis, andcell survival cascades that make important contributionsto lymphomagenesis. Inflammatory chemokines and theirreceptors likely play a central role in these complexinterrelated pathways: CCL2 and CXCR4 in PTCL-NOSand CCL5 and CCR1 in DLBCL. Highly overexpressedoncogenes unique to PTCL-NOS are SPI1, STK6, a-PDGFR, and SH2D1A, whereas in DLBCL they are PIM1,PIM2, LYN, BCL2A1, and RAB13. Oncogenes common toboth lymphomas are MAFB, MET, NF-kB2, LCK, andLYN. Several tumor suppressors are also down-regulated(TPTE, MGC154, PTCH, ST5, and SUI1). This studyillustrates the relevance of tumor-stroma immune traffick-ing and identified potential novel prognostic markers andtargets for therapeutic intervention. [Mol Cancer Ther2005;4(12):1867–79]

IntroductionThe WHO classifies non-Hodgkin’s lymphoma into B-celland T-cell subtypes. B-cell non-Hodgkin’s lymphomas com-prise f85% and T-cell non-Hodgkin’s lymphomas com-prise f15%. Of the T-cell non-Hodgkin’s lymphomas,peripheral T-cell lymphoma–not otherwise specified[PTCL-NOS] comprises f6% to 10% (1). These are raretumors whose pathologic diagnosis has been plagued dueto the absence of immunophenotypic markers of clonality,morphologic heterogeneity, and poor correlation betweencytomorphology and prognosis (2). Large prospectivestudies have shown that PTCL-NOS have a higher relapserate and worse survival than diffuse large B-cell lymphoma(DLBCL; refs. 3–5). Regardless of the differences inoutcome, both types of lymphoma are largely treated thesame, as there are no biological insights to differentiatethese tumors. The chemoresistance of PTCL-NOS com-pared with DLBCL has been attributed to increasedexpression of multidrug resistance proteins and p53 (6, 7).The cell of origin of PTCL-NOS is generally CD4+ T cells

and very rarely CD8+ T cells. Expression of one or morepan-T-cell antigens (CD45RO, CD2, CD3, CD5, and CD7),absence of pan-B-cell antigens, and histopathologic find-ings combine to make the diagnosis (8). In the West, nodalPTCL-NOS predominates, whereas in the East extranodalPTCL is more common, particularly the nasal type. TheKiel classification subdivides PTCL-NOS into low-gradeand high-grade groups. Within each group, distinctpatterns of genetic abnormalities have been identified(9, 10). A second approach analyzed patterns of expressionof chemokines and their receptors. Based on the expressionof CCR4, CXCR3, CD134, and ST2(L), PTCL-NOS has beensubdivided into two prognostic groups (11).Gene expression profiling using cDNA (12–14) and

oligonucleotide microarrays (15) have classified DLBCLinto three subgroups: germinal center B-like, activatedB-like, and type III. The three subgroups had markedlydifferent survival curves after cyclophosphamide-Adria-mycin-vincristine-prednisone (CHOP)–based chemothera-py, providing prognostic information in addition to theinternational prognostic index and independent of clinicalfeatures. A set of 9 genes have been identified that aresufficient to predict the three subgroups and overallsurvival based on immunohistochemistry (16, 17) andquantitative real-time reverse transcription-PCR (RT-PCR;ref. 18). Such an approach applied to PTCL-NOS wouldalso yield a gene expression-based diagnostic classification,prognostic subgroups, and novel targets that may beamenable to therapeutic intervention.The aims of the present study were to perform

transcript profiling of PTCL-NOS and DLBCL to gain

Received 5/10/05; revised 7/21/05; accepted 9/27/05.

Grant support: Southwest Oncology Group grant CA-32102, subcontract03041.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Requests for reprints: Daruka Mahadevan, Department of Medicine,Arizona Cancer Center, University of Arizona, 1515 North CampbellAvenue, Tucson, AZ 85724. Phone: 520-626-4331; Fax: 520-626-3663.E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-05-0146

1867

Mol Cancer Ther 2005;4(12). December 2005

on June 6, 2021. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

http://mct.aacrjournals.org/

biological insights into the similarities and differencesthat exist between B-cell and T-cell malignancies. Pre-vious studies (12, 14) used non-Hodgkin’s lymphoma celllines as controls; however, this study used normalperipheral blood B cells, T cells, and lymph node ascontrols. The classification of the gene expression profileinto the six hallmarks of cancer (19) yielded distinct‘‘tumor profile signatures’’ for PTCL-NOS and DLBCL,respectively. This study has analyzed and evaluated thegene expression profile signature of two phenotypicallydistinct (T cells versus B cells) non-Hodgkin’s lympho-mas and provides a framework for more extensivestudies for diagnostic classification and prognostic sub-group determinations.

Materials andMethodsPatientsA total of nine patients [four DLBCL and five PTCL-NOS]

were studied. The tumor tissue were fixed as snap-frozensamples and stored at �80jC. They were also fixed withbuffered formalin, embedded in paraffin, and stained withH&E. All slides were reviewed (C.M.S.) and the initialhistologic diagnosis was confirmed. These studies wereapproved by the patients or their legal guardians as well asappropriate written consents before biopsy. This study isinstitutional review board exempt.

ImmunohistochemistryImmunohistochemistry was done to determine tumor

lineage on either snap-frozen or formalin fixed, paraffin-embedded tissue samples using a battery of monoclonalantibodies directed against both B cells (CD19, CD20,CD21, and CD22) and T cells (CD1a, CD2, CD3, CD4,CD5, and CD8). Immunoglobulin light (n and E; BectonDickinson Corp., San Jose, CA) and heavy (IgM, IgG, IgA,and IgD; Becton Dickinson) chain status was alsodetermined. In addition, Ki-67 (DAKO, Carpinteria, CA;for proliferation) and CD30 (anaplastic T-cell lymphomaand activated B cells; DAKO) were used. For frozentissues, an indirect immunoperoxidase method using 3,3V-diaminobenzidine as the chromogen was used (20), andfor paraffin sections, the Ventana ES or a benchmarkinstrument (Ventana Medical Systems, Tucson, AZ;ref. 21) was used. All patient samples had sufficienttesting to assure lineage and clonal status, however.Aberrant loss of pan-T lineage antigens and/or T-cellsubsets was used in lieu of receptor gene rearrangementsfor T-cell lesions when such testing was not available orthere was tissue insufficient for study.

RNA Isolation and OligonucleotideMicroarraysTotal RNA was extracted from each frozen PTCL-NOS

and DLBCL specimen using the RNeasy Mini kit (Qiagen,Valencia, CA). The amount of total RNA isolated from thecells was quantified using spectrophotometric A260 meas-urements with yields z25 Ag/sample. Control RNA wasobtained from normal peripheral B cells (AllCells, Berke-ley, CA), normal peripheral T cells (AllCells), and a snap-frozen normal lymph node (Department of Pathology,

University of Arizona). mRNA (5 Ag) was used to generatefirst-strand cDNA by using a T7-linked oligo(dT) primer.After second-strand synthesis, in vitro transcription(Ambion, Austin, TX) was done with biotinylated UTPand CTP (Enzo Diagnostics, Farmingdale, NY), resulting in40- to 80-fold linear amplification of RNA. BiotinylatedRNA (40 Ag) was fragmented to 50- to 150-nucleotide sizebefore overnight hybridization at 45jC to HG-U133A 2.0Affymetrix (Santa Clara, CA) array comprising f18,400transcripts and 22,000 probe sets. After washing, arrayswere stained with streptavidin-phycoerythrin (MolecularProbes, Invitrogen, Carlsbad, CA) and scanned on aHewlett-Packard (Raleigh, NC) scanner. Intensity for eachfeature of the array was captured using GeneChip software(Affymetrix), and a single raw expression level for eachgene was derived from the 10 to 20 probe pairs represent-ing each gene by using a trimmed mean algorithm.Intensity values were scaled such that overall intensityfor each chip of the same type was equivalent. The mean FSD difference between the scaling factors of all GeneChipswas 0.75 F 0.15. Other additional measures of quality, thepercentage of gene present (55.0 F 2.5), the ratio of actin3V to 5V (1.25 F 0.15), and the ratio of glyceraldehyde-3-phosphate dehydrogenase 3V to 5V (1.02 F 0.10) indicateda high overall quality of the samples and assays. Well-measured genes were defined genes that had a ratio ofsignal intensity to background noise of >2 in >80% of thesamples hybridized.

