Roberta Maggio

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgVH unmutated chronic lymphocytic leukemia (CLL) cells

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

Spontaneous regression of chronic lymphocytic leukemia: clinical and biologic features of 9 cases

In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year follow-up, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V(H)3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V(kappa)4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCR-related and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-gamma, TNF-alpha, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion, spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V(H)3-30 and V(kappa)4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression.

Phenotypic and functional characterization of monocyte-derived dendritic cells in chronic lymphocytic leukaemia patients: influence of neoplastic CD19 + cells in vivo and in vitro

Dendritic cells (DCs) are the most potent antigen‐presenting cells and are therefore an attractive option as antigen carriers in vaccination protocols. Chronic lymphocytic leukaemia (CLL) represents a potential good target for these approaches. The present study was designed to investigate the feasibility of generating in vitro fully functional DCs from peripheral blood (PB) monocytes of CLL patients at different phases of the disease. Although functional DCs could be obtained from CLL samples, in patients with active disease the expression of some co‐stimulatory molecules appeared to be reduced. In contrast, DCs from CLL patients in remission showed no difference from those of normal controls. Moreover, patients with active disease produced DCs with reduced allostimulatory ability when compared with normal ones, whereas the functional capacities appeared to be restored in CLL DCs from remission patients. To more precisely assess the possible inhibitory effect of CLL cells on DC development, the influence of autologous leukaemic CD19+ cells on the generation of monocyte‐derived CLL DCs in vitro was investigated. The addition of CLL neoplastic cells markedly affected monocyte‐derived DC maturation. In conclusion, monocytes from CLL patients with active disease give rise to DCs, which show phenotypic and functional defects that are not observed in remission CLL patients. These results need to be taken into account in the design of DC‐based immunotherapeutic approaches in CLL.

FLT3 inhibition in t(4;11)+ adult acute lymphoid leukaemia

The present study was designed to investigate, in t(4;11)+ adult lymphoid leukaemia (ALL) blast cells, the pathogenetic role of the FLT3 protein, its level of mRNA and protein expression, the degree of constitutive phosphorylation, the possible presence of mutations of the sequence, the capacity of signal transduction and the potential therapeutic role of specific inhibitors. We evaluated nine adult ALL patients carrying this translocation. The increased FLT3 mRNA levels, determined by oligonucleotide microarray analysis, was in agreement with the increased protein expression evaluated by Western blot. The protein was constitutively phosphorylated in all cases analysed. Polymerase chain reaction detected no internal tandem duplication or point mutations. The signal transduction apparatus, after stimulation with the specific ligand, was preserved. We then investigated the effect of specific FLT3 inhibition on signal transduction and survival. The PKC412 inhibitor specifically inhibited ligand‐induced phosphorylation; the same inhibitor reduced the survival of leukaemic cells when compared with untreated cells. These data indicate that the FLT3 protein might play a role in this subgroup of ALL with a particularly poor prognosis. Specific inhibition of the kinase receptor must be hypothesised as an innovative therapeutic tool for t(4;11)+ ALL patients.

Environmental stimuli shape microglial plasticity in glioma

In glioma, microglia and infiltrating macrophages are exposed to factors that force them to produce cytokines and chemokines, which contribute to tumor growth and to maintaining a pro-tumorigenic, immunosuppressed microenvironment. We demonstrate that housing glioma-bearing mice in enriched environment (EE) reverts the immunosuppressive phenotype of infiltrating myeloid cells, by modulating inflammatory gene expression. Under these conditions, the branching and patrolling activity of myeloid cells is increased, and their phagocytic activity is promoted. Modulation of gene expression depends on interferon-(IFN)-γ produced by natural killer (NK) cells. This modulation disappears in mice depleted of NK cells or lacking IFN-γ, and was mimicked by exogenous interleukin-15 (IL-15). Further, we describe a key role for brain-derived neurotrophic factor (BDNF) that is produced in the brain of mice housed in EE, in mediating the expression of IL-15 in CD11b+ cells. These data define novel mechanisms linking environmental cues to the acquisition of a pro-inflammatory, anti-tumor microenvironment in mouse brain.

Immunocompetent cell functions in Ph + acute lymphoblastic leukemia patients on prolonged Imatinib maintenance treatment

Imatinib mesylate (Imatinib) is a potent inhibitor of defined tyrosine kinases and is effectively used for the treatment of malignancies characterized by the constitutive activation of these tyrosine kinases, such as Philadelphia chromosome-positive (Ph+) leukemias and gastrointestinal stromal tumors. Suppressive as well as stimulating effects of this drug on T lymphocytes or dendritic cells (DC), which play a major role in immune tumor surveillance, have been reported. For this reason, we questioned whether Imatinib could also affect the phenotypic and functional properties of these subpopulations in Ph+ acute lymphoblastic leukemia (ALL) patients on prolonged Imatinib maintenance treatment. Circulating T lymphocytes and NK cells from Imatinib-treated Ph+ ALL patients showed a subset distribution comparable to that of healthy donors. In addition, T-cell immunomodulant cytokine production (IFN-γ, TNF-α) and proliferative responses were not impaired. A normal monocyte-derived DC differentiation and apoptotic body loading capacity was also observed in the majority of Imatinib-treated patients. In contrast, an impairment in the DC intracellular production of IL-12 was recorded, although this was not observed when normal DC were exposed in vitro to Imatinib. Finally, in vivo Imatinib treatment did not affect the T-lymphocyte proliferation and IFN-γ production induced by leukemic apoptotic body–loaded DC, underling the potential capability of these cells to generate a specific immune response against tumoral antigens. Taken together, these findings provide evidence that immunotherapeutic approaches aimed at controlling residual disease in Ph+ ALL patients in hematologic remission are not jeopardized by the long-term administration of Imatinib.

Clinical responses in allografted acute leukaemia patients with resistant disease using a combined chemo‐immunotherapeutic treatment strategy

Induction of a graft-versus-leukaemia (GVL) immunological response is the key element for the success of the allogeneic haematopoietic stem cell transplant (SCT) procedure. This was first shown by the durable remissions induced by donor lymphocyte infusion (DLI) in relapsed chronic myeloid leukaemia (CML) patients (Kolb et al, 1995). For patients affected by non-CML haematological malignancies, however, this immunological approach is not always successful (Collins et al, 1997; Dermime et al, 1997). Different strategies have been conceived to overcome tumour resistance to donor lymphocytes. Chemotherapy is particularly attractive for both its cytoreductive and immune-potentiating activities (Emens et al, 2001), including provision of lymphoid space, elimination of host anti-donor immune reactivity, suppression of regulatory T cells and induction of activating cytokines (Klebanoff et al, 2005; Rapoport et al, 2005). Animal models of tumour-bearing mice demonstrated that the serum peak concentrations of activating cytokines and growth factors was reached 48 h after a cyclophosphamide-based treatment (Proietti et al, 1998; Bracci et al, 2007). In humans, it has been reported that a fully lymphodepleting chemotherapeutic treatment prior to DLI induces a significant increment of high grade acute graft-versushost disease (GVHD) (Miller et al, 2007), suggesting the possibility of enhancing the anti-tumour efficacy of donor lymphocytes. The present study aimed to analyse the immunological and clinical effects of a chemo-immunotherapeutic treatment strategy consisting of chemotherapy followed 48 h later by DLI in allografted acute leukaemia patients. Four patients with acute myeloid leukaemia (AML) that evolved from a myelodysplastic syndrome (MDS) and two