Connective tissue growth factor is expressed in malignant cells of Hodgkin lymphoma but not in other mature B-cell lymphomas.

Connective tissue growth factor (CTGF) has a major role in development of fibrosis and in the wound-healing process. Microarray analysis of 44 classical Hodgkin lymphoma (cHL) samples showed higher CTGF messenger RNA expression in the nodular sclerosis (NS) than in the mixed cellularity (MC) subtype. When analyzed by immunohistochemical analysis, Hodgkin-Reed-Sternberg (H-RS) cells and macrophages in 23 cHLs and "popcorn" cells in 2 nodular lymphocyte predominant Hodgkin lymphomas showed expression of CTGF protein correlating with the extent of fibrosis. In NS, CTGF was also expressed in fibroblasts and occasional lymphocytes. Malignant cells in 32 samples of various non-Hodgkin lymphomas were negative for CTGF. A staining pattern of stromal cells similar to that of NS cHL was seen in anaplastic large cell lymphoma. Macrophages stained positively in Burkitt lymphomas and in some mantle cell lymphomas. The high occurrence of fibrosis in cHL may be related to CTGF expression by malignant H-RS cells.

Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin's lymphoma.

BACKGROUND: Classical Hodgkin's lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment. METHODS: We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry. RESULTS: In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qalpha, C1Qbeta and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages. CONCLUSIONS AND INTERPRETATIONS: We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment.

Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells.

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern.

To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model. We used a novel designer peptide based self-organizing matrix. The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples. To validate our results we also included a gene expression data set of laser captured Hodgkin-Reed - Sternberg (H-RS) cells. The gene expression profiles were analyzed using Affymetrix technology. We found that the 3D culture affected gene expression of a HL derived cell-line inducing a more tumor-related expression profile. 3D culture affected the expression of 500 genes in L1236, upregulating genes involved in immune response and apoptosis and downregulating genes involved in cell division. It also affected genes involved in actin filament polymerization.

Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells.

A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10(7) EBV-specific cytotoxic T-lymphocytes (CTLs)/m(2) starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.

DNA of lymphoma-associated herpesvirus (HVMF1) in SIV-infected monkeys (Macaca fascicularis) shows homologies to EBNA-1, -2 and -5 genes.

We have characterized a new Epstein-Barr-virus(EBV)-like herpesvirus associated with lymphomas of SIV-infected cynomolgus (Macaca fascicularis) monkeys and propose that this virus is designated herpesvirus macaca fascicularis I (HVMFI). Genomic regions in HVMF1 of potential significance for tumor pathogenesis were analyzed by Southern blotting, PCR and sequencing, and compared with human EBV DNA. Virus from 7 SIV-associated lymphomas and one lymphoma-derived cell line were shown to share homology with the EBNA1- and EBNA2-coding regions of EBV, while some homology to EBV-LMP1 was detectable only at low-stringency hybridization. Homologous regions to the long internal repeat (IR1; BamHI W), the EBER1 and 2 and the latent origin of DNA replication (oriP) could also be demonstrated in HVMF1. These coding regions, except IR1, showed restriction-enzyme maps different from those of EBV. Sequencing of the EBNA5 homologous region of HVMF1 DNA, corresponding to exons W1 and W2, showed 65% homology to the EBV exons W1 and W2, and 80% to the whole region including the intron. Since EBNA5 has been reported to bind tumor-suppressor proteins p53 and Rb in vitro, the HVMF1 homology could be important for the lymphomagenic capacity of this monkey herpesvirus.

A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.

High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.

Relationship between clinical stage, histopathology and antibody titers against the second Epstein-Barr virus nuclear antigen (EBNA-2) in non-Hodgkin's lymphoma patients.

