Luciana Bergui

Cytokines involved in the progression of multiple myeloma.

We have investigated which of the cytokines that are relevant in the in vitro growth of multiple myeloma (MM) malignant plasma cells are actually produced in vivo by MM patients. To this end, we have measured the levels of IL‐1β, IL‐3, IL‐4, IL‐6, IL‐7, IL‐8 and tumour necrosis factor‐alpha (TNF‐α) both in sera and in the supernatant of bone marrow (BM) stromal cell cultures from patients with MM and monoclonal gammopathy of undetermined significance (MGUS). The significance of our findings is three‐fold. First, IL‐6 and IL‐8 are produced by MM BM stromal cells, while IL‐1β, TNF‐α, IL‐4 and IL‐7 are not. Second, IL‐3 is the only cytokine consistently raised in serum samples; we have also detected low levels of serum IL‐6 in a minority of cases, usually in advanced stage of the disease. Third, MM BM stromal cells are active IL‐6 and IL‐8 producers, while both normal and MGUS BM stromal cells are low producers, thus suggesting that in the BM of MM a number of environmental cells, that would normally be quiescent, are instead activated and that, in MM, activated BM stromal cells play an active role in supporting the progressive expansion of the B cell clone.

CD38 increases CXCL12-mediated signals and homing of chronic lymphocytic leukemia cells.

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (P<0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (P<0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38− CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P=0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

CD38 gene polymorphism and chronic lymphocytic leukemia: a role in transformation to Richter syndrome?

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

CD38 at the junction between prognostic marker and therapeutic target

CD38 is an ectoenzyme involved in transmembrane signaling and cell adhesion and is used as a disease marker for leukemias and myeloma. CD38 is a dependable negative prognostic marker for chronic lymphocytic leukemia (CLL). Recent evidence indicates that CD38 is a component of a complex network delivering growth and survival signals to CLL cells. In conjunction with chemokines and their receptors, CD38 also influences cell migratory responses. These considerations are the rationale for devising a CLL therapy that uses CD38 as the target. The use of reagents specifically blocking the molecule might provide a new approach for interfering with deleterious growth circuits, therefore increasing the susceptibility of leukemic cells to conventional chemotherapy.

CD38/CD31 interactions activate genetic pathways leading to proliferation and migration in chronic lymphocytic leukemia cells.

Human CD38 is a pleiotropic glycoprotein belonging to a family of enzymes/receptors involved in the catabolism of extracellular nucleotides. CD38-receptor activities are regulated through binding to the nonsubstrate ligand CD31. CD38 expression above a critical threshold is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. Activation of CD38 by means of agonistic monoclonal antibodies or the CD31 ligand induces proliferation and immunoblast differentiation of CLL cells. Here we define the genetic signature that follows long-term in vitro interactions between CD38+ CLL lymphocytes and CD31+ cells. The emerging profile confirms that the CD31/CD38 axis activates genetic programs relevant for proliferative responses. It also indicates a contribution of this pathway to the processes mediating migration and homing. These results further support the notion that the CD31/CD38 axis is part of a network of accessory signals that modify the microenvironment, favoring localization of leukemic cells to growth-permissive sites.

