Reactive Tissues Research Articles

Diagnostic and Therapeutic Implications of Expression and Phosphorylation of the Translation Regulatory Protein 4E-Binding Protein (BP)-1 in B-Cell Lymphomas and Reactive Lymphoid Tissues.

Abstract 3919Poster Board III-855Identifying pathogenic mechanisms that contribute to the development of lymphomas and influence clinical behavior is critical for developing targeted therapies, and selecting patients who may benefit from such drugs. An important level of control of gene expression occurs during initiation of cap-mediated mRNA translation by the eukaryotic initiation factor-4F (eIF-4F) trimolecular complex (eIF-4E, eIF-4G and eIF-4A), in which eIF-4E is rate limiting and oncogenic. eIF-4F hyperactivity plays a key role in human cancers by mediating expression of proteins critical for cell growth, transformation and tumorigenesis. eIF-4F activity is controlled by repressor eIF-4 binding proteins (BPs). 4E-BP1 activity is regulated by phosphorylation. Hypo/non-phosphorylated 4E-BP1 is active, binds eIF-4E and impedes eIF-4F formation, blocking translation and inducing apoptosis. Phosphorylation of 4E-BP1 (p4E-BP1) releases bound eIF-4E, which initiates cap-dependent translation. Because only limited information is available on the expression and phosphorylation of 4E-BP1 in lymphomas, and since agents (e.g., antisense oligonucleotides and small molecules) that target eIF-4E have been developed, we examined the frequency and level of expression of 4E-BP1 and its phosphorylation in various subtypes of mature B cell non-Hodgkin's lymphomas (BCL). Forty-six BCLs (12 follicular [FL], 13 diffuse large B-cell [DLBCL], 7 mantle cell, 5 extranodal marginal zone, and 9 small lymphocytic [SLL] lymphomas), 4 FL with incipient/partial lymph node involvement, and 11 reactive lymphoid tissues were examined using immunohistochemistry for total and phosphorylated 4E-BP1. Staining intensity was graded as from 0 to 3+. Western immunoblotting (WB) was performed on lysates of 5 mature BCLs (2 FL, 3 DLBCL) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E and 4E-BP1 (total and phosphorylated) expression. In reactive lymphoid tissues, there was regional and cellular specificity of expression of 4E-BP1, with either lack of, or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T-cells, 2+ expression in follicular dendritic cells and paracortical zone Langerhan cells, and 3+ expression in mantle and marginal zones. p4E-BP1 expression was inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells and no expression in the mantle and marginal zone cells or T-cells, and 2+ or 3+ immunoreactivity in follicular dendritic cells and paracortical zone Langerhan cells. In BCLs, a consistently high level (2+ or 3+) of cytoplasmic 4E-BP1 expression was seen in neoplastic lymphocytes in 45/46 (98%) cases. In contrast, p4E-BP1 was moderately or strongly expressed in 19/46 (41%) cases of BCL, being negative in 17 (37%) cases, and only dimly expressed in the remaining 10 (22%) cases. Three of 4 cases with incipient/partial involvement by FL were easily distinguishable from reactive germinal centers by strong, diffuse staining with 4E-BP1 (and 1+ staining in the 4th case) in neoplastic follicles, distinct from negative/weak staining of adjacent reactive germinal centers. In SLL, slightly higher 4E-BP1 expression was noted in proliferation centers in comparison to surrounding small mature lymphocytes. WB confirmed that non-phosphorylated and p4E-BP1 were expressed in reactive nodes, FL and DLBCL. Other components of the eIF-4F complex including eIF-4G, total and p-eIF-4E and total 4E-BP1 were detectable in whole tissue lysates from BCL samples. We conclude that (a) while 4E-BP1 is almost uniformly expressed in various subtypes of BCL, its level of phosphorylation (indicative of activity) varies widely and has regional and cellular specificity, and (b) 4E-BP1 expression may identify minimal/early lymphomatous involvement in tissues. We speculate that 4E-BP1 phosphorylation may influence the biological behavior of BCLs, since in other investigations we found that the level of phosphorylation of 4E-BP1 correlates with survival after CHOP-based chemotherapy in DLBCL. Our findings support therapeutic trials targeting the eIF-4E pathway in many BCL subtypes, particularly in patients where immunostaining identifies high levels of 4E-BP1 phosphorylation. Disclosures:No relevant conflicts of interest to declare.

