Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.

In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt's lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC-translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.

In recent years, the outcome of mantle cell lymphoma (MCL) has improved, especially in younger patients, receiving cytarabine-containing chemoimmunotherapy and autologous stem cell transplantation. Nevertheless, a proportion of MCL patients still experience early failure. To identify biomarkers anticipating failure of intensive chemotherapy in MCL, we performed target resequencing and DNA profiling of purified tumor samples collected from patients enrolled in the prospective FIL-MCL0208 phase 3 trial (high-dose chemoimmunotherapy followed by autologous transplantation and randomized lenalidomide maintenance). Mutations of KMT2D and disruption of TP53 by deletion or mutation associated with an increased risk of progression and death, both in univariate and multivariate analysis. By adding KMT2D mutations and TP53 disruption to the MIPI-c backbone, we derived a new prognostic index, the "MIPI-genetic" ("MIPI- g"). The "MIPI-g" improved the model discrimination ability compared to the MIPI-c alone, defining three risk groups: i) low-risk patients (4-year progression free survival and overall survival of 72.0% and 94.5%); ii) inter-mediate-risk patients (4-year progression free survival and overall survival of 42.2% and 65.8%) and iii) high-risk patients (4-year progression free survival and overall survival of 11.5% and 44.9%). Our results: i) confirm that TP53 disruption identifies a high-risk population characterized by poor sensitivity to conventional or intensified chemotherapy; ii) provide the pivotal evidence that patients harboring KMT2D mutations share the same poor outcome as patients harboring TP53 disruption; and iii) allow to develop a tool for the identification of high-risk MCL patients for whom novel therapeutic strategies need to be investigated. (Trial registered at clinicaltrials.gov identifier: NCT02354313).

Mantle cell lymphoma patients have variable clinical courses, ranging from indolent cases that do not require immediate treatment to aggressive, rapidly progressing diseases. Thus, diagnostic tools capable of stratifying patients according to their risk of relapse and death are needed. This study included 83 samples from the Fondazione Italiana Linfomi MCL-0208 clinical trial. Through gene expression profiling and quantitative real-time PCR we analyzed 46 peripheral blood and 43 formalin-fixed paraffin-embedded lymph node samples. A prediction model to classify patients was developed. By analyzing the transcriptome of 27 peripheral blood samples, two subgroups characterized by a differential expression of genes from the B-cell receptor pathway (B-cell receptorlow and B-cell receptorhigh) were identified. The prediction model based on the quantitative real-time PCR values of six representative genes (AKT3, BCL2, BTK, CD79B, PIK3CD, and SYK), was used to classify the 83 cases (43 B-cell receptorlow and 40 B-cell receptorhigh). The B-cell receptorhigh signature associated with shorter progression-free survival (P=0.0074), selected the mantle cell lymphoma subgroup with the shortest progression-free survival and overall survival (P=0.0014 and P=0.029, respectively) in combination with high (>30%) Ki-67 staining, and was an independent predictor of short progression- free survival along with the Mantle Cell Lymphoma International Prognostic Index-combined score. Moreover, the clinical impact of the 6- gene signature related to the B-cell receptor pathway identified a mantle cell lymphoma subset with shorter progression-free survival intervals also in an external independent mantle cell lymphoma cohort homogenously treated with different schedules. In conclusion, this 6-gene signature associates with a poor clinical response in the context of the MCL- 0208 clinical trial. (clinicaltrials.gov identifier: 02354313).

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

Splenic marginal zone lymphoma is a rare lymphoma. Loss of 7q31 and somatic mutations affecting the NOTCH2 and KLF2 genes are the commonest genomic aberrations. Epigenetic changes can be pharmacologically reverted; therefore, identification of groups of patients with specific epigenomic alterations might have therapeutic relevance. Here we integrated genome-wide DNA-promoter methylation profiling with gene expression profiling, and clinical and biological variables. An unsupervised clustering analysis of a test series of 98 samples identified 2 clusters with different degrees of promoter methylation. The cluster comprising samples with higher-promoter methylation (High-M) had a poorer overall survival compared with the lower (Low-M) cluster. The prognostic relevance of the High-M phenotype was confirmed in an independent validation set of 36 patients. In the whole series, the High-M phenotype was associated with IGHV1-02 usage, mutations of NOTCH2 gene, 7q31-32 loss, and histologic transformation. In the High-M set, a number of tumor-suppressor genes were methylated and repressed. PRC2 subunit genes and several prosurvival lymphoma genes were unmethylated and overexpressed. A model based on the methylation of 3 genes (CACNB2, HTRA1, KLF4) identified a poorer-outcome patient subset. Exposure of splenic marginal zone lymphoma cell lines to a demethylating agent caused partial reversion of the High-M phenotype and inhibition of proliferation.