Data Analysis andVisualizationFor each patient PTCL-NOS or DLBCL compared with

the control sample (normal B cells or T cells or normallymph node), lists of ‘‘robust increasers’’ or ‘‘robustdecreasers’’ were generated using the Affymetrix DataAnalysis Program (MAS 5.0). Fundamentals guide wasused to import these lists into GeneSpring version 5.0and obtain the intersection of robust increasers ordecreasers across all patients PTCL-NOS or DLBCL,respectively. Increasers or decreasers common to normalT cells and lymph node or normal B cells and lymphnode were compared with PTCL-NOS or DLBCLpatients. Further, increasers or decreasers common tonormal T cells and B cells or lymph node to PTCL-NOSand DLBCL were also compared. Gene expressionprofiles for PTCL-NOS or DLBCL were further classifiedaccording to the Hallmarks of Cancer (19) to determinelymphoma-specific tumor profile signatures. These aregenes involved in (a) self-sufficiency in growth signals,including oncogenes; (b) insensitivities to growth inhib-itory signals, including absence of tumor suppressors; (c)evasion apoptosis; (d) limitless replicative potential; (e)sustained angiogenesis; and (f) invasion and metastasis.Finally, searches were conducted for potential therapeutictargets by keywords (e.g., protein kinases to generategene lists for each desired target type). Intersection of thegene lists for each target with the commonly up-regulated and down-regulated genes across patientsamples were obtained. This was also done on theintersects of the up-regulated and down-regulated gene

Expression Profiling of T-Cell and B-Cell Lymphomas1868




lists with the GeneSpring ‘‘Simplified GO Ontology’’ asanother method of classifying genes. For the four DLBCLpatients, the measurement of 9 prognostic genes (LMO2,BCL6, FN1, SCYA3, BCL2, CD10, MUM1, FOXP1 , andCYCLIN D2 ; refs. 22, 23) were applied to ascertain whichsubclass they belonged to based on a comparison withthe normal B cells or lymph node.

Real-time Quantitative RT-PCRTotal RNA (100 ng) was used for reverse transcription

reactions (20 mL total volume) carried out using Super-Script III Platinum Reverse Transcriptase (Invitrogen,Carlsbad, CA). Reactions were incubated at 42jC for 50minutes followed by incubation at 37jC with RNase H for20 minutes. An Opticon DNA Engine (MJ Research, Reno,NV) was used to perform real-time fluorescence detectionPCR. cDNA (1 AL) produced from reverse transcriptionreactions was added to 12.5 AL Platinum SYBR Greenquantitative PCR SuperMix-UDG (Invitrogen), 1 AL gene-specific or h-actin-specific primer pair (see below forprimer design), and 10.5 AL distilled H2O (final volumeof 25 AL). Amplification (95jC for 15 seconds, 55jC for30 seconds, and 72jC for 30 seconds) was repeated for 44cycles. Following the PCR reaction, a melting curve assaywas done to determine the purity of the amplified product.Data were provided as a threshold cycle value (CT) for eachsample, which indicated the cycle at which a statisticallysignificant increase in fluorescence was first detected.These data were then normalized to h-actin, which servedas an unaffected control gene, for each data point andcompared with a normal T-cell control to determinerelative expression ratios. Each measurement was done intriplicate.

Primer DesignPCR primers were designed using MacVector (Accelrys,

San Diego, CA) to produce amplicons with lengths rangingfrom 80 to 250 bp to optimize the efficiency of quantitativePCR. The sequences are 5V-TGGGCTCTTTTGAAG-GATTGG-3V and 5V-GCAGTCTGGCTATCTGATTG-AAGC-3V for Lumican , 5V-CTCCTGGCAGATTCCA-CAAAAG-3V and 5V-CACTTCTTATTGGGGTCAGCA-CAG-3V for CCL18 , 5V-ACTCCCTCAATCTGTCGTTCGC-3Vand 5V-TTCCCGTCCAGTGTCAGGTTATCC-3V for CD14 ,5V-TGTCCCCCTCAAAAGTCATCC-3Vand 5V-TTGCTCAG-TTCATACACCTTCCG-3V for CD54 , 5V-GAATGGTGC-TACTTGAAGACTCTGG-3V and 5V-CACTCTCCC-GCTACACTTGTTTTC-3V for CD163 , 5V-CACACA-CAGGTGGGACACAAATAAG-3Vand 5V-GCAAGTCAAT-GAGACGGAGTCAC-3V for CD106 , 5V-TGAAGTCCA-AGTTCCTCCAGGTC-3V and 5V-TGTTGCTGTTGTGGCT-ATTAGGC-3V for HCK , 5V-TGCGTAAGAGCAAAAAGC-GAAG-3V and 5V-AAATAAGCCCCCCCTCAACCTTGG-3Vfor a-PDGFR , 5V-CGCTTTGCTCCTTGTTTTTTCC-3V and5V-CGTCTCTTTCTCCTACCCCTTGAC-3V for ABCA1 ,and 5V-TGCTACCACAAGAAATACCGCTC-3Vand 5V-AT-CCTCCTTCCCAAACACCAGC-3V for TEM6 . h-actin pri-mers from QuantumRNA h-Actin Internal Standards(Ambion) were used to normalize the quantitative PCRdata.

ResultsMorphology and Immunohistochemistry AnalysisThe five PTCL-NOS patients were all large cell lympho-

mas. All were de novo. Each sample contained >50% tumorcells. Four were of nodal origin, whereas one originated asa soft tissue mass in the knee. All PTCL-NOS patients werenegative for CD1 and CD5 and positive for CD2, CD3,and CD7. The Ki-67 ranges from 30% to 50%. Of the fourDLBCL patients, three were nodal in origin and oneoccurred as a soft tissue mass in the right breast. All fourdid not show any follicular areas; had large cell morphol-ogy, uniform expression of CD20, and monoclonal lightchain immunoglobulin; and contained >50% tumor cells.Proliferation as judged by Ki-67 for the four patients was10%, 35%, 60%, and 76%, respectively. No aberrant antigenexpression, specifically no CD5 coexpression, was detected.Figure 1A shows a representative patient with PTCL-NOSand Fig. 1B shows a patient with DLBCL with immuno-histochemistry analysis, distinguishing the two lymphomasubtypes and providing a measure of tumor involvement.

