Phospho-proteomic analysis of mantle cell lymphoma cells suggests a pro-survival role of B-cell receptor signaling

BACKGROUND: Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. METHODS: PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. RESULTS: PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. CONCLUSION: Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes

Follicular lymphoma: validation of a novel approach for the detection of clonality and microRNA expression profiling

1. APPLICATION OF MICROFLUIDIC TECHNOLOGY TO THE BIOMED-2 PROTOCOL FOR DETECTION OF B-CELL CLONALITY Il protocollo BIOMED-2 \ue8 frutto di una collaborazione tra pi\uf9 centri di ricerca volta alla standardizzazione ed ottimizzazione della rilevazione della clonalit\ue0 nei disordini linfoproliferativi. Il protocollo prevede reazioni di PCR multiple che vengono analizzate attraverso l\u2019elettroforesi capillare oppure l\u2019analisi degli heteroduplex mediante elettroforesi su gel di acrilamide. Abbiamo testato uno strumento commerciale basato sulla elettroforesi microfluidica su chip (Agilent 2100 Bioanalyzer) quale possibile alternativa, per l\u2019analisi delle PCR multiple del BIOMED-2, all\u2019alettroforesi capillare e all\u2019analisi degli heteroduplex. Abbiamo usato il protocollo per la rilevazione della clonalit\ue0 delle cellule B che prevede 5 reazioni di PCR, tre per i geni delle catene pesanti delle immunoglobuline e due per i geni delle catene leggere kappa. Abbiamo testato 68 linfomi B: 33 follicolari e 35 nonfollicolari (18 leucemie linfocitiche croniche, 7 linfomi a grandi cellule, 1 linfoma della zona marginale della milza, 1 linfoma linfoplasmacitico, 1 leucemia a tricoleucociti, 1 linfoma di Hodgkin, 3 non-classificati e due casi senza la disponibilit\ue0 dell\u2019istologia), e 16 linfonodi reattivi. L\u2019elettrofresi su chip ha rilevato monoclonalit\ue0 in 62/68 campioni: 30/33 (91%) dei linfomi follicolari e 32/35 (91%) dei linfomi non-follicolari. L\u2019interpretazione della clonalit\ue0 in 19 su 22 campioni (86%) concordava con quella ottenuta mediante Southern blot. L\u2019elettroforesi su chip \ue8 stata confrontata con l\u2019elettroforesi capillare utilizzando 26 campioni. Sono stati ottenuti risultati concordanti per 68/78 riarrangiamenti per le catene pesanti e 48/52 riarrangiamenti per le catene kappa. La concordanza tra l\u2019elettrofresi su chip e l\u2019elettroforesi capillare nell\u2019interpretazione della clonalit\ue0 \ue8 stata del 96%. Possiamo concludere che questo strumento, basato sulla elettroforesi microfluidica su chip, \ue8 adatto per l\u2019analisi della clonalit\ue0 delle cellule B usando il protocollo BIOMED-2. 2. EVALUATION OF microRNA INVOLVEMENT IN FOLLICULAR LYMPHOMA DEVELOPMENT BY EXPRESSION PROFILING Il linfoma follicolare (FL) \ue8 un linfoma indolente che costituisce il 20%-30% dei linfomi non- Hodgkin\u2019s. FL si presenta come un linfonodo (LN) infiltrato da cellule B del centro germinativo (B-GC). I microRNA sono una classe di piccole molecole di RNA non codificanti che svolgono dei ruoli chiave in molte vie metaboliche durante lo sviluppo e l\u2019omeostasi cellulare. L\u2019espressione aberrante dei microRNA \ue8 un aspetto comune nei tumori, compresi quelli ematologici, suggerendo un loro ruolo nello sviluppo di queste patologie. Dal momento che poco si sa sul ruolo che i microRNA coprono nello sviluppo dei FL, abbiamo analizzato i profili di espressione di 26 FL. Come controlli sono stati analizzati 12 linfonodi reattivi e 10 campioni di cellule B-GC purificate. Molti microRNA erano differenzialmente espressi confrontando i FL sia con i LN che con le cellule B-GC 21 purificate. Interessante \ue8 stato notare come l\u2019espressione di alcuni microRNA presentasse un andamento opposto nei due confronti. L\u2019espressione di miR-9/miR-9*, miR-30 family, miR-15-16 family, miR-34a, cluster-17~92 and miR-28 era, infatti, alta nei FL