Transcript Profiling in Peripheral T-Cell Lymphoma, Not Otherwise Specified, and Diffuse Large B-Cell Lymphoma Identifies Distinct Tumor Profile Signatures

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Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures

Daruka Mahadevan,1 Catherine Spier,2 Introduction 1 4 Kimiko Della Croce, Susan Miller, The WHO classifies non-Hodgkin’s lymphoma into B-cell Benjamin George,1 Chris Riley,1 Stephen Warner,3 and T-cell subtypes. B-cell non-Hodgkin’s lymphomas com- Thomas M. Grogan,2 and Thomas P. Miller1 prise f85% and T-cell non-Hodgkin’s lymphomas com- prise f15%. Of the T-cell non-Hodgkin’s lymphomas, Departments of 1Medicine, 2Pathology, and 3Pharmacology and 4 peripheral T-cell lymphoma–not otherwise specified Arizona Research Labs, Arizona Cancer Center, University of f Arizona, Tucson, Arizona [PTCL-NOS] comprises 6% to 10% (1). These are rare tumors whose pathologic diagnosis has been plagued due to the absence of immunophenotypic markers of clonality, Abstract morphologic heterogeneity, and poor correlation between To glean biological differences and similarities of periph- cytomorphology and prognosis (2). Large prospective eral T-cell lymphoma–not otherwise specified [PTCL-NOS] studies have shown that PTCL-NOS have a higher relapse to diffuse large B-cell lymphoma (DLBCL), a transcripto- rate and worse survival than diffuse large B-cell lymphoma some analysis was done on five PTCL-NOS and four (DLBCL; refs. 3–5). Regardless of the differences in DLBCL patients and validated by quantitative real-time outcome, both types of lymphoma are largely treated the reverse transcription-PCR on 10 selected genes. Normal same, as there are no biological insights to differentiate peripheral blood T cells, peripheral blood B cells, and these tumors. The chemoresistance of PTCL-NOS com- lymph node were used as controls. The resultant gene pared with DLBCL has been attributed to increased expression profile delineated distinct ‘‘tumor profile expression of multidrug resistance proteins and p53 (6, 7). + signatures’’ for PTCL-NOS and DLBCL. Several highly The cell of origin of PTCL-NOS is generally CD4 T cells + overexpressed genes in both PTCL-NOS and DLBCL and very rarely CD8 T cells. Expression of one or more involve the immune network, stroma, angiogenesis, and pan-T-cell antigens (CD45RO, CD2, CD3, CD5, and CD7), cell survival cascades that make important contributions absence of pan-B-cell antigens, and histopathologic find- to lymphomagenesis. Inflammatory chemokines and their ings combine to make the diagnosis (8). In the West, nodal receptors likely play a central role in these complex PTCL-NOS predominates, whereas in the East extranodal interrelated pathways: CCL2 and CXCR4 in PTCL-NOS PTCL is more common, particularly the nasal type. The and CCL5 and CCR1 in DLBCL. Highly overexpressed Kiel classification subdivides PTCL-NOS into low-grade oncogenes unique to PTCL-NOS are SPI1, STK6, a- and high-grade groups. Within each group, distinct PDGFR, and SH2D1A, whereas in DLBCL they are PIM1, patterns of genetic abnormalities have been identified PIM2, LYN, BCL2A1, and RAB13. Oncogenes common to (9, 10). A second approach analyzed patterns of expression both lymphomas are MAFB, MET, NF-kB2, LCK, and of chemokines and their receptors. Based on the expression LYN. Several tumor suppressors are also down-regulated of CCR4, CXCR3, CD134, and ST2(L), PTCL-NOS has been (TPTE, MGC154, PTCH, ST5, and SUI1). This study subdivided into two prognostic groups (11). illustrates the relevance of tumor-stroma immune traffick- Gene expression profiling using cDNA (12–14) and ing and identified potential novel prognostic markers and oligonucleotide microarrays (15) have classified DLBCL targets for therapeutic intervention. [Mol Cancer Ther into three subgroups: germinal center B-like, activated 2005;4(12):1867–79] B-like, and type III. The three subgroups had markedly different survival curves after cyclophosphamide-Adria- mycin-vincristine-prednisone (CHOP)–based chemotherapy, providing prognostic information in addition to the Received 5/10/05; revised 7/21/05; accepted 9/27/05. international prognostic index and independent of clinical Grant support: Southwest Oncology Group grant CA-32102, subcontract features. A set of 9 genes have been identified that are 03041. sufficient to predict the three subgroups and overall The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked survival based on immunohistochemistry (16, 17) and advertisement in accordance with 18 U.S.C. Section 1734 solely to quantitative real-time reverse transcription-PCR (RT-PCR; indicate this fact. ref. 18). Such an approach applied to PTCL-NOS would Requests for reprints: Daruka Mahadevan, Department of Medicine, also yield a gene expression-based diagnostic classification, Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724. Phone: 520-626-4331; Fax: 520-626-3663. prognostic subgroups, and novel targets that may be E-mail: [email protected] amenable to therapeutic intervention. Copyright C 2005 American Association for Cancer Research. The aims of the present study were to perform doi:10.1158/1535-7163.MCT-05-0146 transcript profiling of PTCL-NOS and DLBCL to gain