Gene Expression Profiling of PTCL-NOS and DLBCLThe Affymetrix MAS 5.0 software was used to calculate

the average, SD, median, and interquartile range calcula-tions [e.g., the top 25 increases or decreases; PTCL-NOSversus normal T cells or normal lymph node]. Theintersample variability correlated well with the quantitativereal-time RT-PCR data. For example the five patients withPTCL-NOS for CD14 gene expression signals were 1,034.6,3,371.4, 4,720.3, 3,451.1, and 3,130.5. The average was3,141.5, SD was 1,330.46, median was 3,371.4, and inter-quartile range was 320.6. Based on these data, the averagefold change was calculated to be 88-fold compared withnormal peripheral blood T-cell RNA. Well-measured geneswere defined as genes that had a ratio of signal intensity tobackground noise of >2 in >80% of the samples hybridized.Genes that are robustly up-regulated and common to all

five patients with PTCL-NOS are compared with normalperipheral blood T cells or normal lymph node (Table 1). Itseems that the comparison of PTCL-NOS patients tonormal peripheral blood T cells shows that most of thegenes that are overexpressed are involved in tumorinvasion (COL1A1, FN1, CTGF, COL1A2, CCL2, CLU, C1S,LUM , and LYS ), antiapoptosis (CD14 ), proliferation(CCL18, CXCL13 , and IGFBP7), and angiogenesis (TEM6).Similarly, when PTCL-NOS are compared with the normallymph node, most of the genes that are up-regulated andoverexpressed are involved in tumor invasion (CHI3L1,CCL5 , and CORO1A) and proliferation (SH2D1A, FLI1,AIF1, CRIP1, MAP4K1 , and Rac2). Genes known to becausative in tumorigenesis are prominently expressed,indicating that peripheral blood T cells and lymph nodecontrols both provide valuable information regarding theevolution of the tumor from normal T cells and lymphnodes. The robustly down-regulated genes are not includedhere but will be discussed below with regard to the tumorprofile signature.Genes that are robustly up-regulated and common to all

four patients with DLBCL are compared with normal

Molecular Cancer Therapeutics 1869




peripheral blood B cells or normal lymph node (Table 2).The comparison of DLBCL patients to normal peripheralblood B cells shows that the genes that are up-regulatedand overexpressed are also involved in tumor invasion(LYS, CCL5, CHI3L1, CHIT1, MMP9 , and ADAMDEC1),immune cell trafficking or host cell response to tumor(CCL18, RGS13, CXCL9 , and VNN1), and proliferation(PLA2G2D and PLA2G7). Similarly, when DLBCL patientsare compared with the normal lymph node, most of thegenes that are up-regulated and overexpressed are

involved in tumor invasion (CCL5, CORO1A , andCHI3L1), antiapoptosis (BCL2A1), immune trafficking(CCL18, CD79B, CD52 , and POU2AF1), and proliferation(CD20, MAP4K1 , and Rac2). Tumor tissues express genesthat promote and maintain the oncogenic process, and theprofiles obtained based on normal peripheral blood B cellsand lymph node will help guide the elucidation of themechanism initiation and progression. The robustly down-regulated genes are not included and will be discussedwith regard to the tumor profile signature.

Figure 1. A, a representative patient withPTCL-NOS showing H&E staining for mor-phology and immunohistochemistry to de-lineate T-cell phenotype (CD2 ) is CD8negative (staining is due to tumor-infiltratinglymphocytes) with tumor involvement(>50%) and aggressiveness (Ki-67). B, arepresentative patient with DLBCL showingH&E staining for morphology and immuno-histochemistry to delineate B-cell phenotype(CD20 ) is CD8 negative (staining is due totumor-infiltrating lymphocytes) with tumorinvolvement (>50%) and aggressiveness(Ki-67).





Table 3 shows the highly up-regulated genes that arecommon to both PTCL-NOS and DLBCL when comparedwith normal peripheral blood T cells and B cells or lymphnode, respectively. There are only three genes commonto PTCL-NOS and DLBCL when compared with normalperipheral blood T cells and B cells and they are CCL18,UBD , and RARRES1 . However, there are 11 genes commonto PTCL-NOS and DLBCL when compared with the lymphnode and they are CCL18, CD52, MAP4K1, CORO1A,PPP1R16B, PSMB8, UBD, LYZ, TCRb, RAC2, and CCL5 .

These comparisons provide insights into the importanceof differential gene expression profiles that are commonto both types of lymphoma. By including a single cell(peripheral blood B cells or T cells) as well as a complextissue (lymph node), insights into the relative importance ofthe relationships between tumor-stroma immune traffick-ing cells can now be gleaned.

Tumor Profile Signatures of PTCL-NOS and DLBCLWe next did an analysis based on the six hallmarks of

cancer (19) in which a tumor has the following acquired

Table 2. Highly overexpressed genes in DLBCL compared withnormal peripheral blood B cells or lymph node

Fold D Gene name Function

B-non-Hodgkin’s lymphoma vs normal peripheral blood B cells:

top increases

172 CCL18 Chemokine146 VNN1 Lymphoma homing102 UBD Protein degradation102 LYZ ECM92 CCL5 Chemokine89 PLA2G2D Lipid metabolism83 CHI3L1 ECM82 CHIT1 ECM75 RGS13 G protein signaling53 BID Apoptosis48 APOC1 Lipid metabolism46 PLA2G7 Lipid metabolism45 ADAMDEC1 ECM41 MMP9 ECM34 CXCL9 Chemokine25 GZMK Protease17 LR8 Lipid metabolism15 KIAA0101 Unknown13 GPNMB Tumor suppression11 CTSB Protease

B-non-Hodgkin’s lymphoma vs normal lymph node: top increases

172 CCL18 Chemokine151 IGJ Antibody146 VNN1 Lymphoma homing130 CDW52 Proliferation127 MAP4K1 Proliferation126 NCF1 Inflammation123 CORO1A Cytoskeleton121 CD79B Immune response107 BCL2A1 Apoptosis105 PPP1R16B Protein phosphatase104 PSMB8 Proteasome104 PTPRCAP Protein phosphatase102 UBD Protein degradation102 LYZ ECM100 TCRb T-cell receptor98 CD20 Proliferation97 GRP3 GTP signaling96 POU2AF1 Immune response96 NCF1 Inflammation94 RAC2 Proliferation92 CCL5 Chemokine

Table 1. Highly overexpressed genes in PTCL-NOS comparedwith normal peripheral blood T cells or lymph node

Fold D Gene name Function

PTCL-NOS vs normal peripheral blood T cells: top increases

176 COL1A1 ECM154 CCL18 Chemokine135 CXCL13 Chemokine130 IGFBP7 Proliferation128 RARRES1 Tumor suppression123 CLU ECM109 C1S ECM107 FN1 ECM103 CTGF Proliferation102 CCL2 Chemokine101 LUM ECM99 SERPING1 Protease inhibition99 APOC1 Lipid metabolism98 NID ECM96 COL1A2 ECM94 TEM6 Angiogenesis93 COL3A1 ECM89 APOE Lipid metabolism89 UBD Protein degradation89 PLTP Lipid metabolism88 CD14 Antiapoptosis

PTCL-NOS vs normal lymph node: top increases

200 CHI3L1 ECM154 CCL18 Chemokine120 CCL5 Chemokine120 SH2D1A Proliferation120 NKG7 Natural killer receptor113 HLA-DQB1 Immune regulation109 FLI1 Oncogene108 AIF1 Proliferation108 CORO1A Cytoskeleton107 RAC2 Proliferation106 MAP4K1 Proliferation104 PSMB8 Proteasome104 TCRc T-cell receptor102 CARD15 Apoptosis100 LYZ ECM100 TRIM Inflammation100 CRIP1 Proliferation96 CDW52 Proliferation92 TCRb T-cell receptor89 UBD Protein degradation89 PPP1R16B Protein phosphatase





capabilities: (a) self-sufficiency in growth signals, (b)insensitivity to antigrowth signals, (c) evasion of apoptosis,(d) limitless replicative potential, (e) sustained angiogene-sis, and (f) tissue invasion and metastasis. Genes weresorted into each of these categories common to all fivePTCL-NOS patients compared with peripheral bloodT cells, lymph node, and/or both (Table 4). A similaranalysis was done for the four DLBCL patients comparedwith peripheral blood B cells, lymph node, and/or both(Table 5). This provides a tumor profile signature forPTCL-NOS and DLBCL. PTCL-NOS identified genesassociated with proliferation (IGFBP7, SH2D1A, RAC2 ,and TCIRG1), tumor suppressors (RARRES3, GAS1 , andCDKN1A), invasion (COL1A, FN1 , and CCL2), unlimitedreplication (CTGF, CYR61 , and NEK2 ), angiogenesis(VEGF, PDGFR, CSF1R , and HGF), antiapoptosis (CD14and NF-jB), and oncogenes (FLI1, MAFB, SPI1, STK6 , andMET). DLBCL identified genes associated with prolifer-ation (IGFBP7, CD20, CD72, CRIP1 , and RAB13), tumorsuppressors (GPNMB, CD63, BTG2 , and RARRES3), inva-sion (LUM, FN1, MMP1 , and COL6A3), unlimited repli-cation (CCR1 and CCL5), angiogenesis (VEGF, PDGF,PDGFR , and FGF13 ), and antiapoptosis (CD14 andBCL2A1). Therefore, it is apparent that normal peripheralblood T cells or B cells and lymph node in combinationprovides consistent gene expression findings that now canbe evaluated in detail and attempts be made to positionthese in signaling pathways that make the relationshipsmore obvious.

