Lydia Visser

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.

PURPOSEnThe 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.nnnPATIENTS AND METHODSnAll 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.nnnRESULTSnMore 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.nnnCONCLUSIONnbcl-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.

Epstein-Barr virus positivity in Hodgkin's disease does not correlate with an HLA A2-negative phenotype.

Background. Epstein‐Barr virus‐ (EBV) related DNA and RNA can be found in tissues involved with Hodgkins disease, specifically in the Reed‐Sternberg cells. These cells also express the membrane antigens LMP1 and LMP 2A and 2B. Studies in normal individuals indicate that cellular immunity against LMP2 was frequently mediated through human leukocyte antigen (HLA) A2, whereas responses to LMP1 appeared to be relatively infrequent. Assuming that LMP2‐positive Reed‐Sternberg cells would be sensitive to a CD8‐positive cellular immune response, the hypothesis can be made that EBV‐positive Hodgkins disease should be more common in individuals not expressing HLA A2. To test this hypothesis, the authors have studied the frequency of HLA A2 in EBV‐positive versus EBV‐negative patients with Hodgkins disease.

Neoplastic changes involving follicles: morphological, immunophenotypic and genetic diversity of lymphoproliferations derived from germinal center and mantle zone.

The term follicular lymphoma is generally reserved for nodular proliferations of neoplastic B cells that are of germinal center origin. However, there are also lymphomas that are derived from cells normally present in the mantle zones of follicles (Weisenburger et al. 1982). In addition, one sub-type of Hodgkins disease, the so-called nodular lymphocyte predominance sub-type, is a proliferation of germinal center and mantle zone cells (Poppema et al. 1985, Timens et al. 1986). The basis for classifying follicular lymphomas has been the nodular pattern that mimics the architecture of reactive follicles in combination with the cytological recognition of cell types that can be found in normal germinal centers. These cell types were classified as centroblasts and centrocytes by Lennert et al. (1975) and as non-cleaved cells and cleaved cells by Lukes & Collins (1974). In the Working Formulation for the Classification of Lymphomas, the terms non-cleaved cell and cleaved cell were adopted. In general it is possible to define follicular lymphomas based on the relative proportions of cleaved cells and non-cleaved cells and this sub-classification correlates with the biological behavior of the lymphomas (Non Hodgkins Lymphoma Pathologic Classification Project 1982). Table I summarizes the lymphoma types that are derived from follicles. All of these entities may

High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma

Aim: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). Methods: Serial analysis of gene expression (SAGE) was undertaken on the CD4+ALK+ (anaplastic lymphoma kinase positive) ALCL derived cell line Karpas299 and as comparison on CD4+ T cells. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed on five ALCL derived cell lines and 32 tissue samples to confirm the SAGE data. Results: High expression of Mcl-1 was seen in the Karpas299 cell line, whereas the two other antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-XL, were not detected in the SAGE library. Quantitative RT-PCR confirmed the high expression of Mcl-1 mRNA and low expression of Bcl-2 and Bcl-XL in Karpas299 and in four other ALCL cell lines. To expand on these initial observations, primary tissue samples were analysed for Mcl-1, Bcl-XL, and Bcl-2 by immunohistochemistry. All 23 ALK+ and nine ALK− ALCL cases were positive for Mcl-1. Bcl-2 and Bcl-XL were expressed infrequently in ALK+ ALCL cases, but were present in a higher proportion of ALK− ALCL cases. Conclusion: The consistent high expression of Mcl-1 in ALK+ and ALK− ALCL suggests that Mcl-1 is the main antiapoptotic protein in this disease. The high frequency of Mcl-1, Bcl-2, and Bcl-XL positive ALCL cases in the ALK− group compared with the ALK+ group indicates that ALK induced STAT3 activation is not the main regulatory pathway in ALCL.