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biological insights into the similarities and differences University of Arizona). mRNA (5 Ag) was used to generate that exist between B-cell and T-cell malignancies. Pre- first-strand cDNA by using a T7-linked oligo(dT) primer. vious studies (12, 14) used non-Hodgkin’s lymphoma cell After second-strand synthesis, in vitro transcription lines as controls; however, this study used normal (Ambion, Austin, TX) was done with biotinylated UTP peripheral blood B cells, T cells, and lymph node as and CTP (Enzo Diagnostics, Farmingdale, NY), resulting in controls. The classification of the gene expression profile 40- to 80-fold linear amplification of RNA. Biotinylated into the six hallmarks of cancer (19) yielded distinct RNA (40 Ag) was fragmented to 50- to 150-nucleotide size ‘‘tumor profile signatures’’ for PTCL-NOS and DLBCL, before overnight hybridization at 45jC to HG-U133A 2.0 respectively. This study has analyzed and evaluated the Affymetrix (Santa Clara, CA) array comprising f18,400 gene expression profile signature of two phenotypically transcripts and 22,000 probe sets. After washing, arrays distinct (T cells versus B cells) non-Hodgkin’s lympho- were stained with streptavidin-phycoerythrin (Molecular mas and provides a framework for more extensive Probes, Invitrogen, Carlsbad, CA) and scanned on a studies for diagnostic classification and prognostic sub- Hewlett-Packard (Raleigh, NC) scanner. Intensity for each group determinations. feature of the array was captured using GeneChip software (Affymetrix), and a single raw expression level for each gene was derived from the 10 to 20 probe pairs represent- Materials and Methods ing each gene by using a trimmed mean algorithm. Patients Intensity values were scaled such that overall intensity A total of nine patients [four DLBCL and five PTCL-NOS] for each chip of the same type was equivalent. The mean F were studied. The tumor tissue were fixed as snap-frozen SD difference between the scaling factors of all GeneChips samples and stored at À80jC. They were also fixed with was 0.75 F 0.15. Other additional measures of quality, the buffered formalin, embedded in paraffin, and stained with percentage of gene present (55.0 F 2.5), the ratio of actin H&E. All slides were reviewed (C.M.S.) and the initial 3V to 5V (1.25 F 0.15), and the ratio of glyceraldehyde-3- histologic diagnosis was confirmed. These studies were phosphate dehydrogenase 3V to 5V (1.02 F 0.10) indicated approved by the patients or their legal guardians as well as a high overall quality of the samples and assays. Well- appropriate written consents before biopsy. This study is measured genes were defined genes that had a ratio of institutional review board exempt. signal intensity to background noise of >2 in >80% of the Immunohistochemistry samples hybridized. Immunohistochemistry was done to determine tumor Data Analysis and Visualization lineage on either snap-frozen or formalin fixed, paraffin- For each patient PTCL-NOS or DLBCL compared with embedded tissue samples using a battery of monoclonal the control sample (normal B cells or T cells or normal antibodies directed against both B cells (CD19, CD20, lymph node), lists of ‘‘robust increasers’’ or ‘‘robust CD21, and CD22) and T cells (CD1a, CD2, CD3, CD4, decreasers’’ were generated using the Affymetrix Data CD5, and CD8). Immunoglobulin light (n and E; Becton Analysis Program (MAS 5.0). Fundamentals guide was Dickinson Corp., San Jose, CA) and heavy (IgM, IgG, IgA, used to import these lists into GeneSpring version 5.0 and IgD; Becton Dickinson) chain status was also and obtain the intersection of robust increasers or determined. In addition, Ki-67 (DAKO, Carpinteria, CA; decreasers across all patients PTCL-NOS or DLBCL, for proliferation) and CD30 (anaplastic T-cell lymphoma respectively. Increasers or decreasers common to normal and activated B cells; DAKO) were used. For frozen T cells and lymph node or normal B cells and lymph tissues, an indirect immunoperoxidase method using 3,3V- node were compared with PTCL-NOS or DLBCL diaminobenzidine as the chromogen was used (20), and patients. Further, increasers or decreasers common to for paraffin sections, the Ventana ES or a benchmark normal T cells and B cells or lymph node to PTCL-NOS instrument (Ventana Medical Systems, Tucson, AZ; and DLBCL were also compared. Gene expression ref. 21) was used. All patient samples had sufficient profiles for PTCL-NOS or DLBCL were further classified testing to assure lineage and clonal status, however. according to the Hallmarks of Cancer (19) to determine Aberrant loss of pan-T lineage antigens and/or T-cell lymphoma-specific tumor profile signatures. These are subsets was used in lieu of receptor gene rearrangements genes involved in (a) self-sufficiency in growth signals, for T-cell lesions when such testing was not available or including oncogenes; (b) insensitivities to growth inhib- there was tissue insufficient for study. itory signals, including absence of tumor suppressors; (c) RNA Isolation and Oligonucleotide Microarrays evasion apoptosis; (d) limitless replicative potential; (e) Total RNA was extracted from each frozen PTCL-NOS sustained angiogenesis; and (f) invasion and metastasis. and DLBCL specimen using the RNeasy Mini kit (Qiagen, Finally, searches were conducted for potential therapeutic Valencia, CA). The amount of total RNA isolated from the targets by keywords (e.g., protein kinases to generate cells was quantified using spectrophotometric A260 meas- gene lists for each desired target type). Intersection of the urements with yields z25 Ag/sample. Control RNA was gene lists for each target with the commonly up- obtained from normal peripheral B cells (AllCells, Berke- regulated and down-regulated genes across patient ley, CA), normal peripheral T cells (AllCells), and a snap- samples were obtained. This was also done on the frozen normal lymph node (Department of Pathology, intersects of the up-regulated and down-regulated gene