Nine-Gene PredictiveModel of DLBCLTissue microarray using immunoperoxidase staining for

predictive markers to accurately subdivide DLBCL intoprognostically relevant subgroups has been done andcorrelated with cDNA microarray as the gold standard(22). Six proteins were identified: CD10, BCL2, BCL6,MUM1/IRF4, CYCLIN D2, and FOXP1. Similarly, aquantitative real-time RT-PCR analysis identified six genes

to have prognostic significance: BCL2, BCL6, FN1, LMO2,CCND2 , and SCYA3/CCL3 (23). In this study, we combinedthe genes that were used in the tissue microarray andRT-PCR to evaluate the four DLBCL and five PTCL-NOSpatients. When the four DLBCL patients were comparedwith the normal peripheral blood B cells, three of ninegenes were up-regulated (FN1 140-fold, CCL3 4.9-fold, andBCL6 2.2-fold), and when compared with the lymph node,seven of nine genes were up-regulated (MUM1 28-fold,CCL3 11.4-fold, CCND2 10.4-fold, BCL6 6.7-fold, LMO2 5.3-fold, BCL2 4.6-fold, and FN1 2.3-fold). These data placethese four unselected DLBCL patients into the non-germinal center activated B-cell subgroup. Further, theimportance of the control RNA is highlighted, implyingthat the lymph node may be more informative due to itscomplex nature and that a tumor does follow the hallmarksof cancer.

Table 3. Overexpressed genes common to PTCL-NOS andDLBCL compared with normal peripheral blood T cells,peripheral blood B cells, and lymph node

Common increases

Gene nameFold change PTCL-NOS Fold change DLBCL

CCL18 154.4 172.6CDW52 96.7 130.0MAP4K1 106.6 127.1CORO1A 108.1 123.2PPP1R16B 89.1 106.0PSMB8 104.4 105.0UBD 89.8 102.6LYZ 100.5 102.3RAC2 107.9 94.6CCL5 120.9 92.3TCRb 92.6 100.6

Table 4. Hallmarks of cancer for DLBCL: DLBCL vs peripheralblood B cells and lymph node

Gene name Fold D Definitions

ProliferationIGFBP7 107 IGF-I-binding proteinCD20 98 TetraspaninCD72 83 Type II membrane protein

C-type lectinCRIP1 67 Zinc finger motif, group II

LIM protein familyRAB13 23 GTPase

Tumor suppressorsGPNMB 97 Transmembrane

glycoproteinCD63 90 TetraspaninBTG2 67 B-cell translocation gene 2RARRES3 64 Retinoic acid receptor

response gene 3Invasion and metastasisLUM 67 Lumican, ECMFN1 59 Fibronectin, ECMMMP1 40 MMP, ECMCOL6A3 24 Collagen, ECM

Unlimited replicationCCR1 82 Chemokine receptor,

inflammationCCL5 14 Chemokine ligand,

inflammationAngiogenesisVEGF 65 Vascular endothelial

growth factorPDGF-AA 14 Platelet-derived

growth factorPDGFR-b 10 Platelet-derived growth

factor receptorFGF13 6 Fibroblast growth factor

ApoptosisCD14 141 Lipopolysaccharide receptor,

glycosylphosphatidylinisotolanchor

BCL2A1 107 B-cell lymphoma 2





Discovery of New Therapeutic TargetsThe study also analyzed certain key therapeutic targets

that may be amenable to targeting either with small-molecule inhibitors and/or monoclonal antibodies. Forpatients with PTCL-NOS, the following are novel targets:small-molecule inhibitors to protein kinases (STK6, NEK2,c-MET, SYK , and a-PDGFR), monoclonal antibodies tocirculating ligands (CCL2, VEGF, HGF, CTGF, PDGF, FGF7,and IGFBP7), and a small-molecule inhibitor to Rac2 , amember of the Ras family of small GTP-binding proteins.For patients with DLBCL, the following are novel targets:small-molecule inhibitors to protein kinases (LCK, LYN,PIM1, PIM2, MET , and SYK), small-molecule inhibitors tonovel targets (PSMA, BCL2A1, and NF-jB2), monoclonalantibodies to circulating ligands (CCL2, CCL5 , and VEGF),and cell surface markers (CD72, ICAM2 , and CD52).

Validationwith Quantitative Real-time RT-PCRQuantitative real-time RT-PCR on 10 selected, overex-

pressed, functionally relevant genes (Lumican, CCL18,CD14, CD54, CD106, CD163, a-PDGFR, HCK, ABCA1 , andTEM6) common to all five PTCL-NOS patients validatedthe gene expression profile data (Fig. 2). The expressionlevel for each patient is shown and averaged over three

runs of RT-PCR. These correlated well with the intersamplevariability observed from the gene expression signalsobtained from the DNA microarray.

DiscussionPatients with PTCL-NOS have a worse outcome comparedwith patients with DLBCL (24). To distinguish biologicalproperties of these two phenotypic variants of large celllymphoma, we undertook a gene expression profile studyof PTCL-NOS and DLBCL using an Affymetrix platform.The study identified several key elements in lymphomabiology and has led to hypothesis-generating research thatshould guide elucidation of the underlying oncogenicevents in PTCL-NOS and DLBCL. The complexity of thesetumors is represented by 25 to 30 up-regulated cancergenes and several down-regulated tumor suppressors thatprovide a growth advantage and enhance the ability toinvade and metastasize.

PTCL-NOS Tumor Profile SignatureA predictive model for the PTCL-NOS tumor profile is

delineated based on the six hallmarks of cancer (Fig. 3).Chemokines cause the directed migration of leukocytes

Table 5. Hallmarks of cancer for PTCL-NOS: PTCL-NOS vs peripheral blood T cells and lymph node

Gene name Fold D Functions

ProliferationIGFBP7 107 IGF-I-binding proteinSH2D1A 120 Src homology-2 domain protein 1aRAC2 107 GTPaseTCIRG1 54 Seven-transmembrane spanning vacuolar proton pump proteinHGF 23 Hepatocyte growth factor

Tumor suppressorsRARRES3 128 Retinoic acid receptor response gene 3GAS1 58 Growth arrest specific 1CDKN1A 20 Cyclin-dependent protein kinase inhibitor

Invasion and metastasisCOL1A 176 Collagen, ECMFN1 107 Fibronectin, ECMCCL2 102 Chemokine ligand 2, inflammation

Unlimited replicationCTGF 103 Connective tissue growth factorCYR61 72 Cysteine-rich protein of CCN familyNEK2 57 Serine/threonine kinase related to mitotic regulator NIMA

AngiogenesisVEGF 84 Vascular endothelial growth factorPDGFR-A 26 Platelet-derived growth factor receptorCSF1R 19 Colony-stimulating factor receptor 1PDGFR-B 18 Platelet-derived growth factor receptor

ApoptosisCD14 88 Lipopolysaccharide receptor, glycosylphosphatidylinisotol anchorNF-jB 44 Transcription factor