Non-Hodgkin lymphoma (NHL) patients with centroblastic (Cb) or centroblastic-centrocytic (Cb/Cc)-diffuse lymphomas, immunocytoma (IC) and chronic lymphocytic leukemia (CLL) in clinical stages III-IV and with active disease (highly malignant group) were compared to NHL patients with CLL, IC, and centrocytic (Cc) or centroblastic-centrocytic (Cb-Cc)-diffuse/follicular lymphomas, in clinical stages I-II and with inactive disease (low malignant group) based on the presence of antibodies to Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and 2 (EBNA-2). In the highly malignant group, anti-EBNA-1 geometric mean titers (GMT) were 13.2 (range less than 2-80) and anti-EBNA-2 60.6 (range: 20-320). The ratio between the logarithms of anti-EBNA-1 and anti-EBNA-2 antibody titers was less than 1.0 (mean: 0.32) in all the patients examined. In 6 out of 8 patients of the low malignant group, anti-EBNA-1 titers were higher (mean: 30.1; range 10-160) than anti-EBNA-2 titers (mean: 4.3; range less than 2-80) and the EBNA 1/2 ratio was greater than 1.0. In healthy EBV-seropositive individuals, anti-EBNA-1 GMT were 49 (range: 10-320) and only 5 out of 17 individuals had detectable anti-EBNA-2 titers (GMT: 3; range less than 5-20). The EBNA-1/2 ratio was in all cases greater than 1. Among patients of the highly and low malignant groups, patients with follicular-cell-derived lymphomas had elevated antibody titers against the restricted component of early antigens (EA-R), whereas all patients with IC and 2 out of 4 CLL patients had elevated antibody titers against the diffuse component of early antigens (EA-D). The results indicate that the ratio between anti-EBNA-1 and anti-EBNA-2 antibody titers may be of diagnostic importance in patients with immunodeficiencies.

The role of Epstein-Barr virus in lymphomas of HIV-carriers.

A substantial proportion of the lymphomas in HIV-carriers are EBV positive. Together with the fact that there are multiple signs of EBV-activation in AIDS-patients and patients with ARC or PGL, this suggests that these virus carrying tumors develop as results of the immunosuppression, and that EBV has an important role in their pathogenesis.

Analysis of Epstein-Barr virus-specific and non-specific immune functions in a patient during the development of a non-Hodgkin's lymphoma.

A 57-year-old woman who presented with a reactive non-malignant lymphadenopathy was observed subsequently during the development of a nodular centroblastic non-Hodgkin's B-cell lymphoma. The Epstein-Barr virus (EBV)-specific antibody profile and EBV-specific and non-specific cell-mediated immune functions were determined at first presentation, and at various times during progression, in order to determine whether EBV was causally involved in the lymphoma and to assess in general the patient's cell-mediated immune function. At presentation, an immunodeficient status was suggested by an EBV-specific antibody profile indicative of an activated persistent infection; high antibody titers to viral capsid antigen (VCA) and early antigens (EA), but a low level of antibodies to EBV nuclear antigen (EBNA) confirmed by lack of leukocyte migration inhibition in response to EBNA (LMI-EBNA). The number of positive cells reactive with OKIa1 monoclonal antibody was significantly depressed, as was also the natural and interferon-activated killing (NK-IAK). After emergence of the lymphoma, NK-IAK reactivity and spontaneous lymphocyte DNA synthesis augmented in parallel with an increase in the frequency of Leu-7+ blood lymphocytes. The EBV-specific cell-mediated response, reflected by the outgrowth inhibition (OI) test was abolished in parallel with a decrease in the frequency of OKT3- and OKT4-positive lymphocytes.

Capacity of B-lymphocytic lines of diverse tumor origin to produce and respond to B-cell growth factors: a progression model for B-cell lymphomagenesis.

Human cell lines established from cases of acute lymphoblastic leukemia, lymphosarcoma, Burkitt's lymphoma and multiple myeloma and representing stages of B-lymphocyte development ranging from pre-B through to plasma cells, were assessed for their ability to produce and respond to B-cell growth factors (BCGF). All B-cell lines studied were found to be constitutive producers of a growth activity which assisted the S-phase entry of normal activated B-cells and provided growth support for lymphoblastoid cells transformed by Epstein-Barr virus. Furthermore, all lines responded by enhanced proliferation to supernatants from a BCGF-producing T-cell hybridoma. Not all lines, however, displayed autostimulation to their own supernatants and no tumor B-cell line appeared totally dependent on soluble factors for its growth. Non-tumorigenic B-cell lines, by contrast, revealed a strict dependency on homologous growth factor for their continued proliferation in suspension culture. The findings support a progression model of lymphomagenesis based upon the utilization, production and, ultimately, emancipation from growth-promoting soluble factors.

Immunological characterization of Hodgkin's and non-Hodgkin's lymphoma patients with high antibody titers against Epstein-Barr virus-associated antigens.