Increased numbers of tumour-associated macrophages correlate with shortened survival in some cancers. The molecular bases of this correlation are not thoroughly understood. Events triggered by CXCL12 may play a part, as CXCL12 drives the migration of both CXCR4-positive cancer cells and macrophages and may promote a molecular crosstalk between them.

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3-independent growth in Ba/F3 cells and/or IL-9-independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

The relation between Epstein-Barr virus (EBV) DNA load and clinical course of patients with chronic lymphocytic leukemia (CLL) is unknown. We assessed EBV DNA load by quantitative PCR at CLL presentation in mononuclear cells (MNC) of 220 prospective patients that were enrolled and followed-up in two major Institutions. In 20 patients EBV DNA load was also assessed on plasma samples. Forty-one age-matched healthy subjects were tested for EBV DNA load on MNC. Findings were validated in an independent retrospective cohort of 112 patients with CLL. EBV DNA load was detectable in 59%, and high (≥2000 copies/µg DNA) in 19% of patients, but it was negative in plasma samples. EBV DNA load was significantly higher in CLL patients than in healthy subjects (P < .0001). No relation was found between high EBV load and clinical stage or biological variables, except for 11q deletion (P = .004), CD38 expression (P = .003), and NOTCH1 mutations (P = .05). High EBV load led to a 3.14-fold increase in the hazard ratio of death and to a shorter overall survival (OS; P = .001). Poor OS was attributable, at least in part, to shorter time-to-first-treatment (P = .0008), with no higher risk of Richter's transformation or second cancer. Multivariate analysis selected high levels of EBV load as independent predictor of OS after controlling for confounding clinical and biological variables. EBV DNA load at presentation is an independent predictor of OS in patients with CLL.

B lymphocyte stimulator (BLyS) is a novel member of the TNF ligand superfamily that is important in B cell maturation and survival. We demonstrate that human neutrophils, after incubation with G-CSF or, less efficiently, IFN gamma, express high levels of BLyS mRNA and release elevated amounts of biologically active BLyS. In contrast, surface expression of the membrane-bound BLyS was not detected in activated neutrophils. Indeed, in neutrophils, uniquely among other myeloid cells, soluble BLyS is processed intracellularly by a furin-type convertase. Worthy of note, the absolute capacity of G-CSF-stimulated neutrophils to release BLyS was similar to that of activated monocytes or dendritic cells, suggesting that neutrophils might represent an important source of BLyS. In this regard, we show that BLyS serum levels as well as neutrophil-associated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. In addition, serum obtained from two of these patients induced a remarkable accumulation of neutrophil-associated BLyS in vitro. This effect was neutralized by anti-G-CSF antibodies, indicating that G-CSF, present in the serum, stimulated neutrophils to produce BLyS. Collectively, our findings suggest that neutrophils, through the production of BLyS, might play an unsuspected role in the regulation of B cell homeostasis.

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.

small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

In vitro Omics analysis (i.e. transcriptome) is suggested to predict in vivo toxicity and adverse effects in humans, although the causal link between high-throughput data and effects in vivo is not easily established. Indeed, the chemical-organism interaction can involve processes, such as adaptation, not established in cell cultures. Starting from this consideration we investigate the transcriptomic response of immortalized thyrocytes to ethylenthiourea and chlorpyrifos. In vitro data revealed specific and common genes/mechanisms of toxicity, controlling the proliferation/survival of the thyrocytes and unrelated hematopoietic cell lineages. These results were phenotypically confirmed in vivo by the reduction of circulating T4 hormone and the development of pancytopenia after long exposure. Our data imply that in vitro toxicogenomics is a powerful tool in predicting adverse effects in vivo, experimentally confirming the vision described as Tox21c (Toxicity Testing in the 21st century) although not fully recapitulating the biocomplexity of a living animal.

We showed that α-bisabolol is active against primary acute leukemia cells, including BCR-ABL(+) acute lymphoblastic leukemias (ALL). Here we studied the activity of α-bisabolol against BCR-ABL(+) cells using 3 cell lines (K562, LAMA-84, CML-T1) and 10 primary BCR-ABL(+) ALL samples. We found that: (a) α-bisabolol was effective in reducing BCR-ABL(+) cell viabilty at concentrations ranging from 53 to 73 µM; (b) α-bisabolol concentrations in BCR-ABL(+) cellular compartments were 4- to 12-fold higher than in normal cells, thus indicating a preferential intake in neoplastic cells; (c) α-bisabolol displayed a slight to strong synergism with the Tyrosine Kinase Inhibitors (TKI) imatinib and nilotinib: the combination of α-bisabolol+imatinib allowed a dose reduction of each compound up to 7.2 and 9.4-fold respectively, while the combination of α-bisabolol+nilotinib up to 6.7 and 5-fold respectively; (d) α-bisabolol-induced apoptosis was associated with loss of plasma membrane integrity, irreversible opening of mitochondrial transition pore, disruption of mitochondrial potential, inhibition of oxygen consumption and increase of intracellular reactive oxygen species. These data indicate α-bisabolol as a candidate for treatment of BCR-ABL(+) leukemias to overcome resistance to TKI alone and to target leukemic cells through BCR-ABL-independent pathways.