in confronto ai LN, mentre l\u2019espressione era pi\uf9 bassa quando i FL venivano confrontati con le cellule B-GC purificate. Questi risultati suggeriscono che questi microRNA possano appartenere a una firma delle cellule B-GC e che i FL mantengano delle caratteristiche delle cellule B normali da cui derivano. Diversamente miR-21 e miR-219 erano sovra-espressi con un\u2019alta significativit\ue0 nei FL in confronto sia ai LN sia alle cellule B-GC purificate suggerendo un loro contributo nella patogenesi e nella progressione dei FL.1. APPLICATION OF MICROFLUIDIC TECHNOLOGY TO THE BIOMED-2 PROTOCOL FOR DETECTION OF B-CELL CLONALITY The BIOMED-2 protocol is the successful result of a multicenter effort for standardizing and optimizing detection of clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions that are analyzed by either capillary electrophoresis or heteroduplex analysis on polyacrylamide gel-electrophoresis. We tested a commercially available microfluidic chip-electrophoresis apparatus (Agilent 2100 Bioanalyzer) as an alternative to capillary electrophoresis and heteroduplex analysis for the analysis of multiplex BIOMED-2 PCRs. We used the protocol for detection of B-cell clonality that requires 5 PCR reactions, three for immunoglobulin heavy and two for kappa light chain genes. We tested 68 B-cell lymphomas: 33 follicular and 35 non-follicular (18 chronic lymphocytic leukemia, 7 difuse large B-Cell lymphoma, 1 splenic marginal zone lymphoma, 1 extranodal marginal zone lymphoma, 1 lymphoplasmacytic lymphoma, 1 hairy cell leukemia, 1 Hodgkin lymphoma, 3 unclassified and 2 cases with no histology available), and 16 reactive lymph nodes. Chip electrophoresis was conclusive for monoclonality in 62/68 samples: 30/33 (91%) follicular lymphoma and 32/35 (91%) nonfollicular lymphoma samples. The interpretation of clonality was concordant for 19 of 22 samples (86%) analyzed by Southern blot. Chip electrophoresis was compared with capillary electrophoresis using 26 samples. Concordant results were obtained in 68 of 78 heavy chain and 48/52 kappa chain gene rearrangements. The concordance between the chip electrophoresis and capillary electrophoresis in the interpretation of clonality was 96%. We conclude that the chip-based apparatus is suitable for the analysis of B-cell clonality using the BIOMED-2 protocol. 2. EVALUATION OF microRNA INVOLVEMENT IN FOLLICULAR LYMPHOMA DEVELOPMENT BY EXPRESSION PROFILING Follicular lymphoma (FL) is an indolent lymphoma which accounts for 20%-30% of non-Hodgkin\u2019s lymphomas. FL appears as a lymph node (LN) infiltrated by proliferating germinal center B-cells (GC B-cells). MicroRNAs are a class of small non-coding RNAs that play key roles in many cellular pathways during normal development and cellular homeostasis. Aberrant expression of microRNAs is a common feature of cancer and also of the haematological ones, suggesting they carry out an important role in the development of these pathologies. Since little is known on microRNA role in FL, we analyzed expression profile of 26 FL. As controls we profiled 12 LN and 10 purified GC Bcells samples. Many microRNA were differentially expressed when FL was compared to LN and to GC B-cells. Interestingly some of differentially expressed microRNA showed opposite trends in the two comparisons. The expression of miR-9/miR-9*, miR- 30 family, miR-15-16 family, miR-34a, cluster-17~92 and miR-28 was high in FL as compared to reactive LN, while the expression was lower when FL was compared to GC B-cells. Altogether these results suggested that these microRNAs may belong to a specific signature of GC B-cells and, therefore, that FL maintain the characteristics of the normal B-cells that they are derived from. Differently miR-21 and miR-219 were up-regulated with a high significance in FL compared to both FL and GC B-cells indicating their possible association with follicular lymphoma pathogenesis and progression