Over-Expression of IDO in NHL Results in Conversion of CD4+CD25- T Cells Into CD4+CD25+ T Cells.

Abstract 1931Poster Board I-954 Introduction:Indoleamine 2,3-dioxygenase (IDO),the key enzyme in tryptophan metabolism, catalyzes the initial rate-limiting step of tryptophan degradation along the kynurenine pathway. Tryptophan starvation by IDO consumption and products of tryptophan catabolism can inhibit T-cell activation, regulate T-cell proliferation and survival, and participate in tumor-induced tolerance. This effect can be abrogated by IDO inhibitor, 1-methyl tryptophan (1-MT), which has been proved to possess an anti-tumor effect. In this study, we aim to measure the expressions of IDO and Foxp3 in NHL and to investigate in-vitro whether T-cell tolerance in NHL may be induced by directly converting CD4+CD25- T cells into CD4+CD25+ Treg cells through an IDO-dependent mechanism. Patients and Methods:Twenty fresh and fifty-seven paraffin embedded NHL tissues were collected. Reactive lymphadenitis tissues were set as control group. Quantitative expressions of IDO and Foxp3 mRNA in NHL tissues and counterparts were assessed by real-time PCR. IDO and Foxp3 protein expressions were investigated by both immunohistochemistry staining and Western-blot. Murine CD4+CD25- T-cell isolated by MACS and identified by FACS were co-cultured with IDO-expressing A20 cell line in the presence and absence of 1-MT, which specially inhibits IDO activity. Results:IDO mRNA was up-regulated expression in NHL tissues compared to non-neoplastic counterparts(2-dCt: 0.00582∼0.546 v. 0∼0.0103, p<0.01, Fig 1A). A significant correlation was found between higher IDO mRNA level and Foxp3 mRNA expression. In IHC (Table.1), cytoplasmatic IDO staining was positive in 50 NHL specimens, in whch eighteen were strong-stained. The proportion of Foxp3+ cells was also higher in NHL patients than controls (Ö2=5.28,P<0.05). Interestingly, there were more Foxp3+ cells in IDO strong staining tissues than week staining. IDO protein level detected by WB was coincidentally higher in NHL than controls(IDO/b-actin: 0.77±0.25 v. 0.18±0.18, p<0.01) and with elevated IDO protein level, Foxp3 protein expression was markedly increased(Fig.1B). Co-culture of CD4+CD25- T cells with A20 cells increased the surface expression of CD25 and the percentage of CD4+CD25+ T cells (8.21%). The addition of 1-MT attenuated conversion of CD4+CD25- to CD4+CD25+ T cells (2.37%), whereas 1-MT had no solitary effect on CD4+CD25- T cells(Fig. 1C). In addition, it was confirmed by FACS that most of the converted CD4+CD25+ T cells expressed Foxp3, which was the representative transcriptive factor of Tregs(Fig. 1D). [Display omitted] Table. 1GroupIDOP valueStrong staining (positive cell rate≥30%)Week/Negative staining (positive cell rate<30%)Control group018NHL1839P<0.05aFoxp3-strong staining (positive cell rate≥3%)1610Foxp3- week/negative staining (positive cell rate<30%)229P<0.05baChi-square analysis,bFisher's exact probabilities in 2°Á2 table Conclusion:Both mRNA and protein of IDO are increasingly expressed in NHL, synchronously with the over-expression of Foxp3. Up-regulation of IDO may induce T-cell tolerance by directly increasing Treg cells through the conversion of CD25- into CD25+ T cells. This could be regarded as a novel mechanism of NHL escape from immune control and its inhibition may represent a novel anti-tumor therapeutic strategy. Disclosures:No relevant conflicts of interest to declare.

