Molecular aspects of multiple myeloma

Multiple myeloma (MM) is a malignant proliferating disorder of the B lymphocyte lineage, characterized by an increasing proportion of plasma cells in the bone marrow, a high and progressively increasing concentration of a homogeneous immunoglobulin in the blood and the occurrence of osteolytic bone lesions. It is predominantly a disease of the elderly. The incidence of MM in the Netherlands is approximately 3 in 100,000 inhabitants. The prognosis for survival is about 3 years. In this thesis a study was made of genetic defects responsible for the transforming event in MM. The purpose of this study was to increase our insight into the underlying mechanism of tumor formation and to find tumor specific markers that would improve the diagnosis MM in an early stage of the disease. Since the discovery of oncogenes in the early eighties, much progress has been made in understanding tumor development. Oncogenes can play an important role in the process of malignant transformation. Oncogenes are activated proto-oncogenes, which in normal cells regulate growth and differentiation. Alterations in such genes by translocations, amplifications, point-mutations or deletions can disturb the normal growth pattern of the cell leading to malignant conversion

Immunoglobulin Heavy Chain High-Throughput Sequencing in Pediatric B-Precursor Acute Lymphoblastic Leukemia: Is the Clonality of the Disease at Diagnosis Related to Its Prognosis?

peer reviewedHigh-throughput sequencing (HTS) of the immunoglobulin heavy chain (IgH) locus is a recent very efficient technique to monitor minimal residual disease of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). It also reveals the sequences of clonal rearrangements, therefore, the multiclonal structure, of BCP-ALL. In this study, we performed IgH HTS on the diagnostic bone marrow of 105 children treated between 2004 and 2008 in Belgium for BCP-ALL in the European Organization for Research and Treatment of Cancer (EORTC)-58951 clinical trial. Patients were included irrespectively of their outcome. We described the patterns of clonal complexity at diagnosis and investigated its association with patients' characteristics. Two indicators of clonal complexity were used, namely, the number of foster clones, described as clones with similar D-N2-J rearrangements but other V-rearrangement and N1-joining, and the maximum across all foster clones of the number of evolved clones from one foster clone. The maximum number of evolved clones was significantly higher in patients with t(12;21)/ETV6:RUNX1. A lower number of foster clones was associated with a higher risk group after prephase and t(12;21)/ETV6:RUNX1 genetic type. This study observes that clonal complexity as accessed by IgH HTS is linked to prognostic factors in childhood BCP-ALL, suggesting that it may be a useful diagnostic tool for BCP-ALL status and prognosis

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% +/- 1.2% for patients with deletions versus 83.5% +/- 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia

Myeloma clonotypic B cells are hampered in their ability to undergo B-cell differentiation in vitro

In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V(D)J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n = 10) and the ability to differentiate in vitro was determined using the CD40-culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotype-specific allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) after culturing with CD40L and interleukin 4 (IL-4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B-cell differentiation was studied by abrogating CD40 signalling accompanied by IL-10 and IL-2 stimulation, enhancing differentiation towards Ig-secreting cells. The numbers of clonotypic B cells were determined by quantitative ASO-PCR. An increase in cell number was observed upon CD40L and IL-4 stimulation, whereas the relative number of clonotypic B cells was unaltered. In contrast, upon B-cell differentiation the relative number of clonotypic B cells decreased. In conclusion, clonotypic B cells can be cultured and isolated in vitro using the CD40 system. Clonotypic B cells responded to CD40 triggering in a similar fashion as to non-clonotypic normal B cells. However, the ability of clonotypic B cells to undergo in vitro activation and differentiation into Ig-secreting cells is hampere