Diagnosing any of the more than 30 types of T-cell lymphomas is considered a challenging task for many pathologists and currently requires morphological expertise as well as the integration of clinical data, immunophenotype, flow cytometry and clonality analyses. Even considering all available information, some margin of doubt might remain using the current diagnostic procedures. In recent times, the genetic landscape of most T-cell lymphomas has been elucidated, showing a number of diagnostically relevant mutations. In addition, recent data indicate that some of these genetic alterations might bear prognostic and predictive value. Extensive genetic analyses, such as whole exome or large panel sequencing are still expensive and time consuming, therefore limiting their application in routine diagnostic. We therefore devoted our effort to develop a lean approach for genetic analysis of T-cell lymphomas, focusing on maximum efficiency rather than exhaustively covering all possible targets. Here we report the results generated with our small amplicon-based panel that could be used routinely on paraffin-embedded and even decalcified samples, on a single sample basis in parallel with other NGS-panels used in our routine diagnostic lab, in a relatively short time and with limited costs. We tested 128 available samples from two German reference centers as part of our routine work up (among which 116 T-cell lymphomas), which is the largest routine diagnostic series reported to date. Our results showed that this assay had a very high rate of technical success (97%) and could detect mutations in the majority (79%) of tested T-cell lymphoma samples.

We previously reported that t(14;18)-negative follicular lymphomas (FL) show a clear reduction of newly acquired N-glycosylation sites (NANGS) in immunoglobulin genes. We therefore aimed to investigate in-depth the occurrence of NANGS in a larger cohort of t(14;18)-positive and t(14;18)-negative FL, including early (I/II) and advanced (III/IV) stage treatment-naive and relapsed tumors. The clonotype was determined by using a next-generation sequencing approach in a series of 68 FL with fresh frozen material [36 t(14;18) positive and 32 t(14;18) negative]. The frequency of NANGS differed considerably between t(14;18)-positive and t(14;18)-negative FL stage III/IV, but no difference was observed among t(14;18)-positive and t(14;18)-negative FL stage I/II. The introduction of NANGS in all t(14;18)-negative clinical subgroups occurred significantly more often in the FR3 region. Moreover, t(14;18)-negative treatment-naive FL, specifically those with NANGS, showed a strong bias for IGHV4-34 usage compared with t(14;18)-positive treatment-naive cases with NANGS; IGHV4-34 usage was never recorded in relapsed FL. In conclusion, subgroups of t(14;18)-negative FL might use different mechanisms of B-cell receptor stimulation compared with the lectin-mediated binding described in t(14;18)-positive FL, including responsiveness to autoantigens as indicated by biased IGHV4-34 usage and strong NANGS enrichment in FR3.

TNF-like ligand 1A (TL1A) and its unique receptor death receptor 3 (DR3) acts as broad T-cell costimulator involved in regulatory mechanisms of adaptive immune response under physiological and pathological settings. Moreover, we have recently shown that TL1A negatively regulates B-cell proliferation. Despite increasing interest on the TL1A/DR3-axis functions, very little is known on its expression and role in leukemia. In this study, we investigated the expression and function of TL1A/DR3 axis in chronic lymphocytic leukemia (CLL). DR3 was differentially expressed in activated CLL cells and predominantly detected in patients with early clinical stage disease. Soluble TL1A has been revealed in the sera of CLL patients where higher TL1A levels were associated with early stage disease. T cells, monocytes and leukemic B cells have been identified as major sources of TL1A in CLL. The relevance of these findings has been sustained by functional data showing that exogenous TL1A reduces CLL proliferation induced by stimulation of the B cell receptor. Overall, these data document the expression of the TL1A/DR3 axis in early-stage CLL. They also identify a novel function for TL1A as a negative regulator of leukemic cell proliferation that may influence the CLL physiopathology and clinical outcome at an early-stage disease.

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.

To describe CT characteristics of primary pancreatic lymphoma (PPL), a rare disease with features in common with adenocarcinoma.