Risk factors for hematological malignancy in polyneuropathy associated with monoclonal gammopathy

Polyneuropathy associated with monoclonal gammopathy of undetermined significance (MGUS) is a well-known disease entity. Of the patients with monoclonal gammopathy without neuropathy, 25% develop a hematological malignancy during long-term follow-up. Whether the frequency of hematological malignancy is similar in patients with polyneuropathy associated with monoclonal gammopathy and whether hematological screening is necessary in these patients is unknown. To determine the frequency of and risk factors for a hematological malignancy, we investigated 104 patients with polyneuropathy and monoclonal gammopathy. Potential diagnostic variables were obtained from medical history, physical and neurological examination, and laboratory analysis. The associations between potential diagnostic variables and outcome, hematological malignancy, were evaluated by univariable and multivariable logistic-regression analysis. Among our patients, 23 had a hematological malignancy (8 multiple myeloma, 10 low-grade lymphoma, 3 plasmacytoma, 1 Castleman's disease and 1 POEMS syndrome [polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes]). Weight loss, progression of the neuropathy, and an M-protein level > 1 g/L were independent risk factors for malignancy. Extensive screening is indicated in patients with these features.     

Continuous low-dose cyclophosphamide-prednisone is effective and well tolerated in patients with advanced multiple myeloma

BACKGROUND: Multiple myeloma is an incurable disease and after several lines of chemotherapy, patients enter a phase in which no standard treatment options are available. The poor outlook of these patients requires mild, palliative therapy with low toxicity. Previously used regimens either require frequent hospital attendance, lack efficacy or have significant toxicity. METHODS: In the current study, daily low dose, oral cyclophosphamide (100 mg) and prednisone (10-20 mg; CP) were administered to patients with advanced myeloma. Forty-two patients with progressive disease after melphalan-based and VAD treatment were enrolled. RESULTS: Objective responses were observed in 29 of 42 (69%) patients. In responding patients, median overall survival and progression-free survival were 22.2 months and 15.0 months, respectively. In non-responders, median OS was 3.5 months only. Side-effects were limited. Cytopenia was the most frequent event (8/29) prompting dose reduction. CP had to be stopped permanently in four patients (two cytopenia, two infections). CONCLUSION: Orally administered, low dose continuous CP is a feasible, effective and well-tolerated regimen in the management of advanced multiple myeloma.     

Effects of Ethanol on Cultured Fetal Serotonergic Neurons

In utero ethanol exposure impairs the development of several neurotransmitter systems, including the serotonergic system. However, at present the mechanism by which in utero ethanol exposure damages the developing brain is unknown. This research examined the possibility that ethanol directly impairs the development of serotonergic neurons. This hypothesis was assessed by examining the content of serotonin (5-HT), 5-HT uptake, and 5-HT immunopositive neurons in cultures of fetal rhombencephalic neurons that were exposed to ethanol for 4 days in vitro. In addition, the effects of in vitro ethanol exposure on protein and DNA content of cultured rhombencephalic neurons were determined.
These studies demonstrated that a 4-day exposure of cultured rhombencephatic neurons to 50 to 300 mg ethanol/dl did not affect 5-HT content, 5-HT uptake, or the proportion of 5-HT immunopositive neurons. In addition, this ethanol exposure had no significant effect on protein or DNA content. Additional studies, using a 4-day exposure to 450 mg ethanol/dl also did not detect significant differences in 5-HT uptake or in protein or DNA content.
The marked differences in the findings of the present in vitro and previous in vivo studies may be due to the fact that the ethanol exposure in vivo was longer than that in vitro, and included the period of early development of serotonergic neurons and their progenitors. Alternatively, the differences may be due to ethanol-associated alterations in maternal or fetal factors (e.g., hormones, amino acids, and growth factors) that are necessary for the normal development of the serotonergic system in vivo. Normal concentrations of such factors in the serum-containing media may have protected the cultured neurons from the damaging effects of ethanol.     

The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma.