The EML4-ALK transcript but not the fusion protein can be expressed in reactive and neoplastic lymphoid tissues

Rearrangements involving the ALK gene define two distinct entities in the new 2008 WHO classification of lymphoid neoplasms, i.e. ALK+ anaplastic large cell lymphoma and a rare subset of ALK+ diffuse large B-cell lymphoma. Recently, rearrangements involving ALK and the echinoderm microtubule associated protein-like 4 (EML4) gene were described as a specific genetic alteration in about 6% of non-small cell lung cancer (NSCLC). We investigated the expression of EML4-ALK mRNA and protein in 51 reactive and 58 neoplastic lymphoid tissues. EML4-ALK transcripts were detected in 3/51 (5.9%) of reactive lymphoid tissues and 12/58 (20.7%) of lymphomas of different categories, including follicular lymphoma, diffuse large B-cell lymphoma and Hodgkin's disease. Notably, none of these cases expressed the EML4-ALK fusion protein at Western blotting samples and immunohistochemistry. These results indicate that EML4-ALK rearrangements are not specific of NSCLC and raise yet unsolved questions about their role in promoting human neoplasms.

Integrin α1β1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo

Connective tissue cell activation is of importance during reactive conditions such as solid tumour growth, wound healing and pannus formation in rheumatoid arthritis. Here, we have compared connective tissue cells of mesenchymal origin in human tissues from these conditions and their normal counterparts using a panel of cell-type-specific markers. In particular, we investigated variations of integrin expression among connective tissue cell phenotypes. Connective tissue cell populations were defined based on their association with the microvasculature and their expression of activation markers. The phenotype of these cells varied according to the type of pathological connective tissue examined. Our morphological data from human tissues suggested that the α1β1 integrin, a collagen/laminin receptor, is involved in the differentiation of precursor cells into myofibroblasts. To mechanistically investigate this hypothesis, we employed experimental models for carcinoma growth and wound healing utilizing α1 integrin-deficient mice. The data confirmed that the α1β1 integrin is of importance not only for the differentiation of mesenchymal cells into myofibroblasts but also for the neovascularization and connective tissue organization and emphasize the importance of myofibroblasts in the pathophysiology of tissue repair, inflammation and tumour growth.

Expression of Grb2 distinguishes classical Hodgkin lymphomas from primary mediastinal B-cell lymphomas and other diffuse large B-cell lymphomas

Growth factor receptor-bound protein 2 is an adaptor molecule that mediates B-cell receptor (BCR) signaling pathways, but the expression of growth factor receptor-bound protein 2 in lymphoma tissues has not been reported. We sought to characterize growth factor receptor-bound protein 2 protein expression in reactive tonsillar tissues and lymphoma tissues obtained from diagnostic biopsies of classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and 20 low-grade B-cell lymphomas. Growth factor receptor-bound protein 2 expression was assessed in tissues by immunohistochemistry and in lymphoma cell lines by immunoblotting. In reactive lymphoid tissues, growth factor receptor-bound protein 2 was expressed in the cytoplasm of B-cells and histiocytes but not T-cells. Strong, cytoplasmic growth factor receptor-bound protein 2 expression was seen in the neoplastic cells of follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, splenic marginal zone lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, and nodular lymphocyte predominant Hodgkin lymphoma. In contrast, only 10% of the classical Hodgkin lymphomas showed growth factor receptor-bound protein 2 expression in the neoplastic cells. Growth factor receptor-bound protein 2 protein expression was detected by Western blotting in all lymphoma cell lines tested with higher levels in primary mediastinal large B-cell lymphoma compared with classical Hodgkin lymphoma cell lines. These findings support a role for growth factor receptor-bound protein 2 in the diagnostically challenging workup of classical Hodgkin lymphoma versus primary mediastinal large B-cell lymphoma and warrant further studies to evaluate the biologic significance of growth factor receptor-bound protein 2 in the pathogenesis of classical Hodgkin lymphoma.

Activation of the Endoplasmic Reticulum Stress-Associated Transcription Factor X Box-Binding Protein-1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive B-Cell Lymphomas with Prognostic Implications

X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4(+)/Bcl-6(-)/Pax-5(-) B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas.