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

lists with the GeneSpring ‘‘Simplified GO Ontology’’ as Results another method of classifying genes. For the four DLBCL Morphology and Immunohistochemistry Analysis patients, the measurement of 9 prognostic genes (LMO2, The five PTCL-NOS patients were all large cell lympho- BCL6, FN1, SCYA3, BCL2, CD10, MUM1, FOXP1, and mas. All were de novo. Each sample contained >50% tumor CYCLIN D2; refs. 22, 23) were applied to ascertain which cells. Four were of nodal origin, whereas one originated as subclass they belonged to based on a comparison with a soft tissue mass in the knee. All PTCL-NOS patients were the normal B cells or lymph node. negative for CD1 and CD5 and positive for CD2, CD3, Real-timeQuantitativeRT-PCR and CD7. The Ki-67 ranges from 30% to 50%. Of the four Total RNA (100 ng) was used for reverse transcription DLBCL patients, three were nodal in origin and one reactions (20 mL total volume) carried out using Super- occurred as a soft tissue mass in the right breast. All four Script III Platinum Reverse Transcriptase (Invitrogen, did not show any follicular areas; had large cell morphol- Carlsbad, CA). Reactions were incubated at 42jC for 50 ogy, uniform expression of CD20, and monoclonal light minutes followed by incubation at 37jC with RNase H for chain immunoglobulin; and contained >50% tumor cells. 20 minutes. An Opticon DNA Engine (MJ Research, Reno, Proliferation as judged by Ki-67 for the four patients was NV) was used to perform real-time fluorescence detection 10%, 35%, 60%, and 76%, respectively. No aberrant antigen PCR. cDNA (1 AL) produced from reverse transcription expression, specifically no CD5 coexpression, was detected. reactions was added to 12.5 AL Platinum SYBR Green Figure 1A shows a representative patient with PTCL-NOS quantitative PCR SuperMix-UDG (Invitrogen), 1 AL gene- and Fig. 1B shows a patient with DLBCL with immuno- specific or h-actin-specific primer pair (see below for histochemistry analysis, distinguishing the two lymphoma primer design), and 10.5 AL distilled H2O (final volume subtypes and providing a measure of tumor involvement. of 25 AL). Amplification (95jC for 15 seconds, 55jC for Gene Expression Profiling of PTCL-NOS and DLBCL 30 seconds, and 72jC for 30 seconds) was repeated for 44 The Affymetrix MAS 5.0 software was used to calculate cycles. Following the PCR reaction, a melting curve assay the average, SD, median, and interquartile range calcula- was done to determine the purity of the amplified product. tions [e.g., the top 25 increases or decreases; PTCL-NOS Data were provided as a threshold cycle value (CT) for each versus normal T cells or normal lymph node]. The sample, which indicated the cycle at which a statistically intersample variability correlated well with the quantitative significant increase in fluorescence was first detected. real-time RT-PCR data. For example the five patients with These data were then normalized to h-actin, which served PTCL-NOS for CD14 gene expression signals were 1,034.6, as an unaffected control gene, for each data point and 3,371.4, 4,720.3, 3,451.1, and 3,130.5. The average was compared with a normal T-cell control to determine 3,141.5, SD was 1,330.46, median was 3,371.4, and inter- relative expression ratios. Each measurement was done in quartile range was 320.6. Based on these data, the average triplicate. fold change was calculated to be 88-fold compared with Primer Design normal peripheral blood T-cell RNA. Well-measured genes PCR primers were designed using MacVector (Accelrys, were defined as genes that had a ratio of signal intensity to San Diego, CA) to produce amplicons with lengths ranging background noise of >2 in >80% of the samples hybridized. from 80 to 250 bp to optimize the efficiency of quantitative Genes that are robustly up-regulated and common to all PCR. The sequences are 5V-TGGGCTCTTTTGAAG- five patients with PTCL-NOS are compared with normal GATTGG-3V and 5V-GCAGTCTGGCTATCTGATTG- peripheral blood T cells or normal lymph node (Table 1). It AAGC-3V for Lumican,5V-CTCCTGGCAGATTCCA- seems that the comparison of PTCL-NOS patients to CAAAAG-3V and 5V-CACTTCTTATTGGGGTCAGCA- normal peripheral blood T cells shows that most of the CAG-3Vfor CCL18,5V-ACTCCCTCAATCTGTCGTTCGC-3V genes that are overexpressed are involved in tumor and 5V-TTCCCGTCCAGTGTCAGGTTATCC-3V for CD14, invasion (COL1A1, FN1, CTGF, COL1A2, CCL2, CLU, C1S, 5V-TGTCCCCCTCAAAAGTCATCC-3Vand 5V-TTGCTCAG- LUM,andLYS), antiapoptosis (CD14), proliferation TTCATACACCTTCCG-3V for CD54,5V-GAATGGTGC- (CCL18, CXCL13, and IGFBP7), and angiogenesis (TEM6). TACTTGAAGACTCTGG-3V and 5V-CACTCTCCC- Similarly, when PTCL-NOS are compared with the normal GCTACACTTGTTTTC-3V for CD163,5V-CACACA- lymph node, most of the genes that are up-regulated and CAGGTGGGACACAAATAAG-3Vand 5V-GCAAGTCAAT- overexpressed are involved in tumor invasion (CHI3L1, GAGACGGAGTCAC-3V for CD106,5V-TGAAGTCCA- CCL5, and CORO1A) and proliferation (SH2D1A, FLI1, AGTTCCTCCAGGTC-3V and 5V-TGTTGCTGTTGTGGCT- AIF1, CRIP1, MAP4K1, and Rac2). Genes known to be ATTAGGC-3V for HCK,5V-TGCGTAAGAGCAAAAAGC- causative in tumorigenesis are prominently expressed, GAAG-3V and 5V-AAATAAGCCCCCCCTCAACCTTGG-3V indicating that peripheral blood T cells and lymph node for a-PDGFR,5V-CGCTTTGCTCCTTGTTTTTTCC-3V and controls both provide valuable information regarding the 5V-CGTCTCTTTCTCCTACCCCTTGAC-3V for ABCA1, evolution of the tumor from normal T cells and lymph and 5V-TGCTACCACAAGAAATACCGCTC-3V and 5V-AT- nodes. The robustly down-regulated genes are not included CCTCCTTCCCAAACACCAGC-3V for TEM6. h-actin pri- here but will be discussed below with regard to the tumor mers from QuantumRNA h-Actin Internal Standards profile signature. (Ambion) were used to normalize the quantitative PCR Genes that are robustly up-regulated and common to all data. four patients with DLBCL are compared with normal