Induction of B-cell chronic lymphocytic leukaemia and hairy cell leukaemia like phenotypes by phorbol ester treatment of normal peripheral blood B-cells

Summary. To investigate the relationship between normal B‐cells, B‐cell chronic lymphocytic leukaemia (B‐CLL) cells and hairy cell leukaemia (HCL) cells the three cell types were incubated with phorbol myristic acetate (PMA). The parameters studied were morphology, immunophenotype and tartrate resistant acid phosphatase (TRAP). PMA stimulation of B‐cells induced morphological changes as well as CD5, CD11c, B‐ly 7 and TRAP positivity. The cells formed small aggregates, the cell membranes were ruffled and frequently hairy and a small number of cells became plastic adherent and developed dendritic structures. CD5 and CD11c appeared on day 2, imitating a B‐CLL phenotype. On day 3 and 4 a decrease of CD5, an increase of CD11c and the appearance of B‐ly 7 could be seen mimicking an HCL phenotype. The changes in B‐CLL and HCL upon PMA stimulation were mainly morphological; the B‐CLL cells became ruffled and aggregated strongly, while the HCL cells developed dendritic features and became adherent. The immunophenotype of the PMA stimulated HCL cells did not change. The B‐CLL cells remained CD5 positive and did not become B‐ly 7 positive. The findings indicate that although PMA stimulation of normal peripheral blood cells results in CLL‐ as well as HCL‐like phenotypes, similar stimulation of B‐CLL cells does not result in an HCL‐like phenotype.

Postnatal changes of CD45 expression in peripheral blood T and B cells

SUMMARY. One known postnatal change of CD45 expression is the decline of the CD45RAhigh CD45ROlow T subsets and the reciprocal increase of the CD45RAlow CD45ROhigh T subsets in the peripheral blood. Using a panel of monoclonal antibodies reactive with either protein or carbohydrate epitopes on the variable regions of CD45, we were able to detect more postnatal changes of CD45 expression. These changes are largely caused by modulation of the CD45 glycosylation, including: (I) lesser sialylation of the CD45RA region on T cells, and (2) differential sialylation of the CD45RB region leading to the distinction of CD45RBhigh and CD45RBlow T and B subsets. In addition, the existence of the CD45RAdim CD45ROdim labelled as transitional T cells is only found during the postnatal life. These changes may reflect the maturation of the immune system.

The Nature of the Lymphocytes in Hodgkin’s Disease

Hodgkin’s disease (HD) is characterised by the presence of Reed-Sternberg (RS) cells in the proper environment. This environment includes predominantly small lymphocytes and a variable admixture of histiocytes, plasma cells and eosinophils. It should be kept in mind that the RS cells and their variants constitute less than one percent of the cell population in the vast majority of cases. Nevertheless, these cells are the clonal and abnormal population in HD as shown by a combination of conventional karyotyping and chromosome specific in situ hybridisation (Poppema et al., 1992). As well, in a minority of cases it has been possible to demonstrate clonal rearrangements of the immunoglobulin genes, especially in RS cell-enriched cell fractions (Brinker et al., 1987; Weiss et al., 1987; Sundeen et al., 1987). Although no consistent chromosomal abnormality has been identified in HD, it is of interest that chromosome 14q, including the immunoglobulin heavy chain gene containing q32 region, is the area most frequently involved in translocations in HD (Cabanillas et al., 1988; Poppema et al., 1993). However, the non-Hodgkin’s lymphoma associated translocations t(8;14), t(l 1;14) and t(14;18) are exceedingly rare in HD. The translocation t(2;5) that is found in anaplastic large cell lymphomas (Mason et al., 1990) is not found in HD by cytogenetic analysis whereas the use of reverse transcriptase — polymerase chain reaction (PCR) has led to conflicting results (Poppema et al., 1993). The absence of t(14;18) translocations is of interest since, using PCR-based methods, it has been possible to demonstrate rearrangements involving the major breakpoint region of the bcl-2 gene and the immunoglobulin heavy chain gene in 30% of cases (Stetler-Stevenson et al., 1990).

Erratum: The microenvironment of classical Hodgkin lymphoma: heterogeneity by Epstein-Barr virus presence and location within the tumor (vol 6, e417, 2016)

This corrects the article DOI: 10.1038/bcj.2016.26.