Gene Expression Analysis of Angioimmunoblastic Lymphoma Indicates Derivation from T Follicular Helper Cells and Vascular Endothelial Growth Factor Deregulation

Research Article

Pier Paolo Piccaluga,1,2 Claudio Agostinelli,1 Andrea Califano,3 Antonino Carbone,4 Luca Fantoni,6 Sergio Ferrari,5 Anna Gazzola,1 Annunziata Gloghini,6 Simona Righi,1 Maura Rossi,1 Enrico Tagliafico,5 Pier Luigi Zinzani,1 Simonetta Zupo,7 Michele Baccarani,1 and Stefano A. Pileri1

1Institute of Hematology and Medical Oncology ‘‘L. and A. Sera`gnoli,’’ S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy; 2Institute for Cancer Genetics and 3Center for Computational Biology and Biochemistry, Columbia University, New York, New York; 4Department of Pathology, Istituto Nazionale Tumori, Milan, Italy; 5Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 6Division of Experimental Oncology, Centro di Riferimento Oncologico, Aviano, Italy; and 7S.S.D. Diagnostica Malattie Linfoproliferative, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy

Abstract Introduction Angioimmunoblastic lymphoma (AILT) is the second most Peripheral T-cell lymphomas (PTCL) represent 10% to 15% of common subtype of peripheral T-cell lymphoma (PTCL) and all lymphoid neoplasms (1). They are a heterogeneous group of is characterized by dismal prognosis. Thus far, only a fewstu- tumors that in the Revised European-American Lymphoma dies have dealt with its molecular pathogenesis. We performed (REAL)/WHO classification are subdivided into specified and un- gene expression profile (GEP) analysis of six AILT, six ana- specified forms (1, 2). Angioimmunoblastic T-cell lymphoma plastic large cell lymphomas (ALCL), 28 PTCL-unspecified (AILT) is a PTCL characterized by systemic disease, a polymor- (PTCL/U), and 20 samples of normal T lymphocytes (including phous infiltrate primarily involving lymph nodes, with prominent CD4+,CD8+, and activated and resting subpopulations), proliferation of high endothelial venules and follicular dendritic aiming to (a) assess the relationship of AILT with other cells (1). AILT is the second most common PTCL subtype, PTCLs, (b) establish the relationship between AILT and nor- accounting for 15% to 20% of cases (1). It occurs in the middle mal T-cell subsets, and (c) recognize the cellular programs age and elderly, the male/female ratio being 1/1. Patients usually deregulated in AILT possibly looking for novel potential present with generalized peripheral lymphoadenopathy, hepatos- therapeutic targets. First, we found that AILT and other PTCLs plenomegaly, and frequent skin rashwithpruritus. Bone marrow have rather similar GEP, possibly sharing common oncogenic involvement, as well as B symptoms, is common (3). Other pathways. Second, we found that AILTs are closer to acti- + + common clinical signs are edema, pleural effusion, arthritis, vated CD4 , rather than to resting or CD8 lymphocytes. and ascites. Laboratory findings include polyclonal hyper-g- Furthermore, we found that the molecular signature of globulinemia, circulating immunocomplexes, cold agglutinins follicular T helper cells was significantly overexpressed in with hemolytic anemia, positive rheumatoid factor, and anti– AILT, reinforcing the idea that AILT may arise from such smoothmuscle antibodies. Theclinical behavior is very aggressive, cellular counterpart. Finally, we identified several genes the response to therapy is scarce, and the long-term outcome is PDGFRA, REL VEGF deregulated in AILT, including , and . dismal (4). The expression of several molecules was then studied by The tumor morphology is constituted by a polymorphous immunohistochemistry on tissue microarrays containing 45 population of small- to medium-sized lymphocytes, usually with independent AILT cases. Notably, we found that the vascular clear cytoplasm (2, 5–7). The lymph node architecture is largely endothelial growth factor (VEGF) was expressed not only effaced, and regressed follicles may be seen. The lymphocytes show by reactive cells, but also by neoplastic cells, and that nuclear minimal cytologic atipia, and this form of lymphoma may be K K factor- B (NF- B) activation is uncommon in AILT, as sug- difficult to distinguishfrom atypical T-zone hyperplasia.Neo- gested by frequent exclusively cytoplasmic c-REL localization. plastic cells are admixed withsmall reactive lymphocytes,eosino- Our study provides newrelevant information on AILT biology phils, plasma cells, histiocytes, and abundant follicular dendritic and newcandidates for possible therapeutic targets such as cells (1, 2, 7). High endothelium venules are numerous and show A PDGFRA (platelet-derived growth factor ) and VEGF. [Cancer arborization. Cells positive for EBV are found in the majority of Res 2007;67(22):10703–10] cases. However, the EBV cells are mainly B cells and rarely T cells (1). Immunohistochemistry generally shows T-cell–associated molecule expression, although the phenotypic profile is aberrant in the majority of cases, CD5 and CD7 being the most frequently Note: Supplementary data for this article are available at Cancer Research Online defective antigens, whereas CD10 is frequently present (8). CD4 (http://cancerres.aacrjournals.org/). is most commonly expressed rather than CD8, but notably, the Requests for reprints: Pier Paolo Piccaluga, Molecular Pathology Laboratory, Haematopathology Unit–Institute of Hematology and Medical Oncology ‘‘L. and A. latter two antigens are coexpressed or even not expressed in more Sera`gnoli’’, S. Orsola Malpighi Hospital, University of Bologna, Via Massarenti, 9-40138 than 50% of cases (double-positive and double-negative cases, Bologna, Italy. Phone: 39-51-6364043; Fax: 39-51-6364037; E-mail: pierpaolo.piccaluga@ respectively; ref. 8). The differential diagnosis with other PTCLs unibo.it. I2007 American Association for Cancer Research. and, in particular withPTCL unspecified (PTCL/U), is puzzling doi:10.1158/0008-5472.CAN-07-1708 and mainly based on different features of reactive components www.aacrjournals.org 10703 Cancer Res 2007; 67: (22). November 15, 2007