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Figure 1. A, a representative patient with PTCL-NOS showing H&E staining for morphology and immunohistochemistry to delineate T-cell phenotype (CD2)isCD8 negative (staining is due to tumor-infiltrating lymphocytes) with tumor involvement (>50%) and aggressiveness (Ki-67). B, a representative patient with DLBCL showing H&E staining for morphology and immunohistochemistry to delineate B-cell phenotype (CD20) is CD8 negative (staining is due to tumor-infiltrating lymphocytes) with tumor involvement (>50%) and aggressiveness (Ki-67).

peripheral blood B cells or normal lymph node (Table 2). involved in tumor invasion (CCL5, CORO1A,and The comparison of DLBCL patients to normal peripheral CHI3L1), antiapoptosis (BCL2A1), immune trafficking blood B cells shows that the genes that are up-regulated (CCL18, CD79B, CD52, and POU2AF1), and proliferation and overexpressed are also involved in tumor invasion (CD20, MAP4K1, and Rac2). Tumor tissues express genes (LYS, CCL5, CHI3L1, CHIT1, MMP9, and ADAMDEC1), that promote and maintain the oncogenic process, and the immune cell trafficking or host cell response to tumor profiles obtained based on normal peripheral blood B cells (CCL18, RGS13, CXCL9, and VNN1), and proliferation and lymph node will help guide the elucidation of the (PLA2G2D and PLA2G7). Similarly, when DLBCL patients mechanism initiation and progression. The robustly down- are compared with the normal lymph node, most of the regulated genes are not included and will be discussed genes that are up-regulated and overexpressed are with regard to the tumor profile signature.

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Table 3 shows the highly up-regulated genes that are Table 2. Highly overexpressed genes in DLBCL compared with common to both PTCL-NOS and DLBCL when compared normal peripheral blood B cells or lymph node with normal peripheral blood T cells and B cells or lymph Fold D Gene name Function node, respectively. There are only three genes common to PTCL-NOS and DLBCL when compared with normal B-non-Hodgkin’s lymphoma vs normal peripheral blood B cells: peripheral blood T cells and B cells and they are CCL18, top increases UBD, and RARRES1. However, there are 11 genes common 172 CCL18 Chemokine to PTCL-NOS and DLBCL when compared with the lymph 146 VNN1 Lymphoma homing node and they are CCL18, CD52, MAP4K1, CORO1A, 102 UBD Protein degradation PPP1R16B, PSMB8, UBD, LYZ, TCRb, RAC2, and CCL5. 102 LYZ ECM 92 CCL5 Chemokine 89 PLA2G2D Lipid metabolism Table 1. Highly overexpressed genes in PTCL-NOS compared 83 CHI3L1 ECM with normal peripheral blood T cells or lymph node 82 CHIT1 ECM 75 RGS13 G protein signaling Fold D Gene name Function 53 BID Apoptosis 48 APOC1 Lipid metabolism PTCL-NOS vs normal peripheral blood T cells: top increases 46 PLA2G7 Lipid metabolism 176 COL1A1 ECM 45 ADAMDEC1 ECM 154 CCL18 Chemokine 41 MMP9 ECM 135 CXCL13 Chemokine 34 CXCL9 Chemokine 130 IGFBP7 Proliferation 25 GZMK Protease 128 RARRES1 Tumor suppression 17 LR8 Lipid metabolism 123 CLU ECM 15 KIAA0101 Unknown 109 C1S ECM 13 GPNMB Tumor suppression 107 FN1 ECM 11 CTSB Protease 103 CTGF Proliferation 102 CCL2 Chemokine B-non-Hodgkin’s lymphoma vs normal lymph node: top increases 101 LUM ECM 172 CCL18 Chemokine 99 SERPING1 Protease inhibition 151 IGJ Antibody 99 APOC1 Lipid metabolism 146 VNN1 Lymphoma homing 98 NID ECM 130 CDW52 Proliferation 96 COL1A2 ECM 127 MAP4K1 Proliferation 94 TEM6 Angiogenesis 126 NCF1 Inflammation 93 COL3A1 ECM 123 CORO1A Cytoskeleton 89 APOE Lipid metabolism 121 CD79B Immune response 89 UBD Protein degradation 107 BCL2A1 Apoptosis 89 PLTP Lipid metabolism 105 PPP1R16B Protein phosphatase 88 CD14 Antiapoptosis 104 PSMB8 Proteasome 104 PTPRCAP Protein phosphatase PTCL-NOS vs normal lymph node: top increases 102 UBD Protein degradation 200 CHI3L1 ECM 102 LYZ ECM 154 CCL18 Chemokine 100 TCRb T-cell receptor 120 CCL5 Chemokine 98 CD20 Proliferation 120 SH2D1A Proliferation 97 GRP3 GTP signaling 120 NKG7 Natural killer receptor 96 POU2AF1 Immune response 113 HLA-DQB1 Immune regulation 96 NCF1 Inflammation 109 FLI1 Oncogene 94 RAC2 Proliferation 108 AIF1 Proliferation 92 CCL5 Chemokine 108 CORO1A Cytoskeleton 107 RAC2 Proliferation 106 MAP4K1 Proliferation 104 PSMB8 Proteasome These comparisons provide insights into the importance 104 TCRc T-cell receptor of differential gene expression profiles that are common 102 CARD15 Apoptosis to both types of lymphoma. By including a single cell 100 LYZ ECM (peripheral blood B cells or T cells) as well as a complex TRIM 100 Inflammation tissue (lymph node), insights into the relative importance of 100 CRIP1 Proliferation 96 CDW52 Proliferation the relationships between tumor-stroma immune traffick- 92 TCRb T-cell receptor ing cells can now be gleaned. 89 UBD Protein degradation Tumor Profile Signatures of PTCL-NOS and DLBCL 89 PPP1R16B Protein phosphatase We next did an analysis based on the six hallmarks of cancer (19) in which a tumor has the following acquired