OncogenesFLI1 109 Cell cycle progressorMAFB 80 Musculoaponeurotic fibrosarcoma, basic leucine zipper transcription factorSPI1 29 Transcription factorSTK6 16 Aurora kinase, cell cycle regulatorMET 7 Hepatocyte growth factor receptor





along a chemical gradient of ligand. Many cancers,including lymphomas, have a complex chemokine networkthat influences immune cell infiltration, tumor cell growth,survival, migration, and angiogenesis. These cells them-selves express chemokine receptors and can respond tochemokine in an autocrine and/or paracrine mechanism(25). Many different cancers, including PTCL-NOS (26),have different profiles of chemokine and chemokinereceptor expression, but CXCR4 is most commonly found.It has been shown that c-MAF may alter the function of

multiple myeloma cells by up-regulating CCR1 , a receptorfor the chemokine MIP-1. Three MAF target genes overex-pressed in multiple myeloma are CYCLIN D2, integrin b7 ,

and CCR1 . Increased expression of CYCLIN D2 enhancesproliferation, whereas increased integrin b7 promotesadhesion to bone marrow stroma and increases VEGFelaboration (27). In PTCL-NOS, MAFB is 80-fold overex-pressed and is important for IL-4 gene expression by Th2cells. Several of the target genes activated by c-MAF inmultiple myeloma are also up-regulated in PTCL-NOS,which include VEGF (84-fold), ITGA4 (88-fold), andCCR1 (39-fold), implying aberrant proliferation, increasedadhesion to surrounding stroma, tumor angiogenesis, andimmune dysregulation (Fig. 3). Several other chemokinesare also overexpressed and are likely driven byMAFB and/or two other highly overexpressed oncogenic transcription

Figure 2. Quantitative real-time RT-PCR of 10 selectedgenes common to five PTCL-NOS patients (1–5). Yaxis, level of expression compared with control.





factors FLI1 (100-fold) and SPI1 (29-fold). FLI1 promotescell cycle progression by suppressing the expression of CDKinhibitor p27kip1 (28), whereas SPI1 is normally expressedin all hematopoietic cell lineages, except in T-cell lines (29),but its aberrant expression may be an additional mechanismthat maintains the malignant lymphocyte to proliferate.CCL2 is 100-fold overexpressed and a potent chemo-

attractant for monocytes, memory T lymphocytes, andnatural killer cells. CCL2 is a tumor promoter (30), is ableto recruit leukocytes, and provides growth signals fortumor angiogenesis. Several angiogenic receptors andligands are overexpressed in PTCL-NOS, which includesVEGF (84-fold), a-PDGFR (26-fold), CSF1R (19-fold),b-PDGFR (19-fold), c-MET (7-fold), HGF (24-fold),PDGF-CC (5-fold), and FGF7 (2.5-fold), all seem tocontribute to tumor growth. CCL5 is a 120-fold overex-pressed chemoattractant that binds to several receptors,including CCR1 (39-fold), CCR3, CCR4 , and CCR5. CCL5up-regulates transcription of MMP9 , which can contrib-ute to angiogenesis. Further, tumor-derived CCL5 caninhibit the T-cell response and enhance the in vivogrowth of tumors (31). CXCR4 is 79-fold overexpressedand involved in lymphocyte trafficking. When activated,CXCR4 promotes invasion of lymphoma cells into tissues

through activated LFA1 , which in turn activates the JAKkinases and shown to be essential for lymphoma invasionand metastasis (32).Tumor-stroma interactions play an integral role in

lymphoma cell survival. In PTCL-NOS, IGFBP7 is f130-fold overexpressed, enhances IGF activity, and influencescell adhesion and migration (33), a potential mechanismof tumor invasion. CTGF/CCN2 is >100-fold and CYR61/CCN1 is >70-fold overexpressed and belongs to acysteine-rich secreted protein of the CCN family (34)that binds to integrins and modulates invasive behaviorof cancer cells. Several components of the extracellularmatrix (ECM) are elevated and include CHI3L1 (200-fold), COL1A1 (177-fold), FN1 (108-fold), COL1A2 (103-fold), and emilin (44-fold), most likely mediated byCTGF/CYR61 (Fig. 3). CYR61 has been shown to enhancetumorigenicity through activated integrin-linked kinaseto stimulate h-catenin-TCF/Lef and Akt signaling path-ways (34).Aberrant cellular proliferation due to dysregulated

cell cycle control seems to be mediated by the serine/threonine kinase NEK2 , which is 58-fold overexpressed(Fig. 3) and localizes to the centrosome. Overexpression ofactive NEK2 induces splitting of centrosomes, whereas

4

Figure 3. A predictive model delineating the tumor profile signature of PTCL-NOS. Activation of oncogenic transcription factors (MAFB, FLI1 , and SPI1 )promotes overexpression of chemokines (CCL2 and CCL5) and chemokine receptors (CCR1 and CXCR4 ). These acting in an autocrine and paracrinemanner recruit stromal fibroblasts, inflammatory immune cells, and endothelial cells to form a complex network of tumor tissue. The overexpression ofintegrins (LFA1 and VLA4 ) on the tumor cells and their binding partners on stromal cells (ICAM1, ICAM2 , and VCAM1 ) promotes tumor-stroma cross-talkthat activates proliferative signaling pathways (JAK/STAT ), ECM formation (FN1, COL1A1, COL1A2, CHI3L1, and emilin ), and stromal-derived cytokines/growth factors (CTGF and CYR61). Several tumor-derived angiogenesis promoters (VEGF, PDGF-CC, FGF7, PDGFR , and CSF1R ) are overexpressed, whichhelps maintain a complex angiogenic process. The recruitment of inflammatory immune cells that enhance tumor growth and the production of invasivefactors (HGF, MET, CTGF , and IGFBP7 ) bode for an aggressive T-cell lymphoma. Moreover, the overexpression of NF-nB-driven proliferative factors(TCIRG1, CD14, CD52, TNFRSF25 , and CCR1 ) inhibits many forms of apoptosis and contributes to chemoresistance. The overexpression of NEK2 andSTK6 contributes to cell cycle dysregulation and resultant aneuploidy. Several markers of proliferation are also overexpressed (SH2D1A, LCK, Rac2 , andMAP4K1 ), which contributes to the overall survival and antiapoptotic pathways.





prolonged expression of either active or inactive NEK2leads to dispersal of centrosomal material and loss of afocused microtubule-nucleating activity (35). NEK2 hasalso been shown to be overexpressed in transformedfollicular lymphoma and de novo DLBCL (36). STK6(Aurora kinase-A) is 17-fold overexpressed and plays arole in cell cycle regulation during anaphase and/ortelophase, in relation to the function of the centrosome/spindle pole region during chromosome segregation.Defect in STK6 is responsible for numerical centrosomeaberrations, including aneuploidy, which is related to itsrole in microtubule formation and/or stabilization (37).Several markers of proliferation are represented, of

which SH2D1A is 120-fold overexpressed and binds to aspecific motif in the cytoplasmic domains of severalmembers of the CD2 subfamily of cell surface receptors,where it blocks recruitment of the tyrosine phosphataseSHP-2 and prevents inhibition of tyrosine kinase activatedsignaling pathways (38). Rac2 is 107-fold overexpressedand regulates a diverse array of cellular events, such ascell growth, cytoskeletal reorganization, and activation ofprotein kinases. Rac2 is expressed in thymocytes andactivated T cells and could play a role in control ofgrowth and death of T cells (39). LCK is 35-fold overex-pressed and phosphorylates mitogen-activated proteinkinase and L-selectin with subsequent association ofGrb2/Sos with L-selectin. This association correlates withan activation of p21ras, mitogen-activated protein kinase,and Rac2 and a transient increase of O2

� synthesis. A finalcommon path could involve a L-selectin-associated CD3~-chain-driven T-cell antigen receptor-induced cell prolif-eration (40). TCIRG1/TIRC7 is 54-fold overexpressed, anovel seven-transmembrane spanning vacuolar protonpump protein that is up-regulated during the early stagesof T-cell activation in response to alloantigens. InhibitingTIRC7 suppresses proliferation of activated T cells (41)and supports the notion that it may play an important rolein PTCL proliferation.In PTCL-NOS, several tumor suppressors are down-

regulated. TPTE encodes a PTEN-related tyrosine phos-phatase, is 99-fold down-regulated, and plays an importantrole in suppressing signaling via tyrosine-phosphorylatedproteins (42). MGC15419 is 20-fold down-regulated andmay be critical for the controlled degradation of cellularregulatory proteins (43). PTCH is 6-fold down-regulatedand is the receptor for sonic hedgehog, implicated in theformation of embryonic structures and in tumorigenesis.Mutations of this gene have been associated with nevoidbasal cell carcinoma syndrome, squamous cell carcinoma,trichoepitheliomas, and transitional cell carcinoma (44).ST5 is 5-fold down-regulated and contains a COOH-terminal region that shares similarity with the Rab3 familyof small GTP-binding proteins. This protein preferentiallybinds to the SH3 domain of c-Abl kinase and acts as aregulator of mitogen-activated protein kinase 1/extracel-lular signal-regulated kinase 2 kinase, which may contrib-ute to its ability to reduce the tumorigenic phenotype incells (45).

