Hansmann Ml

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK− and cutaneous ALK− ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK− ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK+ and four ALK− systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4+, CD8+ or CD30+ T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFκB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK− ALCL and cHL despite their different cellular origin. ALK+ ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Dysregulation of global microRNA expression in splenic marginal zone lymphoma and influence of chronic hepatitis C virus infection

The precise molecular pathogenesis of splenic marginal zone lymphoma (SMZL) is still unknown. Clinical and epidemiological data suggest that chronic hepatitis C virus (HCV) infection may have an etiological role in a subset of cases.We performed a large-scale microRNA (miRNA) expression profiling analysis of 381 miRNAs by quantitative reverse transcription PCR (Q-RT-PCR) of 26 microdissected splenic tissue samples (7 HCV+ SMZL; 8 HCV− SMZL and 11 non-neoplastic splenic controls). Single assay Q-RT-PCR and miRNA in situ hybridization (miRNA-ISH) were used to confirm the results in an independent cohort. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a distinct signature of SMZL compared with the normal splenic marginal zone. Supervised analysis revealed differentially expressed miRNAs, including miRNAs with previously recognized tumor suppressive or oncogenic potential. Five miRNAs were found significantly overexpressed in SMZL, including miR-21, miR-155 and miR-146a, whereas seven miRNAs showed significantly reduced expression, including miR-139, miR-345, miR-125a and miR-126. Furthermore, we identified miR-26b, a miRNA known to have tumor suppressive properties, as significantly downregulated in SMZL arising in HCV-positive patients (P=0.0016). In conclusion, there is a characteristic dysregulation of miRNA expression in SMZL with a possible implication in its molecular tumorigenesis.

High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma.

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Jaw1/LRMP, a germinal centre-associated marker for the immunohistological study of B-cell lymphomas

Jaw1, also known as lymphoid‐restricted membrane protein (LRMP), is an endoplasmic reticulum‐associated protein. High levels of Jaw1/LRMP mRNA have been found in germinal centre B‐cells and in diffuse large B‐cell lymphomas of ‘germinal centre’ subtype. This paper documents Jaw1/LRMP expression at the protein level in human tissues by immunohistochemical and western blotting analysis using an antibody reactive with paraffin‐embedded tissues. Jaw1/LRMP was highly expressed in germinal centre B‐cells (in keeping with gene expression data), in ‘monocytoid B‐cells’, and in splenic marginal zone B‐cells. It was absent, or present at only low levels, in mature T‐cells, although cortical thymocytes were weakly positive. Among lymphoid neoplasms, Jaw1/LRMP was found in germinal centre‐derived lymphomas (follicle centre lymphoma, Burkitts lymphoma, lymphocyte‐predominant Hodgkins disease) but not in T‐cell neoplasms (with the exception of a single T lymphoblastic lymphoma). Classical Hodgkins disease and myeloma lacked Jaw1/LRMP but many cases of chronic lymphocytic leukaemia (but not mantle zone lymphoma) were Jaw1/LRMP‐positive. Approximately half of the marginal zone lymphomas were Jaw1/LRMP‐positive. In diffuse large B‐cell lymphomas, Jaw1/LRMP was found in three‐quarters (24/32) of the cases classified phenotypically as being of ‘germinal centre’ type, but it was also expressed in almost half (13/28) of the ‘non‐germinal centre’ cases. A similar proportion of ‘non‐germinal centre’ cases were positive for the protein products of two other genes expressed highly in germinal centre cells (HGAL/GCET2 and PAG). The fact that all three of these proteins are expressed in a significant proportion of diffuse large B‐cell lymphomas assigned to the ‘non‐germinal centre’ category indicates that the immunophenotypic categorization of diffuse large B‐cell lymphoma according to cellular origin may be more complicated than currently understood. Finally, the expression of Jaw1/LRMP in other types of lymphoma and in non‐lymphoid tissues/tumours may be of interest in differential diagnosis and research. Copyright

Different biological risk factors in young poor-prognosis and elderly patients with diffuse large B-cell lymphoma