We have studied nine Hodgkin's lymphoma (HD) and ten non-Hodgkin's lymphoma (NHL) patients with extraordinarily high anti-viral capsid antigen (VCA) titers (greater than 5120). Controls were 13 HD and 23 NHL patients with anti-VCA titers between 40 and 2560. High anti-VCA titers were present in NHL patients at the time of diagnosis or within 16 months, whereas the rise of anti-VCA titers in HD patients appeared to be a late event during the clinical course of the disease (mean time from diagnosis, 68 months). In particular, we have asked whether the exceptionally high anti-Epstein-Barr virus (EBV) titers in some HD and NHL patients can be correlated to some of the EBV-specific and -nonspecific parameters of cell-mediated immunity. The battery of non-EBV-specific immunological tests included the assessment of natural killer cell activity and the analysis of T-lymphocyte subclasses according to surface markers, together with spontaneous and mitogen-induced DNA synthesis and their helper or suppressor activity on PWM-generated immunoglobulin synthesis. Outgrowth inhibition (Ol) and leukocyte migration inhibition were used to assess EBV-specific cell-mediated immunity. The majority of the high-titer HD and NHL patients showed a drastically reduced OKT4:OKT8 ratio in their peripheral lymphocyte population. Low-titer HD and NHL patients showed no such reduction. There was no strict correlation between the number of OKT8-positive cells and suppressor activity in the functional PWM-induced immunoglobulin production test. Part of the high-titer HD patients showed defective cellular responses in the outgrowth inhibition test, directed against the proliferation of EBV-transformed (EBV-determined nuclear antigen-positive) cells. Some of them showed also a deficient leukocyte migration inhibition response to EBV-determined nuclear antigen but, interestingly, not to early antigen-VCA. In the NHL group, only one of the high-titer patients showed a similar defect. None of the low-titer HD and NHL patients showed such defects.

Human lymphoma-lymphoma hybrids and lymphoma-leukemia hybrids. II. Epstein-Barr virus induction patterns.

In addition to the spontaneous expression of markers of the early and late phases of the viral cycle [Epstein-Barr virus (EBV), early viral cycle antigens (EA), and viral capsid antigen (VCA)] by a Panel of hybrid cell lines derived by fusion of human hematopoietic cell lines, the induction of these markers by three chemical inducers [5-iodo-2'-deoxyuridine, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), and sodium butyrate] were analyzed. A variant of the prototype producer of lytic EBV, P3HR-1 (PICAT) was observed to show consistent low spontaneous and induced production of EA and VCA as compared to the P3HR-1 line from which it was derived. An "autohybrid" (PICATPO-1) between these two lines showed a low production of VCA. Hybrids between Raji and P3HR-1 sublines (RUD-PICAT-1 and RUDPUT-2) showed reduced expression of EBV antigens. Both hybrids were capable of VCA expression, in contrast to the Raji parent that expressed EA but not VCA. A new Raji-Daudi hybrid (DITRUD-1) expressed spontaneous and induced EA and VCA at levels intermediate between its two parents. A different type of hybrid was derived from a Daudi subline and the K562 leukemia cell line (DUTKO-1) and was found to be capable of spontaneous as well as induced EA and VCA expression. Both DUTKO-1 and DITRUD-1 were similar to Daudi in their induction profile in respect to a very low response to TPA.

Cell-mediated immune reactions in three patients with malignant lymphoproliferative diseases in remission and abnormally high Epstein-Barr virus antibody titers.

Two patients with Hodgkin's disease in remission and one chronic lymphatic leukemia patient with extraordinarily high anti-Epstein-Barr virus (EBV) (viral capsid antigen) antibody titers (greater than 10,000) were selected to study a spectrum of cell-mediated immune responses, including natural killer, interferon-boosted killer, antibody-dependent lymphocytotoxicity, and T-cell-mediated reactions. The purpose was to compare these reactions in patients with immunosuppression and a high EBV load who can hold their EBV-carrying cells under control with the corresponding reactions in patients with EBV-carrying lymphoproliferative disease. In contrast to the latter group, the three patients of the present study showed a less profound and less general suppression of the immune responses. Multiple effector mechanisms probably safeguard against the proliferation of EBV-transformed B-cells. Clinically manifest EBV-carrying lymphoproliferative disease occurs only in very severe immunodeficiencies effecting multiple effectors.