A Mechanical Plant Defense Defines the Opening of a Phenological Window for Gall Induction byAsphondylia aucubae(Cecidomyiidae: Diptera)

Many insect herbivores can only use hosts during a specific phenological stage, i.e., a phenological window. Previous studies have primarily examined the effects of these windows on insect herbivores, but relatively little is known about the mechanisms controlling the phenological windows. In most gall insect systems, phenological windows have been attributed to the short duration of physiologically active plant tissues that induce gall formation (reactive plant tissue). In the fruit gall midge, Asphondylia aucubae Yukawa and Ohsaki, and the host plant (i.e., Aucuba japonica) system, the disappearance of reactive plant tissue closes the phenological window, but its presence does not define the opening of the window. The hard endocarp of the fruit covers most potential oviposition sites just before the midge emergence season, but decreases in proportional cover during the emergence season. We experimentally manipulated the timing of oviposition relative to fruit development. Midges that emerged earliest and attacked fruits during their earliest developmental stages were unable to oviposit because of intact, hard endocarps, whereas their counterparts that emerged later could oviposit more readily through cracks in the endocarp. We noted possible oviposition avoidance behavior and the necessity of more frequent (repeated) ovipositor insertions to intensively stimulate the decreased reactive tissues during the latter half of the emergence season. Overall, our results indicated that the fragmentation of the defensive, hard endocarp of the host plant defines the opening of the phenological window in this plant-herbivore system.

Expression of BAFF‐R and TACI in reactive lymphoid tissues and B‐cell lymphomas

The receptor for B-cell-activating factor belonging to the tumour necrosis factor family (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) have been established as binding molecules to BAFF. The aim of this study was to determine the pathological diagnostic roles and clinical significance of these BAFF-binding receptors in B-cell neoplasms. Expression of BAFF-R and TACI was examined immunohistochemically in reactive lymphoid tissues and B-cell lymphomas including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (MZBCL) and plasma cell myeloma (PCM). In reactive tissues BAFF-R was expressed exclusively in the cells of the mantle zone and within the germinal centres (GCs), whereas TACI appeared positive in the scattered cells in extrafollicular areas. There were variable patterns of expression of BAFF-R and TACI amongst the different types of B-cell lymphomas (MCL and FL: BAFF-R+TACI-; PCM: BAFF-R-TACI+; DLBCL and MZBCL: variable expression). Reverse transcriptase-polymerase chain reaction studies supported these results. The overall survival of the BAFF-R+ DLBCL group was significantly better than that of the BAFF-R- group. These results indicate that the assessment of expression of BAFF-binding receptors aids subclassification and prognostication of DLBCL.

Utility of CD26 in flow cytometric immunophenotyping of T‐cell lymphomas in tissue and body fluid specimens

CD26 is expressed by most CD4+ T cells in normal peripheral blood specimens. Neoplastic T cells are frequently CD26- in mycosis fungoides/Sezary syndrome involving the peripheral blood. However, CD26 expression by reactive and neoplastic T cells in solid tissues and body fluids has not been fully characterized by flow cytometry (FC). Solid tissue and body fluid specimens were assayed for CD26 expression using four-color FC immunophenotyping, by qualitative assessment of population clusters, and by quantitation with comparison with isotype controls. Benign T cells were studied in reactive tissues and in the background of other malignancies. Many T-cell lymphomas were dim or negative for CD26, whereas a few were brightly positive. In the majority of T-cell lymphomas, CD26 expression could potentially help identify aberrant population clusters. T cells in reactive tissue specimens and tumor-infiltrating T cells were commonly dim to negative for CD26. Both T-cell lymphomas and reactive T cells in tissue and body fluid specimens often show low levels of CD26 expression. Therefore, quantitative methods may not reliably distinguish benign from neoplastic T cells in these specimens. However, CD26, in combination with other T-cell markers, can be helpful for identifying aberrant population clusters in T-cell lymphomas.

Expression of the interferon regulatory factor 8/ICSBP-1 in human reactive lymphoid tissues and B-cell lymphomas: a novel germinal center marker.

To assess the role of interferon regulatory factor (IRF) 8 in B-cell development and lymphomagenesis, we studied its expression in reactive lymphoid tissues, its relationship to other B-cell transcription factors, and its expression in a series of 232 B-cell tumors and 30 cell lines representing a variety of B-cell developmental stages. We found that although IRF8 was detectable in most reactive B-cells, its expression levels differed with developmental stage. Germinal center B cells contained the highest levels of IRF8, with lower levels seen in mantle and marginal zone B cells and none in plasma cells. IRF8 was coexpressed with PAX-5, Pu.1, and B-cell lymphoma (BCL)-6, and similar to BCL-6, was absent from the small population of IRF4-positive germinal center B cells thought to be committed to postgerminal center developmental programs. Similarly, IRF8 was most strongly expressed in lymphomas of germinal center origin with lower levels present in mantle cell lymphomas, chronic lymphocytic leukemia, and marginal zone lymphomas, and no expression observed in plasmacytic/plasmablastic neoplasms. The reciprocal expression pattern with IRF4 in reactive tissues was generally maintained in lymphomas with some exceptions. These results suggest an important role for IRF8 during germinal center B-cell development and in related lymphomas, and provide a new diagnostic marker helpful in distinguishing B-cell non-Hodgkin lymphoma subtypes.