(i.e., prominent vascular structures and abundant follicular den- ing the phosphorylated activated form of the platelet-derived growth factor dritic cells) and phenotype (CD10 and BCL6 expression). Notably, receptor (PDGFR)], CYR61, LIFR-1, IGFBP7, BCL10, L-caldesmon, CXCL13, no unique markers can reliably discriminate between these two vascular endothelial growth factor (VEGF), VEGFR2, c-Rel; RelB, nuclear n n entities. Recently, CXCL13 has been proposed as a possible candi- factor- B (NF- B) p65, selected because of their potential biological and date to distinguishthetwo diseases (7, 9–11). clinical relevance. For additional details, see Supplementary File 1. Bound antibodies were visualized either by the alkaline phosphatase The molecular pathogenesis of AILT, as in general of all anti-alkaline phosphatase (APAAP) complexes technique or the EnVision peripheral T-cell neoplasms, is poorly understood. The karyotype method (8). For negative controls, the primary antibodies were omitted. is often characterized by complex abnormalities, but specific Notably, eachTMA was also tested withanti-CD20 and CD3 antibodies to alterations have not been identified (12), and only few reports define the amount of reactive B cells comprised within the neoplastic T-cell focused on gene expression profile (GEP) of nodal PTCLs (13–16). population. In one study, an AILT molecular signature has been identified The adopted equipment included an Olympus BX41 microscopy with together with a molecular link between AILT and T follicular helper UPlanFl objectives (20Â/0.50 and 40Â/0.75), an Olympus Camedia C-7070 (TFH) lymphocytes (16). Recently, we analyzed the GEP of a large digital camera and the Olympus Master 1.1 software for images acquisition panel of PTCLs and normal T lymphocytes by focusing on PTCL/U and evaluation. (17). We identified a molecular signature typical of PTCL/U and Criteria for immunohistochemical marker evaluation. Eachsection was independently evaluated by at least two experienced hematopatholo- many tumor-associated pathways shared by both PTCL/U and gists. Cases were considered positive if 30% or more of the tumor cells were AILT (17). stained withan antibody. Thenumber of positive cells was estimated by In the present study, we analyzed the GEP of AILT, PTCL/U, ana- eachobserver. Theintensity of staining was also evaluated, but was not plastic large cell lymphoma (ALCL), and normal T-cell subpopu- used to determine positivity because it can vary depending on tissue lations aiming to (a) assess the relationship of AILT with other fixation (8, 17). PTCLs, (b) establish the relationship between AILT and normal T- cell subsets, and (c) recognize the cellular programs that are altered as a consequence of malignant transformation in AILT and identify Results genes that might be suitable as novel potential therapeutic targets. GEPs of AILT and PTCL/U are similar. We performed a gene expression analysis on six angioimmunoblastic lymphomas (AILT) Materials and Methods and, for comparison, on 28 PTCLs/NOS, 6 ALCLs, and 20 samples of purified normal T cells (including CD4+, CD8+, HLA-DR+, and Case selection. Cryopreserved samples of 28 PTCL/U, 6 AILT, and 6 À À ALCL (4 ALK and 2 ALK+) were collected on the basis of stringent criteria HLA-DR cells). First, we applied unsupervised analysis to AILT, to guarantee the homogeneity of the series studied: (a) lymphnode biopsy PTCL/U, and ALCL to assess whether they are clearly distinct performed at disease presentation in the absence of any previous treat- entities. With this method, no complete separation was feasible ment, (b) excellent RNA preservation, and (c) presence of more than 70% among the three PTCL histologic subtypes. In particular, AILT and neoplastic cells in the specimen. In addition, 