Several antiapoptotic proteins are represented in PTCL-NOS. CD14 is 88-fold overexpressed and acts via MyD88,TIRAP, and TRAF6, leading to NF-nB activation, cytokinesecretion, and inflammatory response (46). NF-jB2 is 44-fold overexpressed and is a transcription factor containingrel-polyG-ankyrin domains. NF-jB2/p52 gene rearrange-ments have been observed in patients with lymphoma andhave been shown to contribute to tumorigenesis bysuppressing apoptosis and increasing proliferation inresponse to inflammatory cytokines (47–49). A recentexpression profiling study showed that the NF-nB signalingpathway is deregulated in PTCL-NOS (50), implicating thisinflammatory transcription factor in maintaining cellsurvival pathways. IL-18 is 39-fold overexpressed andinduces IFN-g production in T cells, and dysregulatedIL-18 and/or IL-18R in chronic B-cell lymphoproliferativedisorders may contribute to tumor escape from the hostimmune system by reducing the expression of Fas ligand.Moreover, it has been shown to function as a novelangiogenic mediator by promoting endothelial cell migra-tion (51).Several proapoptotic genes are down-regulated. c-Jun is a

27-fold down-regulated oncoprotein that blocks the ubiq-uitination of tumor suppressor p53 and thus increases thestability of p53 in nonstressed cells. Studies of the mousecounterpart of this gene suggest a key role in T-celldifferentiation (52). CED-6 is 22-fold down-regulated andis an evolutionarily conserved adaptor protein required forefficient engulfment of apoptotic cells and that promotesprogrammed cell death. Inactivating mutations in engulf-ment genes enhance the frequency of cell survival (53, 54).TNFRSF25 is 12-fold down-regulated, is expressed prefer-entially in tissues enriched in lymphocytes, and plays a rolein regulating lymphocyte homeostasis. This receptor hasbeen shown to stimulate NF-nB activity and regulate cellapoptosis. The alternative splicing of this gene in B cellsand T cells encounters a programmed change on T-cellactivation, which predominantly produces full-length,membrane-bound isoforms and is thought to be involvedin controlling lymphocyte proliferation induced by T-cellactivation (55). Therefore, a loss of function would lead touncontrolled T-cell proliferation.

DLBCL Tumor Profile SignatureA predictive model for the DLBCL tumor profile is

delineated based on the six hallmarks of cancer (Fig. 4). Achemokine network seems to be in operation and is likelymodulated by transcription factors MAFB (83-fold overex-pressed), FLI1 (86-fold overexpressed), and CRIP1 (67-foldoverexpressed), a cysteine-rich intestinal protein 1 that con-tains a double zinc finger motif. Overexpression of CRIP inmice produces an imbalance in cytokine pattern favoringTh2 cytokines: low IFN-g and high IL-6 and IL-10, whichalter the immune response and likely favor escape fromimmune attack (56). CCL5 is 147-fold overexpressed andbinds its receptor CCR1 , which is 82-fold overexpressed.Tumor-derived CCL5 can inhibit the inhibitory T-cellresponse and enhance in vivo lymphoma growth. Severalproangiogenic ligands and receptors are overexpressed,





which include VEGF (66-fold), PDGF-AA (15-fold), CSF1R(14-fold), b-PDGFR (11-fold), MET (7-fold), FGF13 (6-fold),and PSMA (4-fold), and are likely to be induced by CCL5-CCR1 . Tumor stroma–derived factors, such as IGFBP7 ,which is 107-fold overexpressed (33) and likely elaboratesseveral components of the ECM, such as CHI3L1 (84-fold),FN1 (60-fold), COL6A3 (49-fold), COL18A1 (47-fold), andICAM2 (30-fold).Markers of proliferation represented include CD20 (99-

fold), a member of the membrane-spanning 4A gene familythat functions as a Ca2+ channel resident on B lymphocytesand plays a role in the development and differentiation ofB cells into plasma cells. It is the target for rituximabin B-cell non-Hodgkin’s lymphoma and shows excellentcorrelation between gene expression profile and immuno-histochemistry (57). CD72 is a 83-fold overexpressed type IImembrane protein of the C-type lectin superfamily and isthe ligand for CD5. It plays a role in pre-B-cell and B-cellproliferation and differentiation. Among 83 malignant non-Hodgkin’s lymphomas evaluated by immunohistochemis-try, all 54 B-cell lymphomas, including precursor B-celllymphomas, were CD72 positive, but staining was notobserved in T-cell lymphomas. Therefore, CD72 is aproliferative marker that cooperates with CD20 in DLBCL

(58). RAB13 , a GTPase, is 29-fold overexpressed and regu-lates the assembly of functional epithelial tight junctions.Expression of the GTP-bound form of RAB13 inhibitsprotein kinase A–dependent phosphorylation and tightjunction recruitment of the vasodilator-stimulated phos-phoprotein, an actin remodeling protein and thus may playa role in invasion and metastasis (59).In DLBCL, two tumor suppressors are down-regulated.

TPTE is a 100-fold down-regulated PTEN-related tyrosinephosphatase that, if active, would switch off tyrosinekinase–driven oncogenic pathways (42). SUI1/MOF2 is 25-fold down-regulated and functions as a modulator of accu-racy at both initiation and elongation phases of translation(60). Transfection of human SUI1 into HepG2 hepatocarci-noma cells inhibited cell growth in culture in soft agar andpartially inhibited tumor formation in nude mice (61).Of the antiapoptotic proteins that are overexpressed,

CD14 is 141-fold overexpressed and is known to activateNF-nB leading to inflammatory cytokine secretion (46) andan inflammatory response that likely helps avoid immuneattack. BCL2A1 is 107-fold overexpressed, forms hetero-dimers or homodimers, and is involved in a wide variety ofcellular activities, such as embryonic development, homeo-stasis, and tumorigenesis. BCL2A1 is a direct transcription

Figure 4. A predictive model delineating the tumor profile signature of DLBCL. Activation of oncogenic transcription factors (MAFB, FLI1 , and CRIP1 )promotes overexpression of chemokines (CCL3, CCL5 , and CXCL9 ) and chemokine receptor (CCR1 ). The chemokine network acting in an autocrine andparacrine manner recruit stromal fibroblasts, inflammatory immune cells, and endothelial cells to form a complex network of tumor tissue. Theoverexpression of integrin LFA1 on the tumor cells and their binding partners on stromal cells (ICAM2 and ICAM3 ) promotes tumor-stroma cross-talk whichactivates proliferative signaling pathways (JAK/STAT ) and ECM formation (Lumican, FN1, COL6A3, COL18A1 , and CHI3L1 ). Several tumor-derivedangiogenesis promoters (VEGF, PDGF-AA, FGF13, PDGFR, CSF1R , and PSMA ) are overexpressed, which helps maintain a complex angiogenic process.The recruitment of inflammatory immune cells that enhance tumor growth and the production of invasive factors (MET and IGFBP7 ) bode for an activatedB-like DLBCL. Moreover, the overexpression of NF-nB-driven proliferative factors (CD14, CD20, CD52, CD72 , and CCR1 ) inhibits many forms of apoptosisand contributes to chemoresistance. The overexpression of CCND2 and MUM1 contributes to cell cycle dysregulation and uncontrolled proliferation.Several markers of proliferation are also overexpressed (PIM1, PIM2, BCL2A1, RAB13 , and MAP4K1 ) that contribute to the overall survival andantiapoptotic pathways.





