Jennifer C. Paterson

MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy that accounts for nearly 40% of all lymphoid tumors. This heterogeneous disease can be divided into germinal center B cell‐like (GCB) and activated B cell‐like (ABC) subtypes by gene expression and immunohistochemical profiling. Using microarray analysis on prototypic cell lines, we identified microRNAs (miR‐155, miR‐21 and miR‐221) that were more highly expressed in ABC‐type than GCB‐type cell lines. These microRNAs were over‐expressed in de novo DLBCL (n = 35), transformed DLBCL (n = 14) and follicular center lymphoma cases (n = 27) compared to normal B cells. Consistent with the cell line model, expression levels were higher in DLBCL cases with an ABC‐type immunophenotype than those that were GCB‐type (p < 0.05). Moreover, using multivariate analysis we found that expression of miR‐21 was an independent prognostic indicator in de novo DLBCL (p < 0.05). Interestingly, expression levels of both miR‐155 and miR‐21 were also higher in nonmalignant ABC than in GCB cells. As we also demonstrate that expression of microRNAs can be measured reliably from routine paraffin‐embedded biopsies of more than 8‐years‐old (p < 0.001), we suggest that microRNAs could be clinically useful molecular markers for DLBCL as well as other cancers.

Novel markers of normal and neoplastic human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are involved in innate immunity (eg, by secreting interferons) and also give rise to CD4+CD56+ hematodermic neoplasms. We report extensive characterization of human pDCs in routine tissue samples, documenting the expression of 19 immunohistologic markers, including signaling molecules (eg, BLNK), transcription factors (eg, ICSBP/IRF8 and PU.1), and Toll-like receptors (TLR7, TLR9). Many of these molecules are expressed in other cell types (principally B cells), but the adaptor protein CD2AP was essentially restricted to pDCs, and is therefore a novel immunohistologic marker for use in tissue biopsies. We found little evidence for activation-associated morphologic or phenotypic changes in conditions where pDCs are greatly increased (eg, Kikuchi disease). Most of the molecules were retained in the majority of pDC neoplasms, and 3 (BCL11A, CD2AP, and ICSBP/IRF8) were also commonly negative in leukemia cutis (acute myeloid leukemia in the skin), a tumor that may mimic pDC neoplasia. In summary, we have documented a range of molecules (notably those associated with B cells) expressed by pDCs in tissues and peripheral blood (where pDCs were detectable in cytospins at a frequency of <1% of mononuclear cells) and also defined potential new markers (in particular CD2AP) for the diagnosis of pDC tumors.

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation

Background T follicular helper (TFH) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for TFH cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified. Design and Methods In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas. Results Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed TFH-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other TFH-associated molecules. Conclusions ICOS is a useful molecule for identifying TFH cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a TFH-like profile) suggests its inclusion in the antibody panel for diagnosing TFH-derived lymphomas. Our findings provide further evidence that the histological spectrum of TFH-derived lymphomas is broader than previously assumed.

Induction of p53 and up-regulation of the p53 pathway in the human 5q- syndrome

To the editor: There is mounting evidence from the study of animal models of human disorders of defective ribosome biogenesis, including Diamond-Blackfan anemia and Treacher Collins syndrome, that ribosomal stress leads to activation of the p53 pathway.[1][1][⇓][2]–[3][3] Stabilization of p53

Peripheral T-cell lymphoma with a follicular growth pattern: derivation from follicular helper T cells and relationship to angioimmunoblastic T-cell lymphoma.

While our knowledge of the normal cellular counterparts of B-cell lymphomas contributes much to the meaningful classification of these neoplasms, our understanding of the cellular ontogeny of mature (peripheral) T-cell lymphomas (PTCL) is much less advanced and their classification is suboptimal. Thus, while there is considered sufficient evidence to classify some PTCL, such as angioimmunoblastic T-cell lymphoma (AITL), an aggressive lymphoma recently shown to have the immunophenotype of follicular helper T (TFH) cells (Dogan et al, 2003; de Leval et al, 2007), as discrete entities, most are subsumed into the biologically and clinically heterogeneous group of PTCL-unspecified (PTCL-U). Amongst PTCL-U, an uncommon subset with a follicular growth pattern (PTCL-F) is particularly poorly understood. Some PTCL-F harbour a t(5;9)(q33;q22)/ITK-SYK, but their cellular origin is unascertained and their proper classification uncertain, leading to difficulties in diagnosis and clinical management (de Leval et al, 2001; Jiang et al, 2005; Ikonomou et al, 2006; Streubel et al, 2006). This report demonstrates the derivation of a typical case of PTCL-F from germinal centre (GC) TFH cells, highlighting a clinical and pathological overlap with AITL. A 27-year-old man presented with a flu-like illness, jaundice, widespread lymphadenopathy and splenomegaly. Lymph node biopsy showed a T-cell non-Hodgkin lymphoma. There was no skin rash, arthritis, effusion or bone marrow involvement (Ann Arbor stage III). Laboratory investigations showed an auto-immune haemolytic anaemia, mild thrombocytopenia, raised lactate dehydrogenase, positivity for anti-smooth muscle auto-antibody but no hypergammaglobulinaemia. The patient received six courses of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) chemotherapy resulting in a complete remission but relapsed with widespread lymphadenopathy 14 months later. An autologous peripheral blood stem cell transplant induced a second complete remission, but four months later the patient developed a haemophagocytic syndrome. Despite treatment with ciclosporin A, dexamethasone and etoposide, he died of a disseminated mycosis. The lymph node sampled (Fig 1) showed partial architectural effacement by lymphoma. The cortex contained numerous, variably sized follicles, the GCs of which contained varying numbers of residual B cells together with many medium-sized to large atypical T cells with clear cytoplasm, irregular nuclei and frequent mitoses. Similar cells also infiltrated follicular mantle zones. The expanded paracortex contained several smaller follicles, often with depleted GCs, many of which also contained atypical T cells. The atypical cells were not confined to follicles however, and were also found in-between follicles. The paracortical infiltrate was accompanied by reactive lymphocytes, plasma cells and occasional eosinophils, and by a notable proliferation of arborising high endothelial venules. Immunohistochemical staining showed the atypical T cells to express CD2, CD3, CD4, and CD5, but not CD8, CD10 or CD30. In addition, they expressed proteins recently shown to be characteristic of GC TFH cells: CXCL13, PD-1, SAP, NFATc1, Bcl6 and CD57. A minor, dispersed interfollicular population of large reactive B cells showed polytypic expression of immunoglobulin light chains and was Epstein–Barr Virus Early RNA (EBER) negative. Staining for CD21 showed follicular dendritic cells (FDC) to be largely confined to follicles with only subtle and focal expansion and little tendency to encircle vessels. Polymerase chain reaction-based analysis of lymphocyte clonality showed monoclonal rearrangements of TCRB@ and TCRG@ but not IGH@, IGK@ or IGL@. Interphase fluorescence in situ hybridisation showed no evidence of an ITK-SYK translocation. The few reports of PTCL-F describe preferential localisation of neoplastic T cells within either GCs or expanded mantle zones (de Leval et al, 2001; Jiang et al, 2005; Ikonomou et al, 2006; Streubel et al, 2006), but the T cell subset to which these cells belong and their microanatomical origin are matters of debate. While some authors suggested an origin from intrafollicular T cells (Ikonomou et al, 2006), others proposed colonisation of follicles by extrafollicular T cells (Jiang et al, 2005). The application of routinely used immunohistochemical markers alone has shown typical expression of Bcl6 and/or CD10, but has failed to resolve these issues due to both heterogeneity amongst cases and a lack of specificity and sensitivity of these antibodies for intrafollicular T cells. In contrast, we used a panel of novel markers recently identified by gene expression profiling and immunohistochemistry to be characteristically expressed together in TFH cells, but not in other T-cell subsets (Chtanova et al, 2004, 2005; Vinuesa et al, 2005; Roncador et al, 2007), to demonstrate a robust TFH immunophenotype in our case. This, together with the preferential localisation of the neoplastic cells within GCs, is correspondence

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

Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative enhancer region, of the ICOS gene, whereas cutaneous T-cell lymphoma cell lines, which strongly express ICOS, show no methylation of the island. Treatment of the ALK(+) TCL cell lines with DNA methyltransferase inhibitor reversed the CpG island methylation and augmented the expression of ICOS mRNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth promoting receptor. These data also show that the DNA methylation status of the intronic CpG island affects transcriptional activity of the ICOS gene and, consequently, modulates the concentration of the expressed ICOS protein.

B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling

The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.

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

Detection of LIM domain only 2 (LMO2) in normal human tissues and haematopoietic and non-haematopoietic tumours using a newly developed rabbit monoclonal antibody

Agostinelli C, Paterson J C, Gupta R, Righi S, Sandri F, Piccaluga P P, Bacci F, Sabattini E, Pileri S A & Marafioti T 
(2012) Histopathology 61, 33–46