20 samples of normal T PTCL/U showed a quite similar GEP, whereas ALCL tended to lymphocytes (including CD4+, CD8+, activated, and resting T cells) were cluster more closely (Supplementary Fig. S1; ref. 17). isolated from peripheral blood and tonsils of healthy donors. AILT are related to activated peripheral CD4 T cells with The diagnosis of PTCL/U, AILT, and ALCL had previously been confirmed TFH functional differentiation. To evaluate the relationship according to the REAL/WHO Classification (1, 2) by at least two between AILT and different normal T-cell subsets, we first studied experienced hematopathologists. In addition, AILT samples were studied AILT for the expression of genes previously shown to be for the expression of CXCL13, which was detected in all of them. All the À differentially expressed in purified resting (HLA-DR )and samples had been obtained at the time of diagnosis, before any treatment + had been given. Clinical details of these cases were previously reported (17). activated (HLA-DR ) T cells (17). We found that AILT are definitely In addition, the paraffin blocks of 193 PTCLs (148 PTCLs/U and 45 AILT) closer to activated T lymphocytes (Fig. 1A). An analogous were collected. The phenotype was assessed by a large panel of reagents investigation was performed using a profile of genes differentially against CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD20, CD30, CD56, expressed between purified CD4 and CD8 T-cell subsets (17). The CD57, CD79a, TIA-1, Granzyme B, ALK protein, and EBER (8). analysis revealed that AILT are closer to CD4 cells (Fig. 1B). Informed consent had been obtained from all the enrolled patients, and Furthermore, we looked for the expression of genes characteristic the tissue collection was approved by each institutional ethical committee. of different functional stages of T cells, suchas T-central memory Normal T-cell isolation. T-cell subpopulations, CD4+ (n = 5), CD8+ À (TCM), T-effector memory (TEM), and TFH (18, 19), in our AILT. (n = 5), HLA-DR+ (n = 5), and HLA-DR (n = 5), were purified from healthy Interestingly, whereas TCM and TEM signatures were not donors as previously described (17). GEP analysis. GEPs were generated and analyzed as previously reported significantly expressed in AILT in comparison withotherPTCLs, (17) using the HG-U133 2.0 plus microarrays (Affymetrix, Inc.).8 For details the molecular signature typical of TFH was significantly overex- on GEP generation and analyses, see Supplementary File 1. pressed in AILT (P = 0.03; Fig. 1C). Gene expression studies were conducted according to MIAMI guidelines.9 Taken together, our results indicate that AILT are molecularly Tissue microarray construction. Tissue microarray (TMA) construc- related to activated peripheral CD4 T cells with TFH functional tion was performed as detailed in a previous publication referring to the differentiation and strongly support the hypothesis that they are same series (8). derived from the transformation and clonal expansion of such Immunohistochemistry. After appropriate antigen retrieval (8), the cells. sections obtained from eachTMA were tested withspecific antibodies AILT differs from normal T cells for the expression of genes against the following molecules: PDGFRa/CD140A, p-PDGFRa [represent- involved in matrix constitution, adhesion, immune response, response to stress, and homeostasis. To identify the genes and cell programs deregulated in AILT, we used a supervised analysis 8 http://www.affymetrix.com/support/index.affx 9 Raw gene expression and additional data are available at http://www.ncbi.nlm. directly comparing the tumor cases with the closer normal cellular + + nih.gov/projects/geo/ (accession number GSE6338). counterparts (CD4 -activated cells). In addition, CD8 cells were

