Ioannis Anagnostopoulos

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells

CD40 ligand (CD40L, CD154), a transmembrane protein structurally related to the cytokine TNF-α, was originally identified on stimulated CD4+ T cells, and later on stimulated mast cells and basophils. Interaction of CD40L on T cells with CD40 on B cells is of paramount importance for the development and function of the humoral immune system. CD40 is not only constitutively present on B cells, but it is also found on monocytes, macrophages and endothelial cells, suggesting that CD40L has a broader function in vivo. We now report that platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo. Like TNF-α and interleukin-1, CD40L on platelets induces endothelial cells to secrete chemokines and to express adhesion molecules, thereby generating signals for the recruitment and extravasation of leukocytes at the site of injury. Our results indicate that platelets are not only involved in haemostasis but that they also directly initiate an inflammatory response of the vessel wall.

Lymphoproliferative lesions of the ocular adnexa: Analysis of 112 cases

OBJECTIVE Lymphoproliferative lesions of the ocular adnexa were analyzed to examine (1) the suitability of the Revised European-American Lymphoma (REAL) classification for the subtyping of the lymphomas in these sites; (2) the predictive value of the REAL classification for the evolution of these tumors; and (3) the frequency and prognostic impact of tumor type, location, proliferation rate (Ki-67 index), p53, CD5 positivity and the presence of monoclonality within these tumors. DESIGN Retrospective review. METHODS The clinical, histomorphologic, immunohistochemical, and molecular biologic (polymerase chain reaction [PCR]) features of lymphoid proliferations of the ocular adnexa were studied. STUDY MATERIALS: The ocular adnexal lymphoproliferative lesions were located as follows: orbit in 52 patients (46%), conjunctiva in 32 patients (29%), eyelid in 23 patients (21%), and caruncle in 5 patients (4%). RESULTS Reactive lymphoid hyperplasia was diagnosed in 12 cases and lymphoma in 99 cases; 1 case remained indeterminate. The five main subtypes of lymphoma according to the REAL classification were extranodal marginal-zone B-cell lymphoma (64%), follicle center lymphoma (10%), diffuse large cell B-cell lymphoma (9%), plasmacytoma (6%), and lymphoplasmocytic lymphoma (5%). Age, gender, and anatomic localization of the lymphomas did not have prognostic significance during a follow-up period of 6 months to 16.5 years (mean, 3.3 years). Extent of disease at time of presentation was the most important clinical prognostic factor: advanced disease correlated with increased risk ratios of having persistent disease at the final follow-up and with lymphoma-related death (P < 0.001). Histomorphologic features and immunohistochemical markers positively correlating with disseminated disease at presentation, stage at final follow-up, and occurrence of lymphoma-related death included cytologic atypia (P < 0.001), MIB-1 proliferation rate (P < 0.001), and tumor cell p53 positivity (P < 0.001). The MIB-1 proliferation rates greater than 20% in extranodal marginal-zone B-cell lymphoma corresponded to at least stage II lymphoma (P < 0.05). CONCLUSION The REAL classification is suitable for the subdivision of the ocular adnexal lymphomas. The MIB-1 proliferation rate and p53 positivity may aid the prediction of disease stage and disease progression, whereas PCR can support the diagnosis and reduce the number of histologically indeterminate lesions.

Aberrantly expressed c‐Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF‐κB

AP‐1 family transcription factors have been implicated in the control of proliferation, apoptosis and malignant transformation. However, their role in oncogenesis is unclear and no recurrent alterations of AP‐1 activities have been described in human cancers. Here, we show that constitutively activated AP‐1 with robust c‐Jun and JunB overexpression is found in all tumor cells of patients with classical Hodgkins disease. A similar AP‐1 activation is present in anaplastic large cell lymphoma (ALCL), but is absent in other lymphoma types. Whereas c‐Jun is up‐regulated by an autoregulatory process, JunB is under control of NF‐κB. Activated AP‐1 supports proliferation of Hodgkin cells, while it suppresses apoptosis of ALCL cells. Furthermore, AP‐1 cooperates with NF‐κB and stimulates expression of the cell‐cycle regulator cyclin D2, proto‐oncogene c‐met and the lymphocyte homing receptor CCR7, which are all strongly expressed in primary HRS cells. Together, these data suggest an important role of AP‐1 in lymphoma pathogenesis.

