Whole-body MRI with diffusion-weighted imaging: a valuable alternative to contrast-enhanced CT for initial staging of aggressive lymphoma.

AIM: To compare the accuracy of whole-body magnetic resonance imaging (Wb-MRI) with diffusion-weighted imaging (DWI) to that of contrast-enhanced computed tomography (CE-CT) and 2-[(18)F]-fluoro-2-deoxy-d-glucose ((18)F-FDG) positron-emission tomography co-registered with low dose-CT (PET-CT) in defining lymphoma disease stage. MATERIALS AND METHODS: From February 2010 to May 2014, 41 lymphoma patients underwent Wb-MRI-DWI, CE-CT, and (18)F-FDG PET-CT. Histological subtypes included aggressive B-cell (n=11), follicular (n=13), mantle cell (n=3), and Hodgkin's (n=14) lymphoma. To compare the procedures, the reference standard (RS) assessment was defined by combining the results from (18)F-FDG PET-CT, CE-CT, and bone marrow (BM) histology, modifications after therapy, and histological re-assessments of uncertain lesions. RESULTS: Among 1025 nodal sites, 217 had disease involvement according to the RS. CE-CT yielded 23 false-negative and 11 false-positive errors. Wb-MRI-DWI failed to recognise 17 localisations and had six false-positive errors; (18)F-FDG PET-CT had no errors. Among 458 extranodal sites, 37 were positive according to the RS. (18)F-FDG PET-CT yielded four false-negative and two false-positive results. CE-CT yielded 17 false-negative errors. Wb-MRI-DWI yielded a single false-negative error. Wb-MRI-DWI was the most reliable imaging technique for BM evaluation. Considering each procedure alone, the final stage would have been missed in four cases using (18)F-FDG PET-CT, 12 cases using CE-CT, and none using Wb-MRI-DWI. CONCLUSION: The present data support Wb-MRI-DWI as a sensitive and specific imaging technique for lymphoma evaluation, supporting its use in place of CE-CT for staging.

Molecular investigation of the cytokines produced by normal and malignant B lymphocytes.

Different normal and malignant human B-cell populations were studied with a twofold aim: to define which cytokines are produced in vivo, and to assess the relationship between cytokine production and kinetic state. To analyse normal B-cells representative of different stages of activation and proliferation in vivo, we purified germinal centre (GC)-B blasts and mantle B (M-B) cells from tonsils. To compare malignant B lymphocytes with their closest normal equivalent cells, we separated malignant CD5+B lymphocytes from the peripheral blood of patients with B-chronic lymphocytic leukemia (B-CLL) and normal CD5+B lymphocytes from cord blood. The expression of interleukins (IL) IL-1 alpha, IL-1 beta, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), IL-2, IL-4, and IL-6 genes was analysed using Northern and Western blotting techniques. TNF-alpha mRNA is produced by resting (M-B) and actively proliferating (GC-B) normal B lymphocytes. TGF-beta mRNA is present at high levels in resting normal M-B cells, while the transcript levels are lower in proliferating GC-B and in activated CD5+B lymphocytes. IL-2 production is limited to the actively proliferating GC-B blasts, IL-1 beta and IL-6 to resting M-B cells. The cytokine production profile of CD5+ malignant B-CLL cells differs from that of their putative normal counterparts and is more like the profile of M-B cells, since B-CLL cells produce IL-1 beta, TNF-alpha, TGF-beta, and IL-6. These observations lead to the following conclusions: among normal B lymphocyte populations, resting M-B lymphocytes are the most active cytokine producers, and B-CLL malignant B cells reflect the production pattern of normal resting B lymphocytes.

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.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

The role of Bcl-2 in the pathogenesis of B chronic lymphocytic leukemia.

At least three categories of genes are envisaged to be involved in the natural history of B-CLL. First, the genes that are responsible for the transforming event(s) in the (presently unknown) target cell; second, the gene(s) that help the progressive accumulation of malignant cells and finally the gene(s) that cause the progression toward a more aggressive lymphoma. The possibility that the clonal expansion of B-CLL is due to a prolonged life-span of monoclonal B cells rather than to an acceleration of their proliferative activity may now be reinterpreted by taking into account some recent findings on the expression of Bcl-2 gene in B-CLL cells. The Bcl-2 gene product regulates programmed cell death and a number of experiments suggest that Bcl-2 is involved in the selection and maintenance of long-lived memory B cells rescuing them from apoptotic death and leading to their accumulation in the GO phase of the cell cycle. Variant chromosomal translocations have been detected in a small fraction (5-10%) of B-CLL, involving Bcl-2 and the Ig light chain gene. Despite the low percentage of Bcl-2 rearrangements the expression of mRNA and protein is appreciable in most samples of fresh B-CLL cells in an amount comparable to that observed in Karpas 422 cells, which contain a t(14;18).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

CD38 in chronic lymphocytic leukemia: from bench to bedside?

Human CD38 is a cell surface molecule endowed with multiple functions. As an enzyme, it catalyzes the production of Ca2+ active metabolites, predominantly cADPR and ADPR. As a receptor, it regulates the activation of an intracellular signaling pathway, generally linked to lymphocyte activation and proliferation in physiological conditions. The finding that CD38 behaves as an independent negative prognostic factor in CLL patients was the starting point for investigations into the functional role of the molecule in the neoplastic context. Data accumulating in over a decade concur to define a model where CD38 is a central element of a large supramolecular complex that includes surface signaling receptors, chemokine receptors, adhesion molecules and matrix metalloproteases. Expression of CD38 within this supramolecular complex makes signal transduction as well as chemotaxis and homing more efficient, suggesting that the molecule is an integrator of proliferative and migratory signals. These data indicate that CD38 is not only a reliable disease marker but also a functional molecule in the CLL context. The next decade will likely tell whether it can also be a useful therapeutic target.

Malignant melanoma of the gallbladder. Case report and review of the literature.

The authors are presenting a new case of malignant melanoma of the gallbladder to be added to those described in the literature. The tumour appeared as a polypoid mass of the gallbladder and had metastasized to the jejunum and the brain. Review of the literature and analysis of this case require a thorough discussion of the real existence of tumours in this unusual primary site.

Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells.

The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67-positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

The nature of the B lymphocyte in B-chronic lymphocytic leukemia.

We have analyzed phenotypic, functional, and molecular properties of B-chronic lymphocytic leukemia (B-CLL) cells as compared to normal B cell differentiation stages and/or subsets. The possibility that the target B cell population transformed by the I primary oncogenic event(s) belongs to the normal CD5+ B cell subset from B mantle zone of secondary follicles is highly likely on phenotypic grounds. Though the genes responsible for the primary oncogenic event are presently unknown, a number of functional and molecular findings indicate that the end-product of their transforming activity is a cell frozen in the G0 phase of the cell cycle. This cell has several abnormalities that prevent an appropriate mitogenic response and presents a pattern of apoptosis-related gene expression that hinders apoptotic death. Pivotal to this apoptosis-escaping capacity is the expression of Bcl-2. We suggest that the increased expression of Bcl-2 together with an asynchronism between the expression of Bcl-2, c-myc, and APO1/Fas gene products shift the cellular balance away from apoptosis thereby helping the progressive accumulation in G0 of malignant CD5+ B cells.

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis.

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B-cell chronic lymphoid leukaemias. Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase. According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells.

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-beta-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.

Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5(+) B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin(+) cells were actively proliferating and, in contrast to Survivin(+) B cells found in normal GC, were Bcl-2(+). In B-CLL BM biopsies, CD5(+), Survivin(+) cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.