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Supplementary Tables S1 and S2). Subsequently, these genes were classified according to functional categories defined by Gene Ontology. We found some categories to be significantly over- represented also when considering the number of genes belong- ing to each category that were comprised within the GeneChip (P < 0.05; refs. 17, 20), suchas matrix constituents, adhesion, immune response, response to stress, and homeostasis (Supple- mentary Table S3). Among others, the newly identified genes that were notably overexpressed in AILT, included (a) FN1 (fibronectin 1), PCOLCE, COL1A1, COL3A1, COL4A1, COL6A1, COL12A1, COL15A1, COL4A2, and COL8A2 (encoding for collagen components), and MMP9, whose expression might be correlated with tissue invasiveness and angiogenesis (21–23); (b) ABCC3 and NNMT, which may contribute to the resistance to chemotherapy, a common feature of AILT (1); (c) PDGFRA (platelet-derived growthfactor a), a tyrosine kinase possibly involved in neoplastic transformation, whose activity can be inhibited by imatinib mesylate (24, 25); (d) molecules involved in cell adhesion such as EMILIN1, LAMB1, PARVA, TNC, FLRT2, ENG, LAMC1, CDH5, CDH11, and RAB13; and finally, (e) chemokines including CCL2, CCL14, CCL19, CCL21, CXCL9, CXCL11, CXCL12, and CXCL13 (Supplementary Fig. S3), which may be responsible for reactive cell recruitment (26). Within the group of down-regulated genes, there were PPP1R15A, STK17B,and GADD45-A and GADD45-B (growtharrest and DNA damage– inducible a and h), which are involved in the apoptosis control. Notably, expression of the two latter is induced by histone deacetylase inhibitors (27–31). Moreover, we found REL to be down-regulated in AILT, suggesting that NF-nB pathway is not consistently active in these tumors. Supervised comparison among AILT, PTCL/U, and ALCL. The 474 gene signature identified in AILT at supervised analysis (Fig. 2A; Supplementary Tables S1 and S2) was largely shared by ALCL and PTCL/U (Fig. 2A). This finding might reflect either the influence of reactive components or the existence of common tumor-associated pathways (17). The latter hypothesis finds some support in the fact that among the deregulated genes, there were those shown to be tumor cell–specific by immunohistochemistry on TMAs (17). Moreover, they included many (142) down- regulated genes that are unlikely referable to nontumoral components. A further supervised analysis revealed 89 genes differently expressed between AILT on one hand and PTCL/U and ALCL on the other (Supplementary Table S4). Among the latter, there were FGF11, TP53, MMPL1, LIFR, EDNRB, FOXF2, and CLK1. Figure 1. Relatedness of AILT to normal lymphocytes. Relatedness of the GEP Finally, as differential diagnosis of AILT and PTCL/U is actually of AILT to normal T-cell populations. A supervised analysis is used to identify the based on relatively subjective elements, we compared by super- genes differentially expressed between the two groups of samples. HLA-DR+ À + vised analysis these two PTCL subtypes. We found 80 genes (activated) T cells are compared with HLA-DR (resting) T cells (A), and CD4 T CCL19, CD81, cells are compared with CD8+ T cells (B). The expression of the selected genes consistently differentially expressed, including is investigated in AILT represented on the right side of the matrix. A cell-type IFNGR1, metallothionein 1X, 2A, 1G, 1F, and VEGF (Supplementary classification is used to measure the relatedness of PTCL/U to HLA-DR+ and À Table S5). HLA-DR T cells (A) and to CD4+ and CD8+ T cells (B). Gray area, 95% confidence; the P value decreases with increasing distance from the x-axis. In Because we found VEGF among genes overexpressed in AILT, we C, the relatedness of AILT to TFH cells is shown. The median of the mean specifically looked at the expression of VEGFRs (VEGFR1, VEGFR2, expression values of THF signature in AILT and other PTCLs (PTCL/U and ALCL) are reported. t test comparing the mean expression values of THF and VEGFR3). We found VEGFR2/KDR to be significantly overex- signature in the two groups (AILT versus other PTCLs) revealed a significant pressed in AILT versus normal T cells (P = 0.0008, Supplementary difference (P = 0.03). Fig. S2). Immunohistochemical validation on TMAs. To investigate also included in the comparison to abrogate the possible puzzling whether or not the up-regulated mRNA levels of PTCL/U- effect of contaminating normal CD8+ lymphocytes in AILT samples. associated genes corresponded to elevated levels of the encoded Four hundred seventy-four probe sets were differentially expressed, proteins, we stained TMAs constructed from 45 AILT (including corresponding to 346 unique genes. Two hundred and four were the cases subjected to gene expression analysis) using specific up-regulated, and 142 were down-regulated in AILT (Fig. 3A, monoclonal and polyclonal antibodies raised against the selected www.aacrjournals.org 10705 Cancer Res 2007; 67: (22). November 15, 2007