The TNF-Family Cytokine TL1A/Death Receptor 3 System Reduces Metabolic Activity in Chronic Lymphocytic Leukemia B Cells

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily, expressed on dendritic cells, macrophages, lymphocytes, and endothelial cells. It is the only described ligand for death receptor 3 (DR3), a death-domain containing receptor of the TNF-receptor superfamily, mainly expressed on lymphocytes, natural killer (NK) cells, and NK-T cells. TL1A\u2013DR3 interaction results in co-stimulatory signaling for activated T cells, leading to amplification of inflammation and immune responses, correlated with greater pathogenicity in diverse autoimmune diseases. In contrast, in activated B cells the TL1A/DR3 system exerts inhibitory effect on cell proliferation, suggesting that TL1A may also have modulatory and homeostatic functions on B-cell expansion. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults in Western countries. It is well documented that several elements within the tumor microenvironment, including antigens, cytokines, adhesion molecules, and surface receptors, play a fundamental role in supporting the growth of CLL. In contrast, little is known on regulatory mechanisms of CLL growth. In this study, we have investigated the possible regulatory role of the TL1A/DR3 system in B cells from CLL patients. CLL patients from the Hematology Unit at the University Hospital of Verona (Italy) were included in this study (n=37). Disease characteristics and demographic variables were collected on all patients. Purified B cells were obtained by negative selection. DR3 expression was measured on peripheral blood B cells by flow cytometry and western blot at baseline and following B cell receptor (BCR) stimulation with anti-human IgM. DR3 expression was confirmed on lymph-node CLL specimens by immunofluorescence. Metabolic activity of CLL B cells was analyzed by MTT assay. Apoptosis was analyzed by Annexin V assay. TL1A serum levels in CLL patients were measured by ELISA. Baseline analysis in vitro showed that DR3 was expressed at low levels in CLL B cells. Cell activation through stimulation of the BCR induced a significant increase of DR3 expression in a fraction of CLL B cells (p<0.001). Higher levels of DR3 expression were associated with early-stage (Rai 0) disease (p=0.019). The relevance of these findings was confirmed by immuofluorescence analysis of B-CLL lymph-node specimens showing that DR3 was expressed at high levels in some CLL B cells in vivo. Treatment of purified CLL B cells with exogenous recombinant TL1A in vitro, in the presence of BCR stimulation, induced a decreased metabolic activity in 3/8 B-CLL cell samples (37.5%). No change in CLL metabolic activity was observed following treatment with TL1A alone, in the absence of BCR stimulation. Treatment of B-CLL samples with TL1A, either in the presence or absence of BCR stimulation, induced no changes in cell viability, thus ruling out that decreased metabolic activity was due to reduced survival. A soluble form of TL1A was detected in the sera of CLL patients and higher serum levels of TL1A were significantly associated with early-stage (Rai 0) disease (p=0.023) and absence of CD38 expressions (p=0.035). In summary, this study shows that B-CLL metabolic activity can be reduced through the activity of the TL1A/DR3 system, in the presence of the BCR stimulation. Furthermore, TL1A and DR3 levels are higher in patients with early-stage disease. Taken together, these findings suggest that the TL1A/DR3 system in vivo, in the presence of antigen stimulation, may modulate leukemic cell metabolism in early-stage CLL, thus influencing the clinical course of disease

B-cell chronic lymphocytic leukemia cells espress functional death receptor 3 following stimulation of the B cell receptor

Background. In the microenvironments where B-CLL develops, complex molecular networks propagate signals that confer growth advantage to CLL. Within this context, interplaying between TNF superfamily members and their cognate receptors has been shown to play a relevant role in controlling B-CLL growth and survival. TNF-like protein 1A (TL1A) is a recently discovered member of the TNF superfamily expressed on various cell types, including macrophages, dendritic cells, and endothelial cells. The TL1A cognate receptor, death receptor 3 (DR3), is a member of the TNF receptor superfamily expressed on various cell types, including activated T cells and macrophages. TL1A-DR3 interactions have been shown to modulate immune and inflammatory functions. So far, the expression of DR3 on B-lineage cells as well as possible roles of TL1A-DR3 interplaying in CLL biology has not been explored. Aims. The objective of the study was to investigate DR3 expression and function(s) in B-CLL. Methods. B cells were purified from PBMC of 23 B-CLL patients and 13 healthy donors by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and various time points following stimulation with F(ab’)2 anti-human IgM conjugated to latex microspheres. DR3 mRNA was detected by quantitative RT-PCR. Phosphorylation states of NF-kappaB, Erk1/2, and TBK1 were measured by using phospho-specific antibodies and flow cytometry in basal condition and following DR stimulation of cells with agonistic antibody at different time points (15, 30, 60, 180 minutes). Results. Although both healthy and CLL B cells did not express DR3 in basal conditions, stimulation of B cell receptor (BCR) induced a statistically significant increase of DR surface expression in healthy as well as malignant B cells (p<0.001 for both cells). Time course analysis showed that DR3 expression peaked at 24 hour after stimulation. DR3 expression was confirmed also at the mRNA level. Time course analysis showed that DR3 mRNA in B-CLL cells peaked at 2.5 hour following anti-IgM stimulation (4-fold change with respect to basal conditions). Anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells if compared with healthy B cells (p<0.05). Interestingly, when B-CLL patients were stratified by IGVH mutational status, anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells harboring unmutated IGVH genes compared with cells with mutated IGVH genes. To assess whether the anti-IgM-induced DR3 molecule was functionally active in B-CLL cells, we examined the ability of DR3 to trigger phosphorylation events in biologically relevant signaling nodes (i.e. Erk1/2, NF-kappaB, and TBK1) by stimulating cells with anti-DR3 agonistic antibodies. Phospho-specific flow cytometry analysis showed that DR3 engagement induced phosphorylation of Erk1/2 but not NF-kappaB or TBK1. Conclusions. We described for the first time the expression of functional DR3 molecules in B-lineage cells activated by BCR stimulation in healthy and pathological cells (i.e. B-CLL cells). The findings that anti-IgM-induced DR3 expression was higher in B-CLL cells if compared with healthy B cells and that, among B-CLL patients, this expression was higher in cells with unmutated IGVH genes, suggest that DR3 stimulation may have a role in B-CLL pathogenesis