Origin of Nodular Lymphocyte-Predominant Hodgkin's Disease from a Clonal Expansion of Highly Mutated Germinal-Center B Cells

BACKGROUND The atypical cells of nodular lymphocyte-predominant Hodgkins disease, designated lymphocytic and histiocytic (L&H) cells, have a B-cell phenotype. To clarify the clonality of these cells, we studied rearranged immunoglobulin genes for the variable region of the heavy chain (V[H] genes) in individual L&H cells from 11 patients with nodular lymphocyte-predominant Hodgkins disease. We also studied the expression of immunoglobulin light chains by those cells in six of the same patients. METHODS Single CD20+ L&H cells were isolated from frozen sections by a technique of micromanipulation. The rearranged V(H) genes of these cells were amplified by the polymerase chain reaction (PCR), sequenced, and compared with germ-line V(H) genes. Immunoglobulin light-chain messenger RNA (mRNA) was detected by in situ hybridization. RESULTS Of 615 L&H cells isolated from all the frozen sections, 160 yielded PCR products. In each of the 11 patients, the L&H cells that could be evaluated had identically rearranged V(H) genes, whether they were isolated from the same nodule, different nodules, or different blocks of tissue. All the V(H) sequences derived from the L&H cells were highly mutated (7.5 to 27.2 percent). In two cases the coding capacity of the V(H) genes was completely or partially disrupted by mutations. Intraclonal diversity was found in six cases, and monotypic immunoglobulin light-chain mRNA was found in six. CONCLUSIONS The L&H cells of nodular lymphocyte-predominant Hodgkins disease represent a monoclonal expansion of B cells. The high load of V(H) gene mutations and signs of intraclonal diversity suggest a relation between L&H cells and germinal-center B cells at the centroblastic stage of differentiation.

Nuclear Factor κB–dependent Gene Expression Profiling of Hodgkin's Disease Tumor Cells, Pathogenetic Significance, and Link to Constitutive Signal Transducer and Activator of Transcription 5a Activity

Constitutive nuclear nuclear factor (NF)-κB activity is observed in a variety of hematopoietic and solid tumors. Given the distinctive role of constitutive NF-κB for Hodgkin and Reed-Sternberg (HRS) cell viability, we performed molecular profiling in two Hodgkins disease (HD) cell lines to identify NF-κB target genes. We recognized 45 genes whose expression in both cell lines was regulated by NF-κB. The NF-κB–dependent gene profile comprises chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors, the majority of which maintain a marker-like expression in HRS cells. Remarkably, we found 17 novel NF-κB target genes. Using chromatin immunoprecipitation we demonstrate that NF-κB is recruited directly to the promoters of several target genes, including signal transducer and activator of transcription (STAT)5a, interleukin-13, and CC chemokine receptor 7. Intriguingly, NF-κB positively regulates STAT5a expression and signaling pathways in HRS cells, and promotes its persistent activation. In fact, STAT5a overexpression was found in most tumor cells of tested patients with classical HD, indicating a critical role for HD. The gene profile underscores a central role of NF-κB in the pathogenesis of HD and potentially of other tumors with constitutive NF-κB activation.

Effect of anti-CD 20 antibody rituximab in patients with post-transplant lymphoproliferative disorder (PTLD)

Post‐transplant lymphoproliferative disorders (PTLD) are a life‐threatening complication following solid organ transplantation. Treatment with rituximab, a humanized anti‐CD20 monoclonal antibody, has proved to be a promising approach and shown a low toxicity profile. Between February 1999 and April 2002, we conducted a multicentre phase II trial investigating rituximab as single agent in 17 patients with PTLD. Transplanted organs were heart (n = 5), kidney (n = 4), lung (n = 4) and liver (n = 4). Patients were treated with four weekly doses of 375 mg/m2 of rituximab. The mean follow‐up time is 24.2 months. Histology was distributed in 10 diffuse large cell‐, 2 marginal zone‐, 1 Burkitt‐like lymphoma, 1 Hodgkin‐like PTLD and 3 polymorphic lymphoproliferations. Therapy was well tolerated and no severe adverse events were observed. The mean overall survival period is 37.0 months with 11 patients still living. In total, 9 patients (52.9%) achieved a complete remission, with a mean duration of 17.8 months. Partial remission was observed in 1 patient, minor remission in 2 patients, no change in 3 patients and 1 patient experienced progressive disease. Two patients relapsed, at intervals 3 and 5 months after obtaining complete remission. Rituximab proved to be well tolerated and effective in the treatment of PTLD.