Homing mechanisms in the etiopathogenesis of multiple myeloma

Although multiple myeloma (MM) is characterized by a monoclonal expansion of plasma cells, it has been assumed that the tumor close also includes more immature B cells. We could demonstrate by DNA sequence analysis of the variable region in immunoglobulin (Ig) heavy chain genes, that myeloma patients have peripheral blood monoclonal B cells that have not switched their Ig isotype but are somatically hypermutated. This finding suggests that myeloma originates from a germinal center B cell of the lymph node, most probably a memory B cell or B lymphoblast. The identification of these cells in the peripheral blood circulation implies that they must be equipped with homing receptors that allow them to migrate from the lymph node to the marrow environment. Within the marrow compartment these precursors will receive the appropriate differentiation signals to become mature tumor cells. The growth and survival of these bone marrow (BM) plasma cells is believed to be regulated by a functional interplay with the surrounding marrow stroma involving different adhesive mechanisms and the action of several cytokines. We found that myeloma plasma cells express several adhesion molecules (ICAM- 1, N-CAM, CD44, VLA-4). Myeloma cell lines can bind to purified fibronectin (FN) using mostly the VLA-4 receptor. However this interaction contributes only partially tn binding with intact stromal layers. Although the main growth stimulating factor of myeloma cells has been identified as interleukin 6 (IL-6), in vitro experiments revealed that this cytokine can only induce a short-term proliferation of myeloma cells and does not seem to support the self-renewal of the clonogenic cell. Therefore (an) additional stroma- associated factor(s) might be needed. We established a myeloma cell line (MM 5.1) that grows only on stromal layers cultured from autologous or allogeneic BM and not in the presence of exogenously added IL-6. Recently a stroma- independent variant (MM 5.2) of the cell line has been obtained. This cell line offers a useful model to study the growth regulation of myeloma cells by the marrow stroma, and to unravel the mechanisms that lead to stroma- independent tumor growth as can be observed in refractory and end stage disease patients.SCOPUS: ar.jFLWNOinfo:eu-repo/semantics/publishe

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity. © 1994 Rapid Communications of Oxford Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity

The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H-2D(k) molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7- 1. Expression of B7-1 rendered BW-Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK- mediated elimination of metastasizing, NK-resistant tumor cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Co-stimulation lowers the threshold for activation of naive T cells by bacterial superantigens

Staphylococcus enterotoxins bind class II MHC molecules on antigen presenting cells (APC) and stimulate T cells expressing appropriate V beta gene products. Although the role of non-TCR associated co-stimulatory receptors during antigen-specific T cell stimulation has been clearly established, the involvement of co-stimulatory activity in T cell activation by superantigens has been the matter of controversy. In this report, we examine the role of co-stimulation provided by selected APC populations in the response to bacterial exotoxins (staphylococcal enterotoxin A, staphylococcal enterotoxin B and toxic shock syndrome type 1). We demonstrate that the APC population able to activate naive T cells to IL-2 production is heterogeneous, comprising both adherent (presumably dendritic) and non-adherent (mostly B lymphocytes) cells. By stimulating naive T cells in the presence of graded doses of superantigens, we have observed that half-maximal IL-2 production was achieved at lower doses of superantigens in the presence of dendritic cells. Similarly, addition of antibodies to CD28 or B7.1-transfected cell lines increased the sensitivity of naive T cells to lower doses of superantigens. These observations indicate therefore that superantigens can be presented to naive T cells by APC displaying distinct levels of co-stimulatory activity, although with different efficacy. Thus, naive T cells are sensitive to CD28-mediated co-stimulation during superantigen-mediated responses but IL-2 production can be induced by high doses of superantigens in the presence of APC expressing weak co-stimulatory activity. These observations are compatible with the hypothesis that CD28-mediated signals participate in T cell activation by lowering T cell sensitivity to TCR ligands.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Metastasis of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2D(k) antigens

In vivo inoculation of a low metastatic BW 5147 derived T-cell lymphoma variant into syngeneic mice, had led to the generation of a highly metastatic variant. The shift towards a more metastatic phenotype is accompanied by an increase in major histocompatibility class I H-2D(k) antigen expression. This suggests that H-2D(k) antigens may control the metastatic potential of BW T lymphoma cells. Our present findings indicate that H-2D(k) expression is directly correlated with the metastatic potential of BW cells. We have confirmed such correlation by specifically altering the lever of H-2D(k) expression by: 1) FACS analysis, 2) IFNγ treatment, 3) H-2D(k) gene transfection. Cells sorted for low H-2D(k) expression had a significantly reduced metastatic potential. Induction of H-2D(k) expression on these cells by either IFN-γ treatment or H-2D(k) gene transfection concomitantly led to increased metastasis. We also assessed metastatic potential of BW cells in irradiated, immunecompromised recipients. Our results show that the immune system is implicated and we further tested which immune effecters are involved. In vivo depletion of natural killer (NK) and CD8+ T-cells revealed that the difference in metastatic potential of the H-2D(k) variants relies upon an NK-dependent mechanism, whereas CD8+ T-cells are not implicated. Our observations suggest that highly metastatic cells, expressing a high level of H-2D(k) antigens are more resistant to NK-cell-mediated cytotoxicity in vivo. We have confirmed our in vivo results by in vitro cytotoxicity assays using poly I:C induced NK and IL-2 activated LAK cells. We conclude that a NK-dependent mechanism accounts for the association between differential H-2D(k) antigen expression and metastasis.SCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe