Determination of the plasma cell labelling index with bromodeoxyuridine in a double fluorescence technique

A new technique is described in which plasma cells actively synthesizing DNA can be identified in a heterogeneous cell population such as bone marrow. This method uses bromodeoxyuridine (BrdU) and a fluorescent monoclonal antibody against BrdU in combination with cytoplasmic staining for immunoglobulin (Ig). In 26 patients with monoclonal gammopathy (MG) the plasma cell labelling index (LI) as determined by this immunofluorescent method was compared with the tritiated thymidine (3H-TdR) LI. No difference in sensitivity was found between the two methods. As the determination of the plasma cell LI with the BrdU/anti BrdU method is easy to perform and results can be obtained within 4 h, this immunofluorescent method seems to be an attractive alternative to the laborious time-consuming classic 3H-TdR LI.     

Induction therapy with vincristine, adriamycin, dexamethasone (VAD) and intermediate-dose melphalan (IDM) followed by autologous or allogeneic stem cell transplantation in newly diagnosed multiple myeloma

We performed a phase II study to test the efficacy and feasibility of induction therapy with vincristine, adriamycin and dexamethasone (VAD) and intermediate-dose melphalan, 70 mg/m2 (IDM), to autologous or allogeneic stem cell transplantation in newly diagnosed multiple myeloma (MM). A total of 77 patients received two cycles of VAD (n = 62) and/or two cycles of i.v. IDM 70 mg/m2 (n = 15) combined with G-CSF. PBSC were harvested after the first IDM, successfully in 87% of patients. Patients with a response to induction received myeloablative therapy with PBSCT (n = 50) followed by IFN maintenance or allo-BMT (n = 11). Seventy-two per cent of patients achieved a response after VAD which increased to 85% after IDM. Of patients who received PBSCT and allo-BMT, 24% and 45% achieved CR, respectively. Toxicity of induction consisted mainly of bone marrow suppression after IDM (median 8 days) with prolonged aplasia in 11% of patients after the second IDM. Only six infections WHO grade 3 occurred during induction. Treatment-related mortality of PBSCT and allo-BMT was 6% and 18%, respectively. Median time of follow-up is 44 months, and 50% of patients after PBSCT and 60% of patients after allo-BMT are still in remission. Survival rates of all patients were 82%, 75% and 63%, and for transplanted patients 86%, 79% and 68% after 12, 24 and 36 months. Well known prognostic factors, including alpha-IFN maintenance after PBSCT, were not significant for response or survival although patients in CR after allo-BMT had a strong tendency for better outcome. VAD/IDM is an effective and safe induction therapy for autologous and allogeneic stem cell transplantation. Based on these observations a phase III trial was started in October 1995 comparing IFN maintenance with PBSCT and allo-BMT after response to induction with VAD and IDM.     

Malignant transformation of monoclonal gammopathy of undetermined significance: cumulative incidence and prognostic factors.

The cumulative incidence of malignant transformation was studied in 88 patients with monoclonal gammopathy of undetermined significance (MGUS) that had a complete prospective follow-up. At a median follow-up of 6.75 years, 10 patients developed multiple myeloma (MM) (11.4%) and 2 developed immunocytoma (2.3%). The cumulative incidence of malignant transformation was 9.1, 21.3, 38 and 48.3% at 5, 10, 15 and 20 years, respectively. In univariate analysis on 102 MGUS patients, M-component level, bone marrow plasma cell percentage and kappa light chain correlated significantly with the development of a malignancy (p=0.0289, 0.0265 and 0.0013, respectively). In multivariate analysis, light chain type of M-component and plasma cell percentage had independent prognostic significance. A high-risk (M-component level > 10 g/l and/or plasma cell percentage > 2%) and a low-risk group ( M-component level < 10 g/l and/or plasma cell percentage < 2%) of MGUS patients was identified, which differed significantly in the cumulative incidence of developing a malignancy (p<0.001 for M-component level and p=0.007 for plasma cell percentage). These results imply that high-risk patients should receive a more frequent follow-up, in comparison to low-risk patients.     

Lymphocyte function-associated antigen-1 expression on plasma cells correlates with tumor growth in multiple myeloma.

Lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) expression on bone marrow-derived plasma cells from normal individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), and patients with multiple myeloma (MM) was studied by immunofluorescence microscopy and flow cytometry using a new monoclonal antibody (MoAb) F8.8. This MoAb recognizes the alpha-chain (CD11a) of LFA-1 as determined by immunoprecipitation, and inhibits T-cell-induced cytotoxicity. Although the F8.8 MoAb stains unstimulated peripheral blood T cells with the same mean fluorescence intensity as other anti-CD11a MoAbs, it proved to be superior in detecting CD11a on plasma cells as compared with reference MoAbs. Using the anti-CD11a MoAb F8.8, a strong correlation was found between LFA-1 expression and disease activity in MM, as defined by clinical performance and serum M-protein level. Hardly any LFA-1+ plasma cells were detected in normal individuals, patients with MGUS, and MM patients in a nonactive phase of their disease, while plasma cells of some MM patients with active disease and all patients with fulminant disease expressed LFA-1. Plasma cell LFA-1 expression correlated well with the labeling index (LI) of the tumors in the individual patients. The relation between LFA-1 expression and the tumor growth suggests an involvement of this adhesion molecule in cellular interactions resulting in plasma cell proliferation.     

Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.     

Recognition of xeno-(HLA, SLA) major histocompatibility complex antigens by mouse cytotoxic T cells is not H-2 restricted: a study with transgenic mice

Cytotoxic T lymphocytes (CTLs) recognize antigens in the context of major histocompatibility complex (MHC) class I gene products. The T-cell receptor (TCR) that mediates this MHC-restricted antigen recognition recognizes short peptide fragments rather than the intact antigen. Presentation of peptides to the TCR may thus be a major function of the MHC. An intriguing question emerging from this model is whether peptide presentation also applies to foreign MHC antigens and which of the available MHC molecules can present preferentially the peptides of the foreign MHC molecule. Allo- and xenoreactive CTLs might either recognize native MHC class I molecules or peptides presented by self MHC or by the foreign class I MHC itself. The finding that synthetic peptides corresponding to MHC class I regions are recognized by allo- and xenoreactive CTLs suggests that recognition of foreign MHC by CTLs might involve degraded fragments presented by syngeneic class I molecules. We used MHC transgenic mice as a tool to study these questions. The CTL responses against human (HLA) antigen B27 were analyzed by using HLA-B27 transgenic mice with various H-2 haplotypes. We report here that mouse xeno-MHC-specific (anti-B27) CTLs are perfectly able to kill human and mouse cells expressing the appropriate xenoantigen and that in primary and secondary responses to xeno-MHC, the mouse T-cell repertoire does not use self-H-2 as a restriction element. Absence of H-2 restriction was confirmed by the lack (less than 1/10(6] of H-2-restricted HLA-specific CTL precursors. Therefore, H-2-restricted recognition of xeno-MHC antigens cannot be generalized as part of a classical MHC class I-specific response. These results indicate that xenoreactive CTLs usually recognize intact MHC molecules or MHC peptides preferentially presented by their native MHC molecule. We suggest the latter possibility.     

Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.     

Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma

Patients in complete clinical remission after myeloablative allogeneic stem cell transplantation (allo-SCT) were enrolled in a longitudinal study to assess the predictive value of molecular monitoring. Using polymerase chain reaction (PCR) for immunoglobulin gene rearrangements it was possible to generate a clone-specific molecular marker in 48 of 70 patients. Of these 48 patients, 16 (33%) attained durable PCR-negativity after transplantation, whereas 13 (27%) remained persistently PCR-positive and 19 (40%) showed a mixed pattern. The cumulative risk of relapse at 5 years was 0% for PCR-negative patients, 33% for PCR-mixed patients, and 100% for PCR-positive patients. Within the group studied it was not possible to identify any clinical feature predictive of durable PCR-negativity. We believe that these findings could prompt the design of prospective studies to evaluate if the treatment of molecular disease can extend remission duration and survival.     

Comparison of plasma cell infiltration in bone marrow biopsies and aspirates in patients with multiple myeloma

In 54 patients with multiple myeloma plasma cell infiltration was compared in bone marrow biopsies and aspirates. In 48% of cases plasma cell infiltration was comparable, in 48% infiltration in the aspirate was lower than in the biopsy. In only two cases more plasma cells were found in the aspirate. Eleven patients (20%) had less than 20% plasma cells in the aspirate and more than 50% in the biopsy. Underestimation of plasma cell load especially seems to occur in patients with a focal growth pattern of multiple myeloma or when strong fibrosis is present. 69% of patients with stage III, according to Durie & Salmon (1975), and 76% of patients with a high beta 2-microglobulin had more than 50% plasma cells in the biopsy, indicating that these parameters, which are based on tumour load, are influenced by other factors as well. The bone marrow biopsy is of superior value for direct estimation of the tumour load in multiple myeloma compared to bone marrow aspirates. A prospective study is needed to determine its prognostic significance.