The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow (BM) microenvironment, supporting the proliferation and survival of malignant plasma cells. An interesting candidate signal is hepatocyte growth factor/scatter factor (HGF), since its receptor Met is expressed on MM cells, while HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. To explore this hypothesis, we studied the biological effects of HGF stimulation on MM cell lines and on primary MMs. We observed that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. Stimulation of MM cells with HGF led to the activation of the RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathways, signaling routes that have been implicated in the regulation of cell proliferation and survival. Indeed, functional studies demonstrated that HGF has strong proliferative and anti-apoptotic effects on both MM cell lines and primary MM cells. Furthermore, by applying specific signal-transduction inhibitors, we demonstrated that MEK is required for HGF-induced proliferation, whereas activation of PI3K is required for both HGF-induced proliferation and for rescue of MM cells from apoptosis. Taken together, our data indicate that HGF is a potent myeloma growth and survival factor and suggest that the HGF/Met pathway is a potential therapeutic target in MM.     

Frape-1 and Frape-3: two different recombinant retroviruses encoding the same human marker gene

A major goal in retroviral-based gene therapy is to establish methods that allow for the selection and tracking of transduced cell populations. Ex vivo gene marking of normal and malignant hemopoietic cells allows the cells to be followed subsequently in vivo. For in vivo applications, a neutral marker gene that is nonimmunogenic is desirable. To track two distinctively treated cell populations in a single individual, we designed and constructed two retroviral vectors; both of these vectors encode a truncated form of the human low-affinity nerve growth factor receptor, a neutral gene that does not transduce signals and is expected to be nonimmunogenic in humans. The two vectors, named Frape-1 and Frape-3, are identical at the protein level but differ at the DNA level, containing restriction sites that allow easy detection by polymerase chain reaction analysis. We show that cell lines and primary CD34+ cells can be readily transduced with these vectors and that transduced cells can be distinguished by polymerase chain reaction- and vector-specific restriction sites. These vectors will be useful for toxicity studies on in vivo gene therapy and for determining the source of relapse in hematological malignancies.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

Antigens shared by malignant plasma cells and normal B cells may be involved in graft versus myeloma

Cytotoxic T cells play an important role in graft-versus-host-disease (GvHD) and graft-versus-leukaemia/myeloma, which may occur in patients treated with an allogeneic stem cell transplantation (ASCT). Here, we describe the selection of a myeloma reactive CD4+ cytotoxic T cell-line (CTL) and two CD4+ clones from this CTL. The CTL was generated from the blood from a patient with multiple myeloma (MM) with graft versus myeloma/GvHD, following an ASCT. The CTL was stimulated using irradiated peripheral blood mononuclear cells and EBV transformed B cells from the myeloma patient (EBVp), both of which were obtained prior to ASCT. Both the CTL and the two T cell clones specifically lysed EBVp and secreted IFN-gamma after coculture with EBVp and autologous myeloma tumour cells in a class II restricted fashion. These results show that myeloma tumour cells and autologous B cells present a common polymorphic peptide that functions as a target for graft derived cytotoxic T cells. Identification of these proteins will give insight into the relationship between graft versus myeloma (GvM) and GvHD and may provide immunotherapeutical targets in the treatment of MM.     

Anti-adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: Correlation with B cell differentiation stage

We show that major histocompatibility complex (MHC) class II molecules on B cells transmit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-γ-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphopro-liferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.     

Thalidomide in induction treatment increases the very good partial response rate before and after high-dose therapy in previously untreated multiple myeloma

In the prospective phase 3 HOVON-50/GMMG-HD3 trial, patients randomized to TAD (thalidomide, doxorubicin, dexamethasone) had a significantly higher response rate (at least PR) after induction compared with patients randomized to VAD (vincristine, adriamycin, dexamethasone, 72% vs. 54%, p<0.001). Complete remission (CR) and very good partial remission (VGPR) were also higher after TAD. After High Dose melphalan 200mg/m(2) response was comparable in both arms, 76% and 79% respectively. However, CR plus VGPR were significantly higher in the patients randomized to TAD (49% vs. 32%, p<0.001). CTC grade 3-4 adverse events were similar in both arms.