Geert Harms

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

Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma

BackgroundFlat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA expression changes associated with invasive breast cancer, like miR-21, have not been studied in FEA.MethodsWe performed miRNA in-situ hybridization (ISH) on 15 cases with simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was investigated by immunohistochemistry.ResultsTwo out of fifteen cases showed positive staining for miR-21 in normal breast ductal epithelium, seven out of fifteen cases were positive in the FEA component and nine out of twelve cases were positive in the DCIS component. A positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all three components were present in one tissue block and an increase of miR-21 from normal breast to FEA and to DCIS was observed in five cases. In three cases the FEA component was negative, whereas the DCIS component was positive for miR-21. In three other cases, normal, FEA and DCIS components were negative for miR-21 and in the last case all three components were positive. Overall we observed a gradual increase in percentage of miR-21 positive cases from normal, to FEA, DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1 staining decreased from positive in normal breast to negative in most DCIS and IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast tissue.ConclusionUpregulation of miR-21 from normal ductal epithelial cells of the breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No clear relation was observed between the staining pattern of miR-21 and its previously reported target genes.

Dimeric galectin-1 induces IL-10 production in T-lymphocytes: an important tool in the regulation of the immune response

Galectin‐1, a β‐galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune‐regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL‐10 mRNA and protein levels was demonstrated in non‐activated and activated CD4+ and CD8+ T‐cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin‐1 protein. IL‐10 is known to suppress TH1 type immune responses and upregulation of IL‐10 may thus contribute to the immune‐regulatory function of galectin‐1. Galectin‐1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin‐1 may be a useful tool in the treatment of T‐cell‐mediated diseases. Copyright

miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes

MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitts lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

Human immune response to pneumococcal polysaccharides : Complement-mediated localization preferentially on CD21-positive splenic marginal zone B cells and follicular dendritic cells

A functionally intact spleen with a marginal zone, containing B cells with high density of surface C3d-receptors (CD21), is essential for the ability to induce a primary immune response to thymus-independent type 2 (TI-2) antigens. Main representatives of natural TI-2 antigens are capsular pneumococcal polysaccharides (PPSs). In this study the localization of different types of PPS antigen is determined in human spleen tissue. Our findings indicate that a main type of TI-2 antigen, PPS, localizes preferentially in the marginal zone. PPSs show co-localization with C3, presumably C3d, at the surface of strongly CD21+ B cells equipped for rapid activation. This enables a rapid primary humoral response. The other main PPS localization at follicular dendritic cells in germinal centers, relevant for isotype switching of anti-PPS antibodies, does not seem to be dependent on the presence of specific immunoglobulin. This may explain the finding of specific IgG in an early stage after antigenic challenge. It seems likely that complement C3 fragments (likely C3d), bound to PPSs, enable PPS localization at B-cell and follicular dendritic cell surfaces by binding to CD21, the C3d receptor.

Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

Expression of the T-cell transcription factors, GATA-3 and T-bet, in the neoplastic cells of Hodgkin lymphomas

Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.

MiRNA profiling in B non-Hodgkin lymphoma: a MYC-related miRNA profile characterizes Burkitt lymphoma