Prognostically relevant risk factors in patients with diffuse large B-cell lymphoma (DLBCL) have predominantly been evaluated in elderly populations. We tested whether previously described risk factors are also valid in younger, poor-prognosis DLBCL patients. Paraffin-embedded samples from 112 patients with de novo DLBCL, enrolled in the R-MegaCHOEP trial of the German High Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) were investigated using immunohistochemistry (MYC, FOXP1, LMO2, GCET1, CD5, CD10, BCL2, BCL6, IRF4/MUM1) and fluorescence in situ hybridization (MYC, BCL2, BCL6). MYC, BCL2 and BCL6 breaks occurred in 14, 21 and 31%, respectively. In the majority of cases, MYC was simultaneously rearranged with BCL2 and/or BCL6. The adverse impact of MYC rearrangements was confirmed, but the sole presence of BCL2 breaks emerged as a novel prognostic marker associated with inferior overall survival (OS) (P=0.002). Combined overexpression of MYC and BCL2 showed only limited association with inferior OS. All immunohistochemical cell of origin classifiers applied failed to predict survival time. DLBCL tumors with significant proportion of immunoblastic and/or immunoblastic-plasmacytoid cells had inferior OS, independently from from BCL2 break. Younger, poor-prognosis DLBCL patients, therefore, display different biological risk factors compared with an elderly population, with BCL2 translocations emerging as a powerful negative prognostic marker.

Loss of CD19 expression in B-cell neoplasms

Aims : To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas.

T-cell receptor diversity prevents T-cell lymphoma development.

Mature T-cell lymphomas (MTCLs) have an extremely poor prognosis and are much less frequent than immature T-cell leukemias. This suggests that malignant outgrowth of mature T lymphocytes is well controlled. Indeed, in a previous study we found that mature T cells are resistant to transformation with known T-cell oncogenes. Here, however, we observed that T-cell receptor (TCR) mono-/oligoclonal mature T cells from TCR transgenic (tg) mice (OT-I, P14) expressing the oncogenes NPM/ALK or ΔTrkA readily developed MTCLs in T-cell-deficient recipients. Analysis of cell surface markers largely ruled out that TCR tg lymphomas were derived from T-cell precursors. Furthermore, cotransplanted non-modified TCR polyclonal T cells suppressed malignant outgrowth of oncogene expressing TCR tg T lymphocytes. A dominant role of an anti-leukemic immune response or Tregs in the control of MTCLs seems unlikely as naïve T cells derived from oncogene expressing stem cells, which should be tolerant to leukemic antigens, as well as purified CD4 and CD8 were resistant to transformation. However, our results are in line with a model in which homeostatic mechanisms that stabilize the diversity of the normal T-cell repertoire, for example, clonal competition, also control the outgrowth of potentially malignant T-cell clones. This study introduces a new innate mechanism of lymphoma control.

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkins disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed–Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-γ2 (absent from Reed–Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkins disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma

The neoplastic Reed–Sternberg cells characteristic of classical Hodgkins lymphoma (cHL) are of B‐cell origin but they almost always show striking loss of a range of B‐cell‐associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkins lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL‐6 expression, ‘ongoing’ Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down‐regulation of a number of markers associated with the B‐cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down‐regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL. Copyright

Highly recurrent mutations of SGK1 , DUSP2 and JUNB in nodular lymphocyte predominant Hodgkin lymphoma

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)—a subtype of Hodgkin lymphoma (HL)—is characterized by a low content of tumor cells, the lymphocyte predominant (LP) cells. Transformation into diffuse large B-cell lymphoma (DLBCL) occurs in about 10% of patients. We performed whole-genome mutation analysis of the DLBCL components from two composite lymphomas consisting of clonally related NLPHL and DLBCL as a means to identify candidate tumor suppressor genes and oncogenes in NLPHL. The analysis of LP cells for selected mutations of the DLBCL revealed that most mutations are also present in the LP cells, indicating a close relationship between the two components. The analysis of 62 selected genes in NLPHL by targeted ultra-deep sequencing revealed three novel highly recurrently mutated genes (each mutated in ~50% of cases), that is, DUSP2, SGK1 and JUNB. SGK1 was expressed in the LP cells of primary NLPHL cases and in the NLPHL cell line DEV. Administration of an SGK1 inhibitor induced apoptosis in the NLPHL cell line DEV and the DLBCL cell line Farage, suggesting a pathogenetic role of SGK1 in the LP and DLBCL cells. In summary, the present study identifies SGK1, DUSP2 and JUNB as novel key players in the pathogenesis of NLPHL.