c-FLIP Mediates Resistance of Hodgkin/Reed-Sternberg Cells to Death Receptor–induced Apoptosis

Resistance to death receptor–mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkins lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain–containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1β–converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-κB. Despite expression of other NF-κB–dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell–derived Hodgkins lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkins lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.

Immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma: dissociation between phenotype and genotype.

We have determined the tumor cell immunophenotype and the rearrangement configuration of immunoglobulin and T-cell receptor genes in 39 cases of Hodgkins disease (HD), six HD-derived cell lines and 22 cases of Ki-1-positive anaplastic large cell lymphomas (Ki-1-ALC). Rearrangements were observed in 11/39 HD cases, 15/22 Ki-1-ALC, and all cell lines. Epstein-Barr virus DNA was found in five HD cases, one cell line, and one Ki-1-ALC. Both HD and Ki-1-ALC frequently displayed a dissociated genotypic and phenotypic maturation status, i.e. an immature genotype in association with late activation markers. We postulate that the tumor cells in many cases of HD and some cases of Ki-1-ALC may be derived from immature lymphoid cells by a transformation process that superimposes characteristics of mature activated lymphocytes on these cells.

Sequential treatment with rituximab followed by CHOP chemotherapy in adult B-cell post-transplant lymphoproliferative disorder (PTLD): the prospective international multicentre phase 2 PTLD-1 trial.

BACKGROUND Post-transplantation lymphoproliferative disorder (PTLD) develops in 1-10% of transplant recipients and can be Epstein-Barr virus (EBV) associated. To improve long-term efficacy after rituximab monotherapy and to avoid the toxic effects of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy seen in first-line treatment, we initiated a phase 2 trial to test whether the subsequent use of rituximab and CHOP would improve the outcome of patients with PTLD. METHODS In this international multicentre open-label phase 2 trial, treatment-naive adult solid-organ transplant recipients diagnosed with CD20-positive PTLD who had failed to respond to upfront immunosuppression reduction received four courses of rituximab (375 mg/m(2) intravenously) once a week followed by 4 weeks without treatment and four cycles of CHOP every 3 weeks. In case of disease progression during rituximab monotherapy, CHOP was started immediately. Supportive therapy with granulocyte-colony stimulating factor after chemotherapy was mandatory and antibiotic prophylaxis was recommended. The primary endpoint was treatment efficacy measured as response rates in all patients who completed treatment with rituximab and CHOP, per protocol, and response duration, in all patients who completed all planned therapy and responded. Secondary endpoints were frequency of infections, treatment-related mortality, and overall survival. This study is registered at ClinicalTrials.gov, number NCT01458548. FINDINGS 74 patients were enrolled between Dec 12, 2002 and May 5, 2008, of whom 70 patients were eligible to receive treatment. PTLD was of late type in 53 (76%) of 70 patients, monomorphic in 67 (96%) of 70, and histologically EBV associated in 29 (44%) of 66 cases. Four of 70 patients did not receive CHOP. 53 of 59 patients had a complete or partial response (90%, 95% CI 79-96), of which 40 (68%, 55-78) were complete responses. At data cutoff (June 1, 2011) median response duration in the 53 patients who had responded to treatment had not yet been reached (>79·1 months). The main adverse events were grade 3-4 leucopenia in 42 of 62 patients (68%, 55-78) and infections of grade 3-4 in 26 of 64 patients (41%, 29-53). Seven of 66 patients (11%, 5-21) had CHOP-associated treatment-related mortality. Median overall survival was 6·6 years (95% CI 2·8-10·4; n=70). INTERPRETATION Our results support the use of sequential immunochemotherapy with rituximab and CHOP in PTLD. FUNDING F Hoffmann-La Roche, Amgen Germany, Chugaï France.