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capabilities: (a) self-sufficiency in growth signals, (b) Table 4. Hallmarks of cancer for DLBCL: DLBCL vs peripheral insensitivity to antigrowth signals, (c) evasion of apoptosis, blood B cells and lymph node (d) limitless replicative potential, (e) sustained angiogene- Gene name Fold D Definitions sis, and (f) tissue invasion and metastasis. Genes were sorted into each of these categories common to all five Proliferation PTCL-NOS patients compared with peripheral blood IGFBP7 107 IGF-I-binding protein T cells, lymph node, and/or both (Table 4). A similar CD20 98 Tetraspanin analysis was done for the four DLBCL patients compared CD72 83 Type II membrane protein with peripheral blood B cells, lymph node, and/or both C-type lectin (Table 5). This provides a tumor profile signature for CRIP1 67 Zinc finger motif, group II PTCL-NOS and DLBCL. PTCL-NOS identified genes LIM protein family associated with proliferation (IGFBP7, SH2D1A, RAC2, RAB13 23 GTPase Tumor suppressors and TCIRG1), tumor suppressors (RARRES3, GAS1, and GPNMB 97 Transmembrane CDKN1A), invasion (COL1A, FN1, and CCL2), unlimited glycoprotein replication (CTGF, CYR61,andNEK2), angiogenesis CD63 90 Tetraspanin (VEGF, PDGFR, CSF1R, and HGF), antiapoptosis (CD14 BTG2 67 B-cell translocation gene 2 and NF-jB), and oncogenes (FLI1, MAFB, SPI1, STK6, and RARRES3 64 Retinoic acid receptor MET). DLBCL identified genes associated with prolifer- response gene 3 ation (IGFBP7, CD20, CD72, CRIP1, and RAB13), tumor Invasion and metastasis suppressors (GPNMB, CD63, BTG2, and RARRES3), inva- LUM 67 Lumican, ECM sion (LUM, FN1, MMP1, and COL6A3), unlimited repli- FN1 59 Fibronectin, ECM MMP1 40 MMP, ECM cation (CCR1 and CCL5), angiogenesis (VEGF, PDGF, COL6A3 24 Collagen, ECM PDGFR,andFGF13), and antiapoptosis (CD14 and Unlimited replication BCL2A1). Therefore, it is apparent that normal peripheral CCR1 82 Chemokine receptor, blood T cells or B cells and lymph node in combination inflammation provides consistent gene expression findings that now can CCL5 14 Chemokine ligand, be evaluated in detail and attempts be made to position inflammation these in signaling pathways that make the relationships Angiogenesis more obvious. VEGF 65 Vascular endothelial Nine-GenePredictiveModelofDLBCL growth factor PDGF-AA 14 Platelet-derived Tissue microarray using immunoperoxidase staining for growth factor predictive markers to accurately subdivide DLBCL into PDGFR-b 10 Platelet-derived growth prognostically relevant subgroups has been done and factor receptor correlated with cDNA microarray as the gold standard FGF13 6 Fibroblast growth factor (22). Six proteins were identified: CD10, BCL2, BCL6, Apoptosis MUM1/IRF4, CYCLIN D2, and FOXP1. Similarly, a CD14 141 Lipopolysaccharide receptor, quantitative real-time RT-PCR analysis identified six genes glycosylphosphatidylinisotol anchor BCL2A1 107 B-cell lymphoma 2

Table 3. Overexpressed genes common to PTCL-NOS and DLBCL compared with normal peripheral blood T cells, to have prognostic significance: BCL2, BCL6, FN1, LMO2, peripheral blood B cells, and lymph node CCND2, and SCYA3/CCL3 (23). In this study, we combined Common increases the genes that were used in the tissue microarray and Gene name RT-PCR to evaluate the four DLBCL and five PTCL-NOS Fold change PTCL-NOS Fold change DLBCL patients. When the four DLBCL patients were compared with the normal peripheral blood B cells, three of nine CCL18 154.4 172.6 genes were up-regulated (FN1 140-fold, CCL3 4.9-fold, and CDW52 96.7 130.0 BCL6 2.2-fold), and when compared with the lymph node, MAP4K1 106.6 127.1 seven of nine genes were up-regulated (MUM1 28-fold, CORO1A 108.1 123.2 CCL3 11.4-fold, CCND2 10.4-fold, BCL6 6.7-fold, LMO2 5.3- PPP1R16B 89.1 106.0 fold, BCL2 4.6-fold, and FN1 2.3-fold). These data place PSMB8 104.4 105.0 these four unselected DLBCL patients into the non- UBD 89.8 102.6 LYZ 100.5 102.3 germinal center activated B-cell subgroup. Further, the RAC2 107.9 94.6 importance of the control RNA is highlighted, implying CCL5 120.9 92.3 that the lymph node may be more informative due to its TCRb 92.6 100.6 complex nature and that a tumor does follow the hallmarks of cancer.