In Vitro and In Vivo Model of EBV-Positive Non-Hodgkin Plasmablastic Lymphoma with Focal Plasmacytic Differentiation

Assessment of EBV-positive large B-cell lympoma cell line with plasmacytic differentiatio

Phospho-tyrosine profiling of mantle cell lymphoma cells suggests the BCR signalling pathwayn as a potential therapeutic target

The BCR signalling pathway is therapeutically promising, and recent data indicate that it might be important in the pathogenesis of B-CLL and DLBCL. Its importance in MCL was suggested by gene expression studies. We show that tonic BCR signalling might be one of the mechanisms driving cell survival and proliferation in MCL, and that the inhibition of SYK might be a viable therapeutic approach. More studies are needed to verify the importance in vivo of this pathway

MicroRNA signatures and Foxp3+ cell count correlate with relapse occurrence in follicular lymphoma

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3+ cells number. Notably, Foxp3+ cells were significantly less in relapsed patients and lower Foxp3+ cell number associated with shorter time-to-relapse. Foxp3+ cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3+ cells might be predictive of disease relapse

Oral Metronomic Vinorelbine (OMV) in elderly or pretreated patients with advanced non small cell lung cancer: outcome and pharmacokinetics in the real world

Background Oral metronomic therapy (OMV) is particularly suitable for palliative care, and schedules adapted for unfit patients are advisable. This study investigated the effects of oral vinorelbine given every other day without interruption and its pharmacokinetic profile in patients with advanced lung cancer. Materials and Methods Ninety-two patients received OMV at doses of 20, 30 or 50 mg. Toxic events, clinical benefit and overall survival were analysed. Blood pharmacokinetics were evaluated in 82 patients. Results Median treatment duration and overall survival were 15 (range 1.3-144) and 32.3 weeks, respectively; fourty-eight (60%) patients experienced clinical benefit. Outcomes were unrelated to previous therapies, age, histology or comorbidities. Toxicity was associated with higher blood concentrations of the drug. Pharmacokinetics were stable for up to two years, and were not influenced by treatment line or age. Conclusions OMV produced non-negligible survival in patients and also showed stable long-term blood concentrations. The schedule of 20-30 mg every other day without interruption gave good tolerability and clinical benefit

Identification of microRNAs implicated in the late differentiation stages of normal B cells suggests a central role for miRNA targets ZEB1 and TP53

In the late B cell differentiation stages, miRNAs expression modifications promoting or inhibiting key pathways are only partially defined. We isolated 29 CD19+ human B cell samples at different stages of differentiation: B cells from peripheral blood; naïve, germinal center (GC) and subepithelial (SE) B cells from tonsils. SE cells were further split in activated and resting B cell. The miRNA expression profile of these B cells was assessed by microarray analysis and selected miRNAs were validated by quantitative RT-PCR and in situ hybridization on normal tonsils. The comparison of all samples showed changes in 107 miRNAs in total. Among 48 miRNAs differentially expressed in naïve, GC and SE cells, we identified 8 miRNAs: mir-323, mir-138, mir-9*, mir-211, mir-149, mir-373, mir-135a and mir-184, strictly specific to follicular cells that had never been implicated before in late stages of B cell development. Moreover, we unveiled 34 miRNAs able to discriminate between CD5- activated B cells and resting B cells. The miRNAs profile of CD5- resting B cells showed a higher similarity to naïve CD5+ than CD5- activated B cells. Finally, network analysis on shortest paths connecting gene targets suggested ZEB1 and TP53 as key miRNA targets during the follicular differentiation pathway. These data confirm and extend our knowledge on the miRNAs-related regulatory pathways involved in the late B cell maturation