Sibrand Poppema

BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas.

In a previous study we demonstrated high expression of the non‐coding BIC gene in the vast majority of Hodgkins lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA‐155 (miR‐155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR‐155 is also expressed in HL. High levels of miR‐155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non‐HL subtypes were negative for BIC as determined by RNA‐ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell‐like phenotype than in DLBCL with germinal centre B cell‐like phenotype. Differential BIC expression was confirmed by qRT‐PCR analysis. Northern blot analysis showed expression of miR‐155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR‐155 in HL, PMBL and DLBCL. Copyright

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

High expression of the CC chemokine TARC in Reed-Sternberg cells : A possible explanation for the characteristic T-cell infiltrate in Hodgkin's lymphoma

Hodgkins lymphoma is characterized by the combination of Reed-Sternberg (R-S) cells and a prominent inflammatory cell infiltrate. One of the intriguing questions regarding this disease is what is causing the influx of T lymphocytes into the involved tissues. We applied the serial analysis of gene expression (SAGE) technique on the Hodgkins lymphoma-derived cell line L428 and on an Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell line. A frequently expressed tag in L428 corresponded to the T-cell-directed CC chemokine TARC. Reverse transcription polymerase chain reaction analyses demonstrated expression of TARC in nodular sclerosis (NS) and mixed cellularity (MC) classical Hodgkins lymphomas but not in NLP Hodgkins lymphoma, anaplastic large-cell lymphomas, and large-B-cell lymphomas with CD30 positivity. Two of five cases of T-cell-rich B-cell lymphoma (TCRBCL) were TARC positive. RNA in situ hybridization (ISH) showed a strong signal for TARC in the cytoplasm of R-S cells, and immunohistochemical staining confirmed the presence of the TARC protein in the R-S cells of NS and MC Hodgkins lymphomas. The lymphocytic and histiocytic (L&H)-type cells of nodular lymphocyte predominance Hodgkins lymphoma and the neoplastic cells of non-Hodgkins lymphomas with the exception of two cases of TCRBCL did not stain for TARC. TARC is known to bind to the CCR4 receptor, which is expressed on activated Th2 lymphocytes. The immunophenotype of lymphocytes surrounding R-S cells is indeed Th2-like, and by RNA ISH these lymphocytes showed a positive signal for the chemokine receptor CCR4. The findings suggest that production of TARC by the R-S cells may explain the characteristic T-cell infiltrate in classical Hodgkins lymphoma.

High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma.

In a search for genes specifically expressed in Reed–Sternberg (RS) cells of Hodgkin lymphoma (HL), we applied the serial analysis of gene expression (SAGE) technique on the HL‐derived cell line DEV. Genes highly expressed in DEV were subjected to an RT‐PCR analysis to confirm the SAGE results. For one of the genes, a high expression was observed in DEV and other HL‐derived cell lines but not in non‐Hodgkin lymphoma (NHL)–derived cell lines and normal controls, suggesting an HL‐specific expression. This gene corresponds to the human BIC gene, a member of the noncoding mRNA‐like molecules. RNA in situ hybridization (ISH) indicated an exclusive nucleolar localization of BIC transcripts in all RS cells in 91% of HL cases, including nodular lymphocyte predominance (NLP) HL and classical HL. Analyses of normal human tissues revealed BIC transcripts in only a small number of CD20‐positive B‐cells in lymph node and tonsil tissue, albeit at a much lower level compared to that of RS cells. BIC RT‐PCR in the Burkitt lymphoma–derived cell line Ramos demonstrated a significant up‐regulation upon cross‐linking of the B‐cell receptor (BcR). IκBα‐mediated blocking of NF‐κB translocation in Ramos did not effect the up‐regulation of BIC expression upon BcR triggering, suggesting that activation of NF‐κB is not involved in regulation of BIC expression. In summary, our data show that expression of BIC is specific for RS cells of HL. In normal tissue, BIC is expressed weakly in a minority of germinal center B cells. Expression of BIC can be modified/influenced by BcR triggering, indicating that BIC might play a role in the selection of B cells.

Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma.

We previously demonstrated high expression of primary‐microRNA BIC (pri‐miR‐155) in Hodgkin lymphoma (HL) and lack of expression in most non‐Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral‐blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT‐PCR (qRT‐PCR) and of miR‐155 using Northern blotting. Both BIC RNA ISH and qRT‐PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein–Barr virus (EBV)–positive cases] compared to HL and normal controls. In agreement with these findings, no miR‐155 was observed in BL tissues. EBV‐negative and EBV latency type I BL cell lines also showed very low BIC and miR‐155 expression levels as compared to HL cell lines. Higher levels of BIC and miR‐155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR‐155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR‐155 is not a common finding in BL. Expression of BIC and miR‐155 in 3 latency type III EBV–positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

PRIMARY BREAST LYMPHOMA - AN IMMUNOHISTOLOGIC STUDY OF 20 NEW CASES

Primary malignant lymphomas of the breast (PBL) are uncommon. the authors report the clinical, histologic, and immunoperoxidase findings on 20 cases recorded at the Alberta Cancer Registry over the last 23 years. These cases were then added to material on 257 cases abstracted from the literature and analyzed. It was found that there are two clinicopathologic types of PBL. the first affects pregnant or lactating women with bilateral, diffuse disease, is rapidly fatal, and corresponds histologically to a Burkitts‐type lymphoma. the second is unilateral at presentation and afflicts a broad age range, but primarily older women. This has a variable course only part of which is predicted by histologic grade and stage. Tumor size, treatment, and side of presentation were not found to be significant prognostic factors. Histologically, these tumors can be grouped into large cell B‐cell lymphomas, monocytoid B‐cell lymphomas (MBCL), and undifferentiated, some of which may be T‐cell. Evidence suggesting that the MBCL of breast are the equivalent of the malignant lymphomas of the mucosa‐associated lymphoid tissues (MALT) is reviewed. the breast is a hormone‐dependent member of the MALT and therefore it is interesting that two of these tumors were strongly positive for estrogen receptors.