Characterization of bone marrow stromal cells from multiple myeloma

We have cultured multiple myeloma (MM) bone marrow (BM) stromal cells that are able to sustain the in vitro growth of monoclonal B-cells. Our aim was to evaluate which adhesion molecules are expressed and which extracellular matrix proteins are produced by these cells and whether they differ from the stromal cells that can be grown under the same experimental conditions from the BM of monoclonal gammopathies of undetermined significance (MGUS) and of normal donors. MM BM stromal cells that support malignant B-cell development have a striking proliferative ability that is absent in MGUS and normal donors of the same age group and are formed by four major different cell populations. Two kinds of HLA-DR+, CD10+ fibroblast-like cells can be recognized through the expression (or the lack) of alpha-smooth muscle actin isoform; further, macrophages and osteoclasts can be identified. Fibroblast-like cells that express alpha-smooth muscle actin isoform, often organized along stress fibers in a periodic fashion, may be considered as myofibroblasts. Fibroblast-like cells react strongly with antibodies to CD54 (ICAM-1), integrin beta 1, beta 3, beta 5 and some of associated alpha chains. Integrin beta 1 is diffusely exposed on the surface while beta 3 is clustered in focal contacts in association with vinculin. A still undetermined subpopulation of fibroblasts is highly positive for alpha v beta 5 that is clustered at focal contacts as shown by association with stress fiber termini and by interference reflection microscopy. A major difference between MM and normal donor BM stromal cells involves lower deposition and simpler organization of the extracellular matrix proteins (fibronectin, laminin, collagen type IV) deposited by MM fibroblast-like cells. CD14+ macrophages from MM, MGUS and normal donor BM are CD11a+ (alpha L), CD11b+ (alpha M), CD11c+ (alpha X), CD54+ (ICAM-1), CD56+ (N-CAM), beta 1 and beta 2 (CD18) integrin positive. The integrin beta 1 is diffusely expressed on the surface, while beta 2 is concentrated in podosomes. MM osteoclasts show a weak diffuse staining with CD54 and CD56 MoAbs; beta 1 integrin has a diffuse surface expression, while beta 3 integrin is concentrated in the podosomes. Normal donor osteoclasts are CD54- and the staining with CD56 is barely visible. These findings lead us to suggest that the microenvironment provided by MM BM may be significantly different from that of normal BM indicating its potential role in controlling the local proliferation and differentiation of malignant B-lineage cells.

Bone Marrow Microenvironment and the Progression of Multiple Myeloma

The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1 beta and M-CSF activating BM stromal cells to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-1 beta, TNF-beta, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population.

Fludarabine ability to down-regulate Bcl-2 gene product in CD5+ leukaemic B cells: in vitro/in vivo correlations.

CD5+ B‐chronic lymphocytic leukaemia (B‐CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl‐2 family of genes. Fludarabine (9‐β‐D‐arabinofuranosyl‐2‐fluoradenine, F‐ara‐A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F‐ara‐A‐induced apoptosis might be related to Bcl‐2 modifications and to evaluate in vitro/in vivo correlations.  Peripheral blood lymphocytes from eight B‐CLL and four leukaemic MCL were cultured in the presence of different concentrations of F‐ara‐A ±methylprednisolone (MP). F‐ara‐A down‐regulated the expression of Bcl‐2 in 5/12 cases. mRNA down‐regulation was maximal at 48 h; protein down‐regulation was prominent after 48 h. Both events were dose‐dependent. The amount of apoptosis was significantly higher in the samples treated with F‐ara‐A than in those exposed to MP alone. In the seven remaining cases, no Bcl‐2 down‐regulation was observed after exposure to F‐ara‐A and the degree of F‐ara‐A‐induced apoptosis overlapped that induced by MP.  The in vivo outcome after treatment with three to six courses of F‐ara‐A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl‐2 down‐regulation and prominent apoptosis after exposure to F‐ara‐A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl‐2 modulation after exposure to F‐ara‐A, two had a PR, but the other three did not show any in vivo clinical response.

B‐chronic lymphocytic leukaemia patients with stable benign disease show a distinctive membrane phenotype

Summary. The circulating B cells of 98 patients with B‐CLL were analysed with a panel of conventional markers and monoclonal antibodies including RFA‐1 and RFA‐4. A subgroup of patients was defined with a distinctive double membrane marker phenotype (RFA‐1+, RFA‐4+). These patients showed features of clinical stability. Their physical signs, haematological features and lymphocyte counts remained unchanged for 5–10 years: therefore, the RFA‐1+, RFA‐4+ membrane phenotype appears to identify a subgroup of B‐CLL patients with a particularly non‐progressive disease.

Phenotypic, cytogenetic and molecular characterization of a new B-chronic lymphocytic leukaemia (B-CLL) cell line

A lymphoid cell line was established from a patient with B-cell chronic lymphocytic leukaemia (B-CLL) by infecting blood lymphocytes with Epstein-Barr virus (EBV). Immunoglobulin gene rearrangement studies and the presence of a chromosome marker (isochromosome 17q) provided the formal proof that the line has originated from the neoplastic B cells. The morphology and phenotype indicate that the EBV-induced cell line has reached a plasma cell-like stage of differentiation.