ples in Figs. 3 and 4).The number of evaluable cases varied among the analyses. The main reasons for case exclusion were core loss, unrepresentative sample, or suboptimal antigen preservation. PDGFRa was expressed in 36/36 cases (100%); of note, it turned out to be phosphorylated in 21/22 evaluable cases (95%). ILGFBP7

Figure 2. Identification of genes differentially expressed in AILT and normal T lymphocytes. Supervised analysis was performed using six AILT samples versus the three normal T-cell subpopulations identified as the closest normal counterparts (CD4+, CD8+, and HLA-DR+). A, the analysis identified 474 probe sets (346 genes) that are differentially expressed in AILT versus all the other samples (Supplementary Tables S1 and S2). B, the results were confirmed when AILT samples were divided into training (TR, n = 3) and test (TE, n = 3) set. Three AILT cases (TR) were used for supervised comparison versus normal T cells; the obtained signature was tested in the other three (TE), which showed an almost identical profile.

molecules. This method was previously shown to be very effective in identifying tumor-associated molecules and discriminate those Figure 3. Immunohistochemical validation of gene expression results on related to reactive elements (17, 32). TMAs. Examples of the immunostaining patterns for CXCL13 (A), PDGFRa a (B), and p-PDGFRa (C). Original magnification: A, Â200; B and C, The expression in AILT cells was confirmed for PDGFR , VEGF, Â n 400. A, immunoperoxidase staining by the EnVision+ technique; B and C, VEGFR2/KDR, LIFR, ILGFBP7, and BCL10, CALD1, and NF- B immunoalkaline phosphatase staining by the APAAP method; Gill’s hematoxylin components (namely, RELA/p65, RELB/p50, REL/c-rel; see exam- nuclear counterstaining.

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nuclear levels. RELB/p50 was expressed in 18/23 cases, withcyto- plasmic, cytoplasmic/nuclear, and nuclear staining in 14, 2, and 2 cases, respectively. REL/c-rel was expressed in 24/24 cases, with cytoplasmic or cytoplasmic + nuclear staining in 19 and 5 cases, respectively (Table 1). Overall, NF-nB units turned out to be mostly localized in the cytoplasm (39/60 evaluable cores) and, in a minority of cases, at the cytoplasmic and nuclear (14/60 cores) or nuclear (5/60 cores) level. These findings were actually consistent with the gene expression results, not suggesting a constitutive/significant activation of this pathway in the majority of cases (Figs. 4 and 5 and Table 1). The immunohistochemical analysis results are summarized in Table 2. Taken together, these results indicate that there is a rather good correspondence between mRNA and protein expression. In

Figure 4. Immunohistochemical validation of gene expression results on TMAs. Examples of the immunostaining patterns for RELB/p50 (A), VEGF (B), VEGFR2 (C). Original magnification: A and C, Â200; B, Â400; B, immunoperoxidase staining by the EnVision+ technique; A and C, immunoalkaline phosphatase staining by the APAAP method; Gill’s hematoxylin nuclear counterstaining. was expressed in 17/27 cases (63%); LIFR in 12/28 (43%), and

CYR61 in 36/37 cases (97%). Finally, AILT cells seemed to be VEGF Figure 5. Gene expression and protein localization of NF-nB components. positive in 18/18 cases (100%), whereas VEGFR2/KDR was present A, median expression values of RELA, RELB, and REL were compared in AILT in 27/27 samples (100%). Withregard to NF- nB pathway, RELA/p65 and normal samples of T lymphocyte subsets (Student’s t test). B, localization of NF-nB components (RELA/p65, RELB/p50, and REL/c-rel) as detected by was expressed in 16/16; in six cases, the staining was detected at immunohistochemistry. Of note, all the AILT cases showed REL expression the cytoplasmic level and, in seven cases, at the cytoplasmic and levels lower than the lowest one recorded in normal T cells (C). www.aacrjournals.org 10707 Cancer Res 2007; 67: (22). November 15, 2007