Association with HLA class I in Epstein-Barr-virus-positive and with HLA class III in Epstein-Barr-virus-negative Hodgkin's lymphoma.

BACKGROUND Associations of Hodgkins lymphoma with HLA have been reported for many years. In 20-40% of patients with this disorder, Epstein-Barr virus (EBV) is present in the neoplastic cells. Because presentation of EBV antigenic peptides can elicit vigorous immune responses, we investigated associations of the HLA region with EBV-positive and EBV-negative Hodgkins lymphoma. METHODS In a retrospective, population-based study, patients with Hodgkins lymphoma were reclassified according to the WHO classification, and EBV status was assessed by in-situ hybridisation of EBV-encoded small RNAs. Germline DNA was isolated from 200 patients diagnosed between 1987 and 2000 and from their first-degree relatives. Genotyping was done with 33 microsatellite markers spanning the entire HLA region and two single-nucleotide polymorphisms in the genes for tumour necrosis factor alpha and beta. Classic association analysis and the haplotype sharing statistic were used to compare patients with controls. FINDINGS Classic association analysis (but not the haplotype sharing statistic) showed an association of consecutive markers D6S265 and D6S510 (p=0.0002 and 0.0003), located in the HLA class I region, with EBV-positive lymphomas. The haplotype sharing statistic (but not classic association analysis) showed a significant difference in mean haplotype sharing between patients and controls surrounding marker D6S273 (p=0.00003), located in HLA class III. INTERPRETATION Areas within the HLA class I and class III regions are associated with susceptibility to Hodgkins lymphoma, the association with class I being specific for EBV-positive disease. This finding strongly suggests that antigenic presentation of EBV-derived peptides is involved in the pathogenesis of EBV-involved Hodgkins lymphoma. RELEVANCE TO PRACTICE Polymorphisms in the HLA region could explain ethnic variation in the incidence of Hodgkins lymphoma. The association of EBV-positive Hodgkins lymphoma with HLA class I suggests that this polymorphism might affect the proper presentation of EBV antigens to cytotoxic T lymphocytes.

Hodgkin's disease with lymphocytic predominance, nodular type (nodular paragranuloma) and progressively transformed germinal centres—a cytohistological study

The histology, cytology, and enzyme cytochemistry of a nodular variant of Hodgkins disease with lymphocytic predominance, called ‘nodular paragranuloma’, are presented. The histological features of nodular paragranuloma are compared with those of progressively transformed germinal centres, which are enlarged follicles showing a predominance of small lymphocytes and some residual germinal centre cells. Progressively transformed germinal centres are sometimes found in nonspecific lymphadenitis (reactive hyperplasia). The histological similarity and the association between lymph nodes with nodular paragranuloma and lymph nodes with progressively transformed germinal centres in the same patient at different moments or at the same time, suggest that progressively transformed germinal centres are the origin of nodular paragranuloma. Hence, it must be concluded that nodular paragranuloma takes place in B‐cell areas of the lymph node, unlike the other, or at least most of the other, types of Hodgkins disease.

Ectopic expression of human and feline CD9 in a human B cell line confers beta 1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation

Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-β1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving β1 integrins and the activation of protein tyrosine kinases.

Clinical significance of bcl-2-MBR gene rearrangement and protein expression in diffuse large-cell non-Hodgkin's lymphoma: an analysis of 83 cases.

PURPOSE The goal of this study was to assess the prognostic significance of a rearrangement of the major breakpoint region of the bcl-2 gene and/or expression of bcl-2 protein in diffuse large-cell lymphomas of B-cell origin. PATIENTS AND METHODS All 83 patients diagnosed at the Cross Cancer Institute between 1987 and 1992 with malignant lymphoma (ML), diffuse large-cell ML non-cleaved-cell ML or cleaved-cell ML, or with diffuse large-cell immunoblastic ML were studied. bcl-2 rearrangement was identified by a polymerase chain reaction technique. This technique detects the approximately 60% of rearrangements involving the major breakpoint region bcl-2 gene (bcl-2-MBR). bcl-2 protein expression was studied by immunohistochemistry. RESULTS More than 66% of the cases expressed bcl-2 protein, whereas 18% had a detectable bcl-2-MBR gene rearrangement. Overall, cases with bcl-2-MBR rearrangement had shorter disease-free periods. Cases with nodal and extranodal presentation had a similar frequencies of bcl-2-MBR rearrangement; however, the disease-free period of patients with extranodal presentation and bcl-2-MBR rearrangement was significantly shorter than that of those without rearrangement. CONCLUSION bcl-2 protein is frequently expressed in diffuse large-cell lymphomas, but does not influence prognosis. The bcl-2-MBR gene rearrangement may possibly be associated with a shorter disease-free period, particularly in the specific setting of a lymphoma with extranodal presentation.