target of NF-nB in response to inflammatory mediators andis up-regulated by different extracellular signals, such asgranulocyte macrophage colony-stimulating factor, CD40,and inflammatory cytokines (TNF and IL-1 ), whichprovides a cytoprotective function that may be essentialfor lymphocyte activation and cell survival (62). Mice withBCL2A1 overexpression showed extended survival withaltered B lineage development that led to expansion of thepro-B-cell subset at the expense of pre-B cells, suggestingan impairment of the pro-B-cell to pre-B-cell transition (63).Several genes are implicated in the pathogenesis of DLBCL,which seem to have prognostic significance. Patients withgerminal center B-like DLBCL have the most favorable curerate, whereas patients with activated B-like or type IIIDLBCL have a worse survival and this may be due in partto the ability of the NF-nB pathway to inhibit intrinsicapoptosis and/or chemotherapy-mediated apoptosis (64).A transcriptosome analysis of PTCL-NOS and DLBCL

patients has identified a molecular tumor signature profilethat distinguishes B-cell from T-cell non-Hodgkin’slymphoma. In both types of non-Hodgkin’s lymphoma,MAFB-driven inflammatory chemokine signaling path-ways play critical roles in promoting proliferation, angio-genesis, tumor-stroma interactions, and immune escapealbeit with different key players most likely reflectinglineage specificity. Phase I clinical trials are now feasibletargeting the PDGFR (Gleevec), MET (Mab to HGF), STK6(AK inhibitors), and CD52 (CAMPATH-1H) in this setting.

Acknowledgments

We thank Ellen Chase for coordinating this project and Nikhil Shirahatti fortechnical help with article preparation.

References

1. Jaffe ES, Harris NL, Vardiman JW, Stein H. Pathology and genetics:neoplasms of the hematopoietic and lymphoid tissues. In: Kleihues P,Sobin L, editors. WHO classification of tumors. Lyon: IARC Press; 2001.

2. Chan JK. Peripheral T-cell and NK-cell neoplasms: an integratedapproach to diagnosis. Mod Pathol 1999;12:177–99.

3. Lippman SM, Miller TP, Spier CM, Slyman DJ, Grogan TM. Theprognostic significance of the immunotype in diffuse large-cell lymphoma:a comparative study of the T-cell and B-cell phenotype. Blood 1988;742:436–41.

4. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomashave a worse prognosis than B-lymphomas: a prospective study of361 immunophenotyped patients treated with the LNH-84 regimen.Ann Oncol 1990;1:45 –50.

5. Gisselbrecht C, Gaulard P, Lepage E, et al. Prognostic significance ofT-cell phenotype in aggressive non-Hodgkin’s lymphomas. Blood 1998;92:76–82.

6. Drenou B, Amiot L, Lamy T, LePrise PY, Fauchet R. Multidrugresistance in aggressive lymphoproliferative disorders of T and natural-killer origin. Leuk Lymphoma 1988;30:381–7.

7. Pescarmona E, Pignoloni P, Santangelo C, et al. Expression of p53 andretinoblastoma gene in high-grade nodal peripheral T-cell lymphomasimmunohistochemical and molecular findings suggesting different patho-genic pathways and possible clinical implications. J Pathol 1999;1188:400–6.

8. Gorczyca W, Weisberger J, Liu Z, et al. An approach to diagnosis ofT-cell lymphoproliferative disorders by flow cytometry. Cytomtery 2002;50:177–90.

9. Greer JP. T cell and NK cell lymphoproliferative disorders. Shouldperipheral T cell lymphoma be treated differently from large B celllymphoma. Am Soc Hematol Educ Program Book 2001;268–81.

10. Zettl A, Rudiger T, Konrad MA, et al. Genomic profiling of peripheralT-cell lymphoma, unspecified, and anaplastic large T-cell lymphomadelineates novel recurrent chromosomal alterations. Am J Pathol 2004;164:1837–48.

11. Tsuchiya T, Ohshima K, Karube K, et al. Th1, Th2 and activated T-cellmarker and clinical prognosis in peripheral T-cell lymphoma, unspecified:comparison with AILD, ALCL, lymphoblastic lymphoma and ATLL. Blood2004;103:236–41.

12. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse largeB-cell lymphoma identified by gene expression profiling. Nature 2000;403:503–11.

13. Rosenwald A, Wright G, Chan WC, et al. The use of molecularprofiling to predict survival after chemotherapy for diffuse large B-celllymphoma. N Engl J Med 2002;346:1937–47.

14. Wright G, Tan B, Rosenwald A, et al. A gene expression-basedmethod to diagnose clinically distinct subgroups of diffuse large B celllymphoma. Proc Natl Acad Sci U S A 2003;100:9991–6.

15. Shipp MA, Ross KN, Tamayo P, et al. Diffuse large B-cell lymphomaoutcome prediction by gene-expression profiling and supervised machinelearning. Nat Med 2002;8:68–74.

16. Hans CP, Weisenberger DD, Greiner TC, et al. Confirmation of themolecular classification of diffuse large B-cell lymphoma by immunohis-tochemistry using a tissue microarray. Blood 2004;103:275–82.

17. Chang CC, McClintock S, Cleveland RP, et al. Immunohistochemicalexpression patterns of germinal center and activated B-cell markerscorrelate with prognosis in diffuse large B-cell lymphoma. Am J SurgPathol 2004;28:464–70.

18. Lossos IS, Czerwinski DK, Alizadeh AA, et al. Prediction of survival indiffuse large B-cell lymphoma based on the expression of six genes. N EnglJ Med 2004;350:1828–37.

19. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.

20. Jaramillo M, Rangel C, Grogan T. Immunohistochemistry in leukemiasand lymphomas. Methods Mol Med 2000;55:301–19.

21. Grogan TM, Rangel C, Rimsza L, Rybski J, Zehab R. Clinical andresearch applications of rapid, kinetic-mode automated double labeledimmunohistochemistry and in situ hybridization. Adv Pathol Lab Med1995;8:79–99.

22. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of themolecular classification of diffuse large B-cell lymphoma by immunohis-tochemistry using a tissue microarray. Blood 2004;103:275–82.

23. Lossos IS, Czerwinski DK, Alizadeh AA, et al. Prediction of survival indiffuse large-B-cell lymphoma based on the expression of six genes. N EnglJ Med 2004;350:1828–37.

24. Gisselbrecht C, Gaulard P, Lepage E, et al. Prognostic significance ofT-cell phenotype in aggressive non-Hodgkin’s lymphomas. Grouped’Etudes des Lymphomes de l’Adulte (GELA). Blood 1998;92:76–82.

25. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540–50.

26. Ohshima K, Karube K, Kawano R, et al. Classification of distinctsubtypes of peripheral T-cell lymphoma unspecified, identified by chemo-kine and chemokine receptor expression: analysis of prognosis. Int J Oncol2004;25:605–13.

27. Hurt EM, Wiestner A, Rosenwald A, et al. Overexpression of c-maf isa frequent oncogenic event in multiple myeloma that promotes prolifer-ation and pathological interactions with bone marrow stroma. Cancer Cell2004;5:191–9.

28. Matsunobu T, Tanaka K, Matsumoto Y, et al. The prognostic andtherapeutic relevance of p27kip1 in Ewing’s family tumors. Clin CancerRes 2004;10:1003–12.

29. Delgado MD, Hallier M, Meneceur P, Tavitian A, Moreau-Gachelin F.Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleo-tides. Oncogene 1994;9:1723–7.