Table 1. NF-nB pathway evaluation by immunohistochemistry

Antigen Evaluable cores Positive: cytoplasm Positive: nucleus and cytoplasm Positive: nucleus Negative

RELB/p50 23 14 2 2 5 REL/c-rel 24 19 5 0 0 RELA/p65 13 6 7 0 0 Total 60 39 14 2 5 addition, the immunohistochemical determination allows an CD4+ T lymphocytes. Furthermore, we could show that AILT do unambiguous definition of the cellular compartment carrying the express the TFH molecular signature at significantly higher levels phenotypic attribute (Table 1). rather than PTCL/U and ALCL, whether T-central memory and T-effector memory signatures (19) were not significantly Discussion expressed. Together, these data strongly reinforce the hypothesis that AILT are actually derived from TFH cells. Nevertheless, at This paper has addressed critical questions regarding the cell present, it is not possible to conclude that CXCL13 immunohis- of origin and pathogenesis of AILT using the gene expression tochemical expression (confirmed also in our series) represent a profiling technology. Overall, the panel of cases herein described definitive marker for discriminating AILT and PTCL/U cases, but was sufficient to show the distinctive tracts of the molecular rather a useful additional information together with morphology, signature of this disease, although the overall molecular profile of complete phenotype (including CD21, CD10, and BCL6), and AILT is relatively similar to that of PTCL/U and ALCL (Supple- clinical presentation. mentary Fig. S1; ref. 17). This fact may reflect either the influence of Functional alterations in AILT. This study provides new non-neoplastic elements or the existence of common tumor- information concerning the functional alterations in AILT. The associated pathways (17). To definitely solve this problem, gene direct comparison of AILT withtheclosest cellular counterparts expression analysis of purified PTCL cells is necessary. In addition, showed a consistent deregulation of genes that sustain relevant we could identify the closer cellular counterparts and discover cellular functions, suchas matrix remodeling, adhesion,immune alterations in cellular programs that might be of biological and response, response to stress, and homeostasis. In this regard, it clinical relevance. On the other hand, at present, we could not should be considered that the up-regulation of some genes determine the prognostic impact of the novel identified molecules (especially those involved in the extracellular matrix and adhesion) because the available panel was not designed, and, thus, sufficient, may reflect the presence of contaminating stromal elements pre- for suchanalysis. sent in the lymph node tumor tissues. Our results are overall The cellular derivation of AILT. Morphology and general consistent withthosereported by de Leval et al. (16). However, few phenotype of AILT suggests their derivation from mature T differences could be recorded. First, our signature did not include lymphocytes. Interestingly, immunohistochemical studies sug- some molecules known to be expressed in AILT, suchas CD10. It gested the derivation of AILT from germinal center regulatory T was not really surprising as we previously showed that CD10 is not cells (33) and, in particular, T follicular helper cells (TFH) always expressed by AILT (8). Second, de Leval et al. (16) suggested according to the expression of CXCL13 (7, 9–11), a typical TFH that VEGF expression was mainly due to reactive nonneoplastic marker (18). Furthermore, a recent GEP analysis showed that components surrounding AILT cells. On the contrary, using AILT are linked to TFH at the molecular level (16). In our study, immunohistochemistry as further investigation, we could show we provided further evidence of the close relationship between that AILT neoplastic cells also do ectopically express VEGF, which AILT and TFH cells. In particular, we show that the global might be a relevant therapeutic target, in line with a previous molecular profile of AILT is intimately related to that of activated observation (34). Notably, we could also show the expression of VEGFR2 in AILT cells, suggesting the existence of an autocrine loop. On the other hand, other groups investigated PTCLs by GEP Table 2. Immunohistochemical analysis on TMA (14, 15), but did not specifically focus on AILT and did not find an AILT-associated signature. Interestingly, it was proposed that Antigen Evaluable cases Positive Negative some AILT might present constitutive NF-nB activity as some PTCL/U (35). We found REL to be down-regulated in AILT in com- VEGF 18 18 0 parison with normal T cells, suggesting that this pathway is not VEGFR2 27 27 0 constitutively active in this tumor. In addition, we confirmed our PDGFRa 36 36 0 result by immunohistochemical analysis because we found cyto- p-PDGFRa 22 21 1 plasmic localization (i.e., not activation) of NF-nB components IGFBP7 27 17 10 (p50, p65, and c-rel) in the majority of cases in an independent LIFR-1 28 12 16 panel of AILT. In our opinion, it is likely that previous findings CYR61 37 36 0 BCL10 25 4 21 might be related at least in part to non-neoplastic components Caldesmon 43 43* 0 more than to intrinsic characteristics of the neoplastic cells, although rarer AILT cases that do actually express the functional NF-nB pathway might not be excluded. Certainly, further studies on *Stromal reactivity. larger series and especially on purified neoplastic cells are anyway warranted.