Increased expression of interferon-gamma in hyperplastic lymph nodes from HIV-infected patients.

Polyclonal B cell activation is characteristic of HIV infection and occurs in the presence of severe CD4+ lymphocyte depletion. In contrast, CD4+ lymphocytes are the dominant T cell in the reactive lymphoid tissues of patients not infected with HIV. In this study, lymph node biopsies from eight HIV-infected patients with persistent generalized lymphadenopathy syndrome (PGL) were assessed for IL-1 beta, IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) gene expression using the polymerase chain reaction (PCR). The cytokine gene expression of two cases of reactive adenopathy in patients not infected with HIV was assessed for comparison. IFN-gamma was expressed much more strongly in the PGL samples than in control reactive lymphoid tissues, whereas the other cytokines were expressed to a similar extent in both types of tissues. IFN-gamma may have an important role in maintaining the adenopathy of HIV-infected patients. Expression of cytokines such as IL-2, IL-4 and IL-10 in HIV nodes may be adequate to allow the recruitment of naive B cells to the reactive process.

Expression of the cap-mediated translation pathway in non-Hodgkins lymphomas: diagnostic and therapeutic implications

8595 Background: The eukaryotic initiation factor (eIF) complex controls cap-mediated mRNA translation. This plays a key role in cancer by mediating expression of proteins critical for cell growth, transformation and tumorigenesis (e.g., cyclin D1, survivin, c-myc, Bcl-2 and VEGF). eIF4 complex (eIF4E, eIF4G and eIF4A) activity is inhibited by eIF4E binding protein-1 (4E-BP1) which thus has tumor suppressor activity. Hyper-phosphorylated 4E-BP1 is unable to bind eIF4E and promotes translation initiation and protein synthesis. Methods: We analyzed eIF complex expression in B-cell non-Hodgkins lymphomas (NHL) using immunohistochemistry (IHC) for total 4E-BP1 and Western immunoblotting (WB) for eIF4G (total), eIF4E and 4E-BP1 (total and phosphorylated). IHC was performed on 37 NHL (9 follicular [FL], 9 diffuse large cell [DLCL], 2 Burkitt’s, 5 mantle cell, 5 extranodal marginal zone/lymphoplasmacytoid, 7 small lymphocytic) and 8 reactive tissues (6 nodes, 2 tonsils). Staining intensity was graded from 0 to 3+. WB was performed on 6 NHL (2 FL, 4 DLCL) and 2 reactive tissues. Results: IHC showed a consistently high level (2–3+) of cytoplasmic 4E-BP1 expression in malignant cells in 36 of 37 NHL samples. In contrast, there was marked regional specificity of cytoplasmic expression in reactive tissues (3+ in the mantle and marginal zone cells but absent/minimal (0- 1+) in germinal centers and T cells). Nuclear expression was seen in 4 NHL samples but not in reactive tissues. Incipient/focal involvement by FL showed 3+ staining in neoplastic follicles. WB showed that phosphorylated:total 4E-BP1 ratio was increased (2-fold over reactive samples) in 3 of 4 DLCL, but not in FL. The levels of expression of eIF4G, total and phospho-eIF4E and total 4E-BP1 were comparable in reactive and NHL samples, as expected in whole tissue lysates. Conclusion: (1) 4E-BP1 detection by IHC offers a sensitive diagnostic tool for distinguishing reactive follicles from neoplastic B-cell proliferations. (2) 4E-BP1 hyper-phosphorylation may be a mechanism that can increase expression of growth and proliferation inducing proteins in aggressive NHL. (3) These findings provide the rationale for evaluating cap-dependent translation inhibition as a novel therapeutic strategy in NHL. No significant financial relationships to disclose.