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Discovery of New Therapeutic Targets runs of RT-PCR. These correlated well with the intersample The study also analyzed certain key therapeutic targets variability observed from the gene expression signals that may be amenable to targeting either with small- obtained from the DNA microarray. molecule inhibitors and/or monoclonal antibodies. For patients with PTCL-NOS, the following are novel targets: small-molecule inhibitors to protein kinases (STK6, NEK2, Discussion c-MET, SYK, and a-PDGFR), monoclonal antibodies to Patients with PTCL-NOS have a worse outcome compared circulating ligands (CCL2, VEGF, HGF, CTGF, PDGF, FGF7, with patients with DLBCL (24). To distinguish biological and IGFBP7), and a small-molecule inhibitor to Rac2,a properties of these two phenotypic variants of large cell member of the Ras family of small GTP-binding proteins. lymphoma, we undertook a gene expression profile study For patients with DLBCL, the following are novel targets: of PTCL-NOS and DLBCL using an Affymetrix platform. small-molecule inhibitors to protein kinases (LCK, LYN, The study identified several key elements in lymphoma PIM1, PIM2, MET, and SYK), small-molecule inhibitors to biology and has led to hypothesis-generating research that novel targets (PSMA, BCL2A1, and NF-jB2), monoclonal should guide elucidation of the underlying oncogenic antibodies to circulating ligands (CCL2, CCL5, and VEGF), events in PTCL-NOS and DLBCL. The complexity of these and cell surface markers (CD72, ICAM2, and CD52). tumors is represented by 25 to 30 up-regulated cancer Validation with Quantitative Real-time RT-PCR genes and several down-regulated tumor suppressors that Quantitative real-time RT-PCR on 10 selected, overex- provide a growth advantage and enhance the ability to pressed, functionally relevant genes (Lumican, CCL18, invade and metastasize. CD14, CD54, CD106, CD163, a-PDGFR, HCK, ABCA1, and PTCL-NOS Tumor Profile Signature TEM6) common to all five PTCL-NOS patients validated A predictive model for the PTCL-NOS tumor profile is the gene expression profile data (Fig. 2). The expression delineated based on the six hallmarks of cancer (Fig. 3). level for each patient is shown and averaged over three 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

Proliferation IGFBP7 107 IGF-I-binding protein SH2D1A 120 Src homology-2 domain protein 1a RAC2 107 GTPase TCIRG1 54 Seven-transmembrane spanning vacuolar proton pump protein HGF 23 Hepatocyte growth factor Tumor suppressors RARRES3 128 Retinoic acid receptor response gene 3 GAS1 58 Growth arrest specific 1 CDKN1A 20 Cyclin-dependent protein kinase inhibitor Invasion and metastasis COL1A 176 Collagen, ECM FN1 107 Fibronectin, ECM CCL2 102 Chemokine ligand 2, inflammation Unlimited replication CTGF 103 Connective tissue growth factor CYR61 72 Cysteine-rich protein of CCN family NEK2 57 Serine/threonine kinase related to mitotic regulator NIMA Angiogenesis VEGF 84 Vascular endothelial growth factor PDGFR-A 26 Platelet-derived growth factor receptor CSF1R 19 Colony-stimulating factor receptor 1 PDGFR-B 18 Platelet-derived growth factor receptor Apoptosis CD14 88 Lipopolysaccharide receptor, glycosylphosphatidylinisotol anchor NF-jB 44 Transcription factor Oncogenes FLI1 109 Cell cycle progressor MAFB 80 Musculoaponeurotic fibrosarcoma, basic leucine zipper transcription factor SPI1 29 Transcription factor STK6 16 Aurora kinase, cell cycle regulator MET 7 Hepatocyte growth factor receptor

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

along a chemical gradient of ligand. Many cancers, and CCR1. Increased expression of CYCLIN D2 enhances including lymphomas, have a complex chemokine network proliferation, whereas increased integrin b7 promotes that influences immune cell infiltration, tumor cell growth, adhesion to bone marrow stroma and increases VEGF survival, migration, and angiogenesis. These cells them- elaboration (27). In PTCL-NOS, MAFB is 80-fold overex- selves express chemokine receptors and can respond to pressed and is important for IL-4 gene expression by Th2 chemokine in an autocrine and/or paracrine mechanism cells. Several of the target genes activated by c-MAF in (25). Many different cancers, including PTCL-NOS (26), multiple myeloma are also up-regulated in PTCL-NOS, have different profiles of chemokine and chemokine which include VEGF (84-fold), ITGA4 (88-fold), and receptor expression, but CXCR4 is most commonly found. CCR1 (39-fold), implying aberrant proliferation, increased It has been shown that c-MAF may alter the function of adhesion to surrounding stroma, tumor angiogenesis, and multiple myeloma cells by up-regulating CCR1, a receptor immune dysregulation (Fig. 3). Several other chemokines for the chemokine MIP-1. Three MAF target genes overex- are also overexpressed and are likely driven by MAFB and/ pressed in multiple myeloma are CYCLIN D2, integrin b7, or two other highly overexpressed oncogenic transcription