Several studies have indicated the importance of miRNAs in B cell maturation and in the development of B cell lymphomas. The oncogene MYC plays an important role in B cell lymphomagenesis, particularly in Burkitt lymphoma (BL). Several recent publications have shown that MYC regulates expression of up to 60 miRNAs (Tables I and SI). The impact of the translocation and overexpression of MYC on the miRNA profile in BL has not yet been explored. We determined the miRNA expression profile of paediatric t(8;14) positive and high MYC expressing BL in comparison to MYC translocation negative mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). As a control we included normal B cell subsets obtained from hyperplastic tonsils. Hierarchical clustering showed that the B cell subsets and the non-Hodgkin lymphomas (NHLs) formed two distinct sub-clusters (Fig 1). Unsupervised clustering of the 23 miRNAs significantly differentially expressed between the four B cell subsets (>4-fold) revealed one cluster for the naı̈ve and memory B cells and two additional clusters for the germinal centre (GC) B cells and plasma cells. These results were consistent with previously published data (Appendix S1). 76 miRNAs were differentially expressed (>4-fold) between the four NHL subtypes. Most differences were observed between BL and the other NHLs (CLL n = 58, FL n = 32 and MCL n = 36 miRNAs). Unsupervised hierarchical clustering analysis revealed a unique miRNA profile in BL (Appendix S2). In contrast, a maximum of eight miRNAs were differently expressed between MCL, FL and CLL. A list of validated target genes of the differentially expressed miRNAs is presented in Table SII. Comparing each malignancy to its normal counterpart (MCL with naı̈ve, FL and BL with GC B cells and CLL with memory cells), 54–77 miRNAs were differentially expressed (Figure S1). MYC expression has been shown to be a dominant factor in the regulation of many miRNAs. In the present series of lymphomas, quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a much higher expression in BL than all other NHL (Fig 1C). Comparison of the MYChigh BL to all other MYC-low lymphomas revealed 122 differentially expressed miRNAs (Table SIII), including 39 of the 50 evaluable (78%) known targets of MYC (10 miRNAs were not expressed in our cases) (Fig 1D and Table I). The expression level of these 39 miRNAs was always consistent with the expected upor downregulation, with most miRNAs being downregulated. This indicates a dominant MYC-induced miRNA profile in primary BL. This signature included 39 of the known MYC-regulated miRNAs. The 83 other miRNAs were also differentially expressed between MYC-high BL and MYC-low NHL samples. At present, there are no data supporting a direct MYC-dependent regulation for those 83 miRNAs. A MYC-dependent miRNA signature has also been suggested for diffuse large B cell lymphoma based on a differential expression of MYC targets (Li et al, 2009). Four BL cases (B4, B5, B6 and B7) and two MCL cases (M2 and M3) harboured a 13q31.3 amplification (determined by fluorescence in situ hybridization, results not shown). These six cases showed high expression of the miR-17-92 cluster (Fig 1D). Both in BL and MCL, this association was independent of the MYC expression level (Fig 1C). In line with the findings of Tagawa et al (2007) this indicates that genomic amplification and not MYC overexpression is instrumental in the expression of the miR-17-92 cluster. The miR-17-92 cluster is positively regulated by MYC and acts with MYC to accelerate tumour development (He et al, 2005). MiR-17 and miR-20, two members of this cluster, promote cell cycle progression via E2F1 (O’Donnell et al, 2005). One interesting MYC-repressed gene is MIRLET7A. As MYC is a direct target of MIRLET7A this suggests a positive stimulatory loop for MYC (Sampson et al, 2007). Induction of MIRLET7A in the Namalwa BL cell line resulted in reduced MYC expression and reduced proliferation, whereas downregulation of MYC resulted in increased expression of MIRLET7A. In addition to a significant downregulation of MIRLET7A, we also observed a significant downregulation for MIRLET7E in BL compared to the three other NHL subtypes. MiR-150 was also significantly downregulated in BL and targets MYB, which has an essential role in haematopoietic and lymphoid development and apoptosis (Xiao et al, 2007). Overexpression of miR-150 in BL cells resulted in reduced MYB levels and increased apoptosis (Tan et al, 2009). Interestingly miR-15a has recently also been shown to repress the MYB oncogene in leukemic cell lines (Zhao et al, 2009). Low expression of miR-150 in BL compared to the other three NHL subtypes and of miR-15a in comparison to CLL might thus result in enhanced MYB levels in BL. Ectopic expression of miR-26a in BL cell lines impaired cell cycle progression via its target EZH2, a member of the polycomb-group of genes (Sander et al, 2008). The low expression of miR-26a in BL is thus consistent with the previously observed high expression of correspondence

The CD4+CD26-T-cell population in classical Hodgkin's lymphoma displays a distinctive regulatory T-cell profile

Little is known about the gene expression profile and significance of the rosetting CD4+CD26− T cells in classical Hodgkins lymphoma (cHL). To characterize these T cells, CD4+CD26− and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26− T cells in NSHL than in reactive LNs. The resting CD4+CD26− T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2-related cytokines IL-2, IFN-γ, IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro, lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26− T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.

High expression of calcium‐binding proteins, S100A10, S100A11 and CALM2 in anaplastic large cell lymphoma

Anaplastic large cell lymphomas (ALCL) are characterised by the presence of CD30‐positive large cells, which usually are of T‐cell type. Based on the presence or absence of translocations involving the anaplastic lymphoma kinase (ALK) locus, ALCL cases can be divided into two groups. To gain more insight in the biology of ALCL, we applied serial analysis of gene expression (SAGE) on the Karpas299 cell line and identified 25 up‐ and 19 downregulated genes. Comparison of the differentially expressed genes with DNA copy number changes in Karpas299 revealed that two overexpressed genes, S100A10 and S100A11, were located in an amplicon suggesting that the increased mRNA levels were caused by DNA amplification. Quantitative reverse transcription polymerase chain reaction on 5 ALCL cell lines and 12 ALCL tissues confirmed the SAGE data for 13 out of 14 up‐ and one out of four downregulated genes. Immunohistochemical staining confirmed the presence of S100A10, a calcium‐binding protein, in three out of five ALK+ and all 7 ALK− ALCL cases. S100A11 staining was confirmed in all ALK+ and six of seven ALK− ALCL cases. Three of the upregulated genes represented calcium‐binding proteins, which suggest that altered intracellular signaling might be associated with the oncogenesis of ALCL.