Intracellular Versican Expression in Mesenchymal Spindle Cell Tumors Contrasts With Extracellular Expression in Epithelial and Other Tumors—A Tissue Microarray-based Study

Versican is a large chondroitin sulfate proteoglycan that is an integral component of the extracellular matrix protein. It regulates cell proliferation, adhesion, and migration, and is expressed in a variety of normal tissues and tumors. We studied the pattern of versican expression in various epithelial, mesenchymal, neural, and hematopoietic tumors using immunohistochemistry on tissue microarrays. The primary antibody used was mouse monoclonal antibody to versican (clone 8S270, 1:4000, US Biological). Sections from 3 healing wounds were also included to demonstrate versican expression in reactive tissues. The extracellular matrix in all tissues including all tumors (epithelial and nonepithelial) was positive for versican. However, intracellular cytoplasmic expression of versican was seen only in spindle cells, for example, fibroblasts in healing wounds, 11 of 16 (69%) gastrointestinal stromal tumors and 12 of 42 (28%) smooth muscle tumors. Intracellular versican was not seen in any other tumor [0/344 carcinomas (64 breast, 63 prostate, 61 colorectal, 59 lung, 68 ovarian, and 29 thyroid), 0/22 glioblastoma multiforme, 0/46 lymphomas, and 0/21 melanomas]. As versican plays a role in cell proliferation, differentiation, adhesion, and migration, its differential expression in spindle cell tumors may be associated with the differentiation, progression, and spread of these tumors, which is different from epithelial tumors.

S2025 Expression of the EGFR Family of Receptors (EGFR, HER-2, HER-3 and HER-4) in Neuroendocrine Tumours

The study of human leukocyte antigens, predominantly by monoclonal antibody (mAb) techniques, has become a fundamental part of basic research and clinical investigation. In particular, mAbs have allowed a more precise phenotypic dissection of lymphocyte subsets and have increased our understanding of the mechanisms that regulate humoral immunity and tumour transformation.In the present study we have investigated the expression, in both reactive and neoplastic lymphoid tissues, of a panel of HLDA9 mAbs (TRAIL-R2 (CD262), CCR6 (CD196), HVEM (CD270), Galectin-3 and BAFF-R (CD268)) capable of recognizing their target molecules in paraffin-embedded tissue sections. A series of reactive lymphoid tissues and B and T cell lymphomas (151 cases) were studied, using whole sections and tissue microarrays (T.M.A.).The most interesting results were obtained from the Galectin-3 study. In human lymphomas our data are consistent with the results previously described that showed that Galectin-3 is expressed in anaplastic large cell lymphoma (ALCL). Moreover, we provide additional information of Galetin-3 expression in other lymphoma types. In T cell lymphomas, Galectin-3 was strongly expressed by a significant number of peripheral (PTCL 12/43) and cutaneous T cell lymphomas (CTCL 6/24) while in B cell lymphoma only a small proportion of follicular (FL 2/10) and diffuse large B cell lymphomas (DLBCL 3/10) were positives.Our study encourage further investigations into the potential role that TRAIL-R2, CD196, HVEM, Galectin-3 and BAFF-R proteins may play in lymphocyte development and differentiation, but also constitute an additional tool for the study of lymphoid subpopulations and lymphoproliferative disorders.

Therapeutic anti-inflammatory mechanism of action of Type I interferons

Interferon-beta is used for the treatment of relapsing-remitting multiple sclerosis and other Type I interferons are being studied as potential immune therapies for immune disorders. Yet, the therapeutic anti-inflammatory mechanism of action remains to be clearly elucidated. Herein described is a novel model for Type I interferon therapeutic anti-inflammatory mechanism of action. When administered orally or subcutaneously at a site distal to inflammation, Type I interferons induce a partial state of immune activation through the expression of proinflammatory factors counter-regulated by the expression of tolerance inducing factors in antigen presenting cells proximal to the site of administration. Disease-associated T-cells are recruited to and interact with interferon affected antigen presenting cells, and in the context of non-immune reactive lymphoid tissues, the immature antigen presenting cells induce T-cell apoptosis, anergy, and the conversion to and the de novo expansion of T-regulatory cell populations. The result is a systemic reduction of disease-associated T-cells with a proportional increase in T-regulatory cells and tolerance mediating antigen presenting cells. T-regulatory cells are then recruited to sites of tissue inflammation and engage inflammation associated activated antigen presenting cells, down-regulating activation to produce a therapeutic benefit.