30. Conti I, Rollins BJ. CCL2 (monocyte chemoattractant protein-1) andcancer. Semin Cancer Biol 2004;14:149–54.

31. Adler EP, Lemken CA, Katchen NS, Kurt RA. A dual role fortumor-derived chemokine RANTES (CCL5). Immunol Lett 2003;90:187–94.

32. Opdam FJ, Kamp M, de Bruijn R, Roos E. Jak kinase activity isrequired for lymphoma invasion and metastasis. Oncogene 2004;23:6647–53.





33. Chen Y, Abraham DJ, Shi-Wen X, et al. CCN2 (connective tissuegrowth factor) promotes fibroblast adhesion to fibronectin. Mol Biol Cell2004;15:5635–46.

34. Xie D, Yin D, Tong X, et al. Cyr61 is overexpressed in gliomas andinvolved in integrin-linked kinase-mediated Akt and h-catenin-TCF/Lefsignaling pathways. Cancer Res 2004;64:1987–96.

35. Fry AM, Meraldi P, Nigg EA. A centrosomal function for the humanNek2 protein kinase, a member of the NIMA family of cell cycle regulators.EMBO J 1998;17:470–81.

36. de Vos S, Hofmann WK, Grogan TM, et al. Gene expression profile ofserial samples of transformed B-cell lymphomas. Lab Invest 2003;83:271–85.

37. Vankayalapati H, Bearss DJ, Saldanha JW, et al. Targeting aurora2kinase in oncogenesis: a structural bioinformatics approach to targetvalidation and rational drug design. Mol Cancer Ther 2003;2:283–94.

38. Lewis J, Eiben LJ, Nelson DL, et al. Distinct interactions of theX-linked lymphoproliferative syndrome gene product SAP with cytoplas-mic domains of members of the CD2 receptor family. Clin Immunol 2001;100:15–23.

39. Lores P, Morin L, Luna R, Gacon G. Enhanced apoptosis in the thymusof transgenic mice expressing constitutively activated forms of humanRac2GTPase. Oncogene 1997;15:601–5.

40. Brenner B, Gulbins E, Schlottmann K, et al. L-selectin activates theRas pathway via the tyrosine kinase p56lck. Proc Natl Acad Sci U S A1996;93:15376–81.

41. Kumamoto Y, Tomschegg A, Bennai-Sanfourche F, et al. Monoclonalantibody specific for TIRC7 induces donor-specific anergy and preventsrejection of cardiac allografts in mice. Am J Transplant 2004;4:505–14.

42. Tapparel C, Reymond A, Girardet C, et al. The TPTE gene family:cellular expression, subcellular localization and alternative splicing. Gene2003;323:189–99.

43. Powell JA, Gardner AE, Bais AJ, et al. Sequencing, transcriptidentification, and quantitative gene expression profiling in the breastcancer loss of heterozygosity region 16q24.3 reveal three potential tumor-suppressor genes. Genomics 2002;80:303–10.

44. Taipale J, Cooper MK, Maiti T, Beachy PA. Patched actscatalytically to suppress the activity of Smoothened. Nature 2002;418:892–7.

45. Majidi M, Gutkind JS, Lichy JH. Deletion of the COOH terminusconverts the ST5 p70 protein from an inhibitor of RAS signaling to anactivator with transforming activity in NIH-3T3 cells. J Biol Chem 2000;275:6560–5.

46. Bazil V, Baudys M, Hilgert I, et al. Structural relationship between thesoluble and membrane-bound forms of human monocyte surface glyco-protein CD14. Mol Immunol 1989;26:657–62.

47. Neri A, Chang CC, Lombardi L, et al. B cell lymphoma-associatedchromosomal translocation involves candidate oncogene lyt-10, homolo-gous to NF-nB p50. Cell 1991;67:1075–87.

48. Greten FR, Eckmann L, Greten TF, et al. IKKh links inflammation andtumorigenesis in a mouse model of colitis-associated cancer. Cell 2004;118:285–96.

49. Pikarsky E, Porat RM, Stein I, et al. NF-nB functions as a tumourpromoter in inflammation-associated cancer. Nature 2004;431:461–6.

50. Martinez-Delgado B, Melendez B, Cuadros M, et al. Expressionprofiling of T-cell lymphomas differentiates peripheral and lymphoblasticlymphomas and defines survival related genes. Clin Cancer Res 2004;10:4971–82.

51. Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE.Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2001;167:1644–53.

52. Yoon HS, Kim HA. Prologation of c-Jun N-terminal kinase isassociated with cell death induced by tumor necrosis factor a in humanchondrocytes. J Korean Med Sci 2004;19:567–73.

53. Liu QA, Hengartner MO. Human CED-6 encodes a functionalhomologue of the Caenorhabditis elegans engulfment protein CED-6.J Curr Biol 1999;9:1347–50.

54. Reddien PW, Cameron S, Horvitz HR. Phagocytosis promotesprogrammed cell death in C. elegans. Nature 2001;412:198–202.

55. Screaton GR, Xu XN, Olsen AL, et al. LARD: a new lymphoid-specificdeath domain containing receptor regulated by alternative pre-mRNAsplicing. Proc Natl Acad Sci U S A 1997;94:4615–9.

56. Lanningham-Foster L, Green CL, Langkamp-Henken B, et al. Over-expression of CRIP in transgenic mice alters cytokine patterns and theimmune response. Am J Physiol Endocrinol Metab 2002;282:1197–203.

57. Plosker GL, Figgitt DP. Rituximab: a review of its use in non-Hodgkin’slymphoma and chronic lymphocytic leukemia. Drugs 2003;63:803–43.

58. Schwarting R, Castello R, Moldenhauer G, et al. Human Lyb-2homolog CD72 is a marker for progenitor B-cell leukemias. Am J Hematol1992;41:151–8.

59. Kohler K, Louvard D, Zahraoui A. Rab13 regulates PKA signalingduring tight junction. J Cell Biol 2004;165:175–80.

60. Cui Y, Dinman JD, Kinzy TG, Peltz SW. The Mof2/Sui1 protein isa general monitor of translational accuracy. Mol Cell Biol 1998;18:1506–16.

61. Lian Z, Pan J, Liu J, et al. The translation initiation factor, hu-Sui1may be a target of hepatitis B X antigen in hepatocarcinogenesis.Oncogene 1999;18:1677–87.

62. Wang CY, Guttridge DC, Mayo MW, Baldwin AS, Jr. NF-nB inducesexpression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppresschemotherapy-induced apoptosis. Mol Cell Biol 1999;19:5923–9.

63. Chuang PI, Morefield S, Liu CY, Chen S, Harlan JM, Willerford DM.Perturbation of B-cell development in mice overexpressing the Bcl-2homolog A1. Blood 2002;99:3350–9.

64. Shaffer AL, Rosenwald A, Staudt LM. Lymphoid malignancies: thedark side of B-cell differentiation. Nat Rev Immunol 2002;2:920–32.





2005;4:1867-1879. Mol Cancer Ther Daruka Mahadevan, Catherine Spier, Kimiko Della Croce, et al. identifies distinct tumor profile signaturesotherwise specified, and diffuse large B-cell lymphoma Transcript profiling in peripheral T-cell lymphoma, not

Updated version

http://mct.aacrjournals.org/content/4/12/1867

Access the most recent version of this article at:

Cited articles

http://mct.aacrjournals.org/content/4/12/1867.full#ref-list-1

This article cites 62 articles, 19 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/4/12/1867.full#related-urls

This article has been cited by 10 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/4/12/1867To request permission to re-use all or part of this article, use this link

http://mct.aacrjournals.org/content/4/12/1867http://mct.aacrjournals.org/content/4/12/1867.full#ref-list-1http://mct.aacrjournals.org/content/4/12/1867.full#related-urlshttp://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/4/12/1867http://mct.aacrjournals.org/

transcript profiling in peripheral t-cell lymphoma, not otherwise specified… · ptcl-nos...

Documents