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

factors FLI1 (100-fold) and SPI1 (29-fold). FLI1 promotes through activated LFA1, which in turn activates the JAK cell cycle progression by suppressing the expression of CDK kinases and shown to be essential for lymphoma invasion inhibitor p27kip1 (28), whereas SPI1 is normally expressed and metastasis (32). in all hematopoietic cell lineages, except in T-cell lines (29), Tumor-stroma interactions play an integral role in but its aberrant expression may be an additional mechanism lymphoma cell survival. In PTCL-NOS, IGFBP7 is f130- that maintains the malignant lymphocyte to proliferate. fold overexpressed, enhances IGF activity, and influences CCL2 is 100-fold overexpressed and a potent chemo- cell adhesion and migration (33), a potential mechanism attractant for monocytes, memory T lymphocytes, and of tumor invasion. CTGF/CCN2 is >100-fold and CYR61/ natural killer cells. CCL2 is a tumor promoter (30), is able CCN1 is >70-fold overexpressed and belongs to a to recruit leukocytes, and provides growth signals for cysteine-rich secreted protein of the CCN family (34) tumor angiogenesis. Several angiogenic receptors and that binds to integrins and modulates invasive behavior ligands are overexpressed in PTCL-NOS, which includes of cancer cells. Several components of the extracellular VEGF (84-fold), a-PDGFR (26-fold), CSF1R (19-fold), matrix (ECM) are elevated and include CHI3L1 (200- b-PDGFR (19-fold), c-MET (7-fold), HGF (24-fold), fold), COL1A1 (177-fold), FN1 (108-fold), COL1A2 (103- PDGF-CC (5-fold), and FGF7 (2.5-fold), all seem to fold), and emilin (44-fold), most likely mediated by contribute to tumor growth. CCL5 is a 120-fold overex- CTGF/CYR61 (Fig. 3). CYR61 has been shown to enhance pressed chemoattractant that binds to several receptors, tumorigenicity through activated integrin-linked kinase including CCR1 (39-fold), CCR3, CCR4, and CCR5. CCL5 to stimulate h-catenin-TCF/Lef and Akt signaling path- up-regulates transcription of MMP9, which can contrib- ways (34). ute to angiogenesis. Further, tumor-derived CCL5 can Aberrant cellular proliferation due to dysregulated inhibit the T-cell response and enhance the in vivo cell cycle control seems to be mediated by the serine/ growth of tumors (31). CXCR4 is 79-fold overexpressed threonine kinase NEK2, which is 58-fold overexpressed and involved in lymphocyte trafficking. When activated, (Fig. 3) and localizes to the centrosome. Overexpression of CXCR4 promotes invasion of lymphoma cells into tissues active NEK2 induces splitting of centrosomes, whereas

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 paracrine manner recruit stromal fibroblasts, inflammatory immune cells, and endothelial cells to form a complex network of tumor tissue. The overexpression of integrins (LFA1 and VLA4) on the tumor cells and their binding partners on stromal cells (ICAM1, ICAM2, and VCAM1) promotes tumor-stroma cross-talk that 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, which helps maintain a complex angiogenic process. The recruitment of inflammatory immune cells that enhance tumor growth and the production of invasive factors (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 and STK6 contributes to cell cycle dysregulation and resultant aneuploidy. Several markers of proliferation are also overexpressed (SH2D1A, LCK, Rac2, and MAP4K1), which contributes to the overall survival and antiapoptotic pathways.

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

prolonged expression of either active or inactive NEK2 Several antiapoptotic proteins are represented in PTCL- leads to dispersal of centrosomal material and loss of a NOS. CD14 is 88-fold overexpressed and acts via MyD88, focused microtubule-nucleating activity (35). NEK2 has TIRAP, and TRAF6, leading to NF-nB activation, cytokine also been shown to be overexpressed in transformed secretion, and inflammatory response (46). NF-jB2 is 44- follicular lymphoma and de novo DLBCL (36). STK6 fold overexpressed and is a transcription factor containing (Aurora kinase-A) is 17-fold overexpressed and plays a rel-polyG-ankyrin domains. NF-jB2/p52 gene rearrange- role in cell cycle regulation during anaphase and/or ments have been observed in patients with lymphoma and telophase, in relation to the function of the centrosome/ have been shown to contribute to tumorigenesis by spindle pole region during chromosome segregation. suppressing apoptosis and increasing proliferation in Defect in STK6 is responsible for numerical centrosome response to inflammatory cytokines (47–49). A recent aberrations, including aneuploidy, which is related to its expression profiling study showed that the NF-nB signaling role in microtubule formation and/or stabilization (37). pathway is deregulated in PTCL-NOS (50), implicating this Several markers of proliferation are represented, of inflammatory transcription factor in maintaining cell which SH2D1A is 120-fold overexpressed and binds to a survival pathways. IL-18 is 39-fold overexpressed and specific motif in the cytoplasmic domains of several induces IFN-g production in T cells, and dysregulated members of the CD2 subfamily of cell surface receptors, IL-18 and/or IL-18R in chronic B-cell lymphoproliferative where it blocks recruitment of the tyrosine phosphatase disorders may contribute to tumor escape from the host SHP-2 and prevents inhibition of tyrosine kinase activated immune system by reducing the expression of Fas ligand. signaling pathways (38). Rac2 is 107-fold overexpressed Moreover, it has been shown to function as a novel and regulates a diverse array of cellular events, such as angiogenic mediator by promoting endothelial cell migra- cell growth, cytoskeletal reorganization, and activation of tion (51). protein kinases. Rac2 is expressed in thymocytes and Several proapoptotic genes are down-regulated. c-Jun is a activated T cells and could play a role in control of 27-fold down-regulated oncoprotein that blocks the ubiq- growth and death of T cells (39). LCK is 35-fold overex- uitination of tumor suppressor p53 and thus increases the pressed and phosphorylates mitogen-activated protein stability of p53 in nonstressed cells. Studies of the mouse kinase and L-selectin with subsequent association of counterpart of this gene suggest a key role in T-cell Grb2/Sos with L-selectin. This association correlates with differentiation (52). CED-6 is 22-fold down-regulated and an activation of p21ras, mitogen-activated protein kinase, is an evolutionarily conserved adaptor protein required for À and Rac2 and a transient increase of O2 synthesis. A final efficient engulfment of apoptotic cells and that promotes common path could involve a L-selectin-associated CD3 programmed cell death. Inactivating mutations in engulf- ~-chain-driven T-cell antigen receptor-induced cell prolif- ment genes enhance the frequency of cell survival (53, 54). eration (40). TCIRG1/TIRC7 is 54-fold overexpressed, a TNFRSF25 is 12-fold down-regulated, is expressed prefer- novel seven-transmembrane spanning vacuolar proton entially in tissues enriched in lymphocytes, and plays a role pump protein that is up-regulated during the early stages in regulating lymphocyte homeostasis. This receptor has of T-cell activation in response to alloantigens. Inhibiting been shown to stimulate NF-nB activity and regulate cell TIRC7 suppresses proliferation of activated T cells (41) apoptosis. The alternative splicing of this gene in B cells and supports the notion that it may play an important role and T cells encounters a programmed change on T-cell in PTCL proliferation. activation, which predominantly produces full-length, In PTCL-NOS, several tumor suppressors are down- membrane-bound isoforms and is thought to be involved regulated. TPTE encodes a PTEN-related tyrosine phos- in controlling lymphocyte proliferation induced by T-cell phatase, is 99-fold down-regulated, and plays an important activation (55). Therefore, a loss of function would lead to role in suppressing signaling via tyrosine-phosphorylated uncontrolled T-cell proliferation. proteins (42). MGC15419 is 20-fold down-regulated and DLBCL Tumor Profile Signature may be critical for the controlled degradation of cellular A predictive model for the DLBCL tumor profile is regulatory proteins (43). PTCH is 6-fold down-regulated delineated based on the six hallmarks of cancer (Fig. 4). A and is the receptor for sonic hedgehog, implicated in the chemokine network seems to be in operation and is likely formation of embryonic structures and in tumorigenesis. modulated by transcription factors MAFB (83-fold overex- Mutations of this gene have been associated with nevoid pressed), FLI1 (86-fold overexpressed), and CRIP1 (67-fold basal cell carcinoma syndrome, squamous cell carcinoma, overexpressed), a cysteine-rich intestinal protein 1 that con- trichoepitheliomas, and transitional cell carcinoma (44). tains a double zinc finger motif. Overexpression of CRIP in ST5 is 5-fold down-regulated and contains a COOH- mice produces an imbalance in cytokine pattern favoring terminal region that shares similarity with the Rab3 family Th2 cytokines: low IFN-g and high IL-6 and IL-10, which of small GTP-binding proteins. This protein preferentially alter the immune response and likely favor escape from binds to the SH3 domain of c-Abl kinase and acts as a immune attack (56). CCL5 is 147-fold overexpressed and regulator of mitogen-activated protein kinase 1/extracel- binds its receptor CCR1, which is 82-fold overexpressed. lular signal-regulated kinase 2 kinase, which may contrib- Tumor-derived CCL5 can inhibit the inhibitory T-cell ute to its ability to reduce the tumorigenic phenotype in response and enhance in vivo lymphoma growth. Several cells (45). proangiogenic ligands and receptors are overexpressed,