Activation of the Endoplasmic Reticulum (ER) Unfolded Protein Response (UPR) in Aggressive B-Cell Lymphomas.

BACKGROUND The UPR is a prosurvival pathway activated in cells under ER stress induced by the accumulation of unfolded proteins. UPR activation in B cells normally occurs during the differentiation to antibody secreting plasma cells and requires XBP1activation. XBP-1 is a member of the TREB family of transcription factors that exists in the endoplasmic reticulum (ER) as a 33kDa protein, and in the nucleus as an active 50kDa transcription factor. The UPR stimulates two different ER proteins, ATF-6 and Ire-1, to increase XBP-1 transcription and XBP-1 mRNA splicing resulting in the accumulation of the active 50kDa nuclear protein. Moreover XBP1 is a target of proteosome inhibitors and is related to the aggressive behaviour of some carcinomas. The role of the activation of XBP-1 in lymphomas is still unknown.DESIGN: Reactive lymphoid tissues and 25 neoplastic human B-cell lines representing different stages of B-cell development were studied for XBP-1 expression by western blot and XBP-1, PAX-5, Blimp-1/prdm1, MUM-1/IRF-4 and ICSBP1/IRF-8 by immunohistochemistry. XBP-1 activation was assessed in 225 B-cell lymphomas from the archives of the laboratory of pathology by western blot, RT-PCR and immunohistochemistry . To further evaluate whether XBP-1 activation was related to the plasmacytic program or to ER stress signals we analyzed the cell lines by Western blot for XBP-1 and ATF-6 expression.RESULTS We characterize XBP-1 expression in reactive lymphoid tissues, 25 human cell lines and 225 B-cell tumors. In nearly all tonsillar lymphoid cells XBP-1 was detected as a cytoplasmic protein with a paranuclear dot pattern. Nuclear positivity was observed only in scattered centrocytes in the light zone of the germinal centers and in plasma cells, always coexpressed with plasma cell related transcription factors as MUM-1/IRF-4 and Blimp1/prdm1. Active p50XBP-1 was found in 24/25 cell lines by western blot regardless ATF-6 expression and confirmed by immunohistochemistry . Moreover p50XBP1 was found in 27/31(87%) plasmacytomas, 36/64(56%) DLBCL-ABC and in 3/10(30%) DLBCL-GCB and 22/43(51%) plasmablastic lymphomas. Intriguingly, p50XBP1 was detected also in 2/11(18%)BL and 4/25(16%)MCL with blastic features.CONCLUSIONS.XBP-1 is activated in a subset of follicular centre cells committed to plasma cell differentiation and in plasma cells.UPR prosurvival pathways in the neoplastic cell lines are activated independently of the extent of the ATF-6 activation.p50XBP1 is mostly activated in aggressive B-cell lymphomas regardless to the plasmacytic differentiation of the tumours.Thus, p50XBP-1 may be a new molecular target in the treatment of aggressive B-cell malignancies.

Aberrant Expression of IL-2R in Pediatric Anaplastic Large Cell Lymphoma: Cellular and Proteomic Analysis of Denileukin Diftitox (ONTAK) as a Potential Therapeutic Agent.