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Biological and clinical implications. The present study independent manner (30) and were shown as effective treatment in confirmed our previous observation that different PTCLs can a PTCL/U both ex vivo and in vivo (17, 31). share molecular abnormalities (17). On the other hand, different Finally, we documented the aberrant expression PDGFRa in PTCL subtypes seemed to present significant differences to what AILT as previously recorded for PTCL/U (17, 32), and we found their relationship with different cellular counterparts is VEGF to be aberrantly expressed by AILT cells. Of note, recently, concerned. We and others showed that AILT is closely related a few articles reported on the clinical efficacy in AILT of thali- to FHC (16), whereas PTCL/U and ALCL are more frequently domide (37–39) and especially of bevacizumab (40), two com- related to TCM and TEM, respectively (36).10 These observations pounds withantiangiogenic properties. Surely, thepractical impact may suggest the intriguing hypothesis that different PTCLs may of inhibiting VEGF and PDGFRa may be the goal of future pilot arise from different T cells, but through largely common clinical studies (25). oncogenic events. In addition, our gene expression analysis identified several genes Acknowledgments deregulated in AILT, which may be interesting for the clinical practice. For instance, AILT are characterized by really dismal Received 5/18/2007; revised 8/1/2007; accepted 9/18/2007. Grant support: Associazione Italiana per la Ricerca sul Cancro, BolognAIL, prognosis (4). We found several chemokines to be deregulated in Fondo per gli Investimenti della Ricerca di Base (FIRB), Ricerca Fondamentale this tumor, which might represent possible therapeutic targets (26). Orientata (RFO) (S.A. Pileri and P.L. Zinzani), Fondazione del Monte di Bologna e Ravenna, and Fondazione Cassa di Risparmio in Bologna. P.P. Piccaluga was also Furthermore, we identified molecules involved in drug resistance, supported by a Rotary International–The Rotary Foundation Academic Year suchas ABCC3 and NNMT, whichmay sustain thetypical Scholarship, and Vanda Vanini e Sandro Cavagnino and Cristina Bassi Association chemoresistance of this tumor. In addition, our gene expression grants. The costs of publication of this article were defrayed in part by the payment of page study showed the down-regulation in AILT of genes that positively charges. This article must therefore be hereby marked advertisement in accordance regulate apoptosis, suchas MOAP1, PPP1R15A, STK17B,and with18 U.S.C. Section 1734 solely to indicate thisfact. GADD45-A and GADD45-B.Inparticular,GADD45-A and The following clinicians and/or pathologists, listed according to their respective GADD45-B institutions, also contributed to the study: Stefano Ascani, (Department of Pathology, regulate apoptosis through p38 activation and c-jun- Terni Hospital, Italy), Fabio Facchetti (Department of Pathology, University of Brescia, NH2-kinase inhibition (27–29). Intriguingly, histone deacetylase Italy), Manlio Ferrarini (Division of Medical Oncology C, National Cancer Research GADD45-A/B Institute, Genoa University, Italy), Domenico Novero (Department of Biomedical inhibitors can induce the expression of in a p53- Science and Human Oncology–Pathologic Anatomy Section, Torino University, Italy), Marco Paulli (Department of Human and Genetic Pathology–Pathologic Anatomy Section, Pavia University, Italy). We are grateful to Prof. Riccardo Dalla Favera, Dr. Katia Basso, and Dr. Ulf Klein for precious advice and discussion, and to Vladan Milkjovic, Yonghui Zang, and Federica 10 P.P. Piccaluga and S.A. Pileri, unpublished data. Sandri for their skilled technical assistance.

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