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

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 and paracrine manner recruit stromal fibroblasts, inflammatory immune cells, and endothelial cells to form a complex network of tumor tissue. The overexpression of integrin LFA1 on the tumor cells and their binding partners on stromal cells (ICAM2 and ICAM3) promotes tumor-stroma cross-talk which activates proliferative signaling pathways (JAK/STAT) and ECM formation (Lumican, FN1, COL6A3, COL18A1, and CHI3L1). Several tumor-derived angiogenesis 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 activated B-like DLBCL. Moreover, the overexpression of NF-nB-driven proliferative factors (CD14, CD20, CD52, CD72, and CCR1) inhibits many forms of apoptosis and 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 and antiapoptotic pathways. which include VEGF (66-fold), PDGF-AA (15-fold), CSF1R (58). RAB13, a GTPase, is 29-fold overexpressed and regu- (14-fold), b-PDGFR (11-fold), MET (7-fold), FGF13 (6-fold), lates the assembly of functional epithelial tight junctions. and PSMA (4-fold), and are likely to be induced by CCL5- Expression of the GTP-bound form of RAB13 inhibits CCR1. Tumor stroma–derived factors, such as IGFBP7, protein kinase A–dependent phosphorylation and tight which is 107-fold overexpressed (33) and likely elaborates junction recruitment of the vasodilator-stimulated phos- several components of the ECM, such as CHI3L1 (84-fold), phoprotein, an actin remodeling protein and thus may play FN1 (60-fold), COL6A3 (49-fold), COL18A1 (47-fold), and a role in invasion and metastasis (59). ICAM2 (30-fold). In DLBCL, two tumor suppressors are down-regulated. Markers of proliferation represented include CD20 (99- TPTE is a 100-fold down-regulated PTEN-related tyrosine fold), a member of the membrane-spanning 4A gene family phosphatase that, if active, would switch off tyrosine that functions as a Ca2+ channel resident on B lymphocytes kinase–driven oncogenic pathways (42). SUI1/MOF2 is 25- and plays a role in the development and differentiation of fold down-regulated and functions as a modulator of accu- B cells into plasma cells. It is the target for rituximab racy at both initiation and elongation phases of translation in B-cell non-Hodgkin’s lymphoma and shows excellent (60). Transfection of human SUI1 into HepG2 hepatocarci- correlation between gene expression profile and immuno- noma cells inhibited cell growth in culture in soft agar and histochemistry (57). CD72 is a 83-fold overexpressed type II partially inhibited tumor formation in nude mice (61). membrane protein of the C-type lectin superfamily and is Of the antiapoptotic proteins that are overexpressed, the ligand for CD5. It plays a role in pre-B-cell and B-cell CD14 is 141-fold overexpressed and is known to activate proliferation and differentiation. Among 83 malignant non- NF-nB leading to inflammatory cytokine secretion (46) and Hodgkin’s lymphomas evaluated by immunohistochemis- an inflammatory response that likely helps avoid immune try, all 54 B-cell lymphomas, including precursor B-cell attack. BCL2A1 is 107-fold overexpressed, forms hetero- lymphomas, were CD72 positive, but staining was not dimers or homodimers, and is involved in a wide variety of observed in T-cell lymphomas. Therefore, CD72 is a cellular activities, such as embryonic development, homeo- proliferative marker that cooperates with CD20 in DLBCL stasis, and tumorigenesis. BCL2A1 is a direct transcription

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Downloaded from mct.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures

Daruka Mahadevan, Catherine Spier, Kimiko Della Croce, et al.

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