Constitutive expression of the NPM/ALK tyrosine kinase is a key oncogenic event in the pathogenesis of anaplastic large cell lymphoma (ALCL). Our previous cDNA microarray analysis of pediatric ALCLs obtained from the Cooperative Human Tissue Network (CHTN) demonstrated overexpression of IL-2Rγ in NPM/ALK-positive ALCLs relative to NPM/ALK-negative lymphomas and reactive lymphoid tissues. Furthermore, the mRNA levels of IL-2Rγ were overexpressed in Jurkat cells transfected with NPM/ALK relative to the vector-transfected cells.To determine the feasibility of targeting IL-2R by ONTAK for treatment of pediatric ALCLs, we determined the prevalence of IL-2R expression in pediatric ALCLs and correlated its expression with that of the ALK protein. We used formalin-fixed paraffin-embedded tissues of ALCLs obtained from the CCG5941 clinical trial involving the pediatric population and determined the expression of ALK and CD25 (IL-2R) using immunohistochemistry. The effect of ONTAK on the viability of 5 cell lines derived from t(2;5)-positive ALCLs was assessed using an assay based on the cleavage of the tetrazolium salt WTS-1 by mitochondrial dehydrogenases. Western blot analysis was performed to determine the effect of ONTAK on cell cycle proteins and apoptosis. We utilized an endoproteinase-catalyzed 16O/18O differential isotopic strategy to quantitatively determine the global proteomic sequelae of IL-2R inhibition using ONTAK. Proteins were collected from the cell lysates of treated and non-treated SUDHL1 cells, subjected to 16O/18O labeling and then analyzed by reverse-phase liquid chromatography and electrospray ionization tandem mass spectrometry.Analysis of 40 cases of ALCL revealed nuclear and cytoplasmic expression of ALK in the majority of cases. Strong membranous and cytoplasmic expression of CD25 was present in 27/40 (77%) cases while the reactive lymphocytes showed negligible or weak expression. Treatment of SUDHL-1 cells with ONTAK resulted in time and dose-dependent decrease in cell proliferation as measured by the WTS assay which was associated with induction of p27Kip1 and cleavage of PARP. A total of 253 proteins with 2 or more unique peptides were identified as being differentially expressed between treated and non-treated SUDHL-1 cells. Proteins of diverse location and function were identified. Importantly a large number of proteins known to be altered by diphtheria toxin and important in protein translation were underexpressed in treated cells (eIF2C, hnRNP, BRCA1, CREG, NuMA, 60S ribosomal protein L8). In addition novel proteins with transcriptional and cell signaling activities were identified, representing unique pathways that may be affected by IL-2 signaling.Our studies demonstrate the constitutive expression of CD25 in the majority of pediatric ALK positive ALCLs. Our quantitative proteomic studies reveal novel insights into the signaling pathways involved in IL-2 signaling in ALCL cells. Our in vitro studies indicate that targeting of CD25 by ONTAK may represent a rational approach for treatment of pediatric patients with ALCL.

Retinoblastoma gene family expression in lymphoid tissues

It appears more and more clear that retinoblastoma (RB) family of proteins represents key molecules in tumour suppression. This family consists of pRb/p105, p107 and pRb2/p130, which participate in a gene regulatory network that governs the cellular response to antimitogenic signals, and whose deregulation constitutes one of the hallmarks of cancer. Irrespective of their structural and biochemical similarities, RB proteins carry out different functional tasks. The expression of RB gene family in the reactive lymphoid tissues again confirms the different role of each member in cell cycle control and differentiation of normal cells. These different functional properties appear to be maintained in tumours lymphoid tissues, where alterations of the RB/p105 gene appear to be relatively rare. In this review, we will summarize the current knowledge about the role of the RB proteins in reactive and neoplastic lymphoid tissue.

PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: Diagnostic value of immunoglobulin κ and λ light chain gene rearrangement investigation

Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Ig κ) and lambda (Ig λ) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Ig κ, and Ig λ PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

FISH detection of t(14;18) in follicular lymphoma on Papanicolaou-stained archival cytology slides

The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou-stained archival cytology material as a practical ancillary technique for diagnosing FL. Cases included 35 FL, 6 small lymphocytic lymphomas/chronic lymphocytic leukemias (SLL/CLL), 4 mantle cell lymphomas (MCL), 4 marginal zone lymphomas (MZL), 1 lymphoplasmacytic lymphoma (LPL), and 10 reactive lymphoid tissues (RLT). FISH was performed on Papanicolaou-stained archival cytology slides using probes for immunoglobulin heavy chain (IGH) on chromosome 14 and BCL2 on chromosome 18. In all, 25 of 32 (81%) FL cases exhibited the t(14;18) translocation, whereas 7 of 32 (19%) lacked the translocation. No cases of non-FL were positive for t(14;18). This series shows a sensitivity of 81% and specificity of 100% for detecting the t(14;18) translocation as a diagnostic tool in FL. When performed on Papanicolaou-stained cytology slides, FISH for t(14;18) is relatively sensitive and quite specific for FL. These findings are similar to those reported on other specimens, such as paraffin-embedded tissue and unstained cytology slides. The authors proposed that their technique would allow the pathologist and clinician the flexibility to utilize previously stained fine-needle aspiration slides for FISH evaluation.