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Multiple myeloma (MM) is a haematological malignancy characterised by the clonal proliferation of plasma cells in the bone marrow. More than 80% of patients with MM display evidence of myeloma bone disease (MBD), characterised by the formation of osteolytic lesions throughout the axial and appendicular skeleton. MBD significantly increases the risk of skeletal-related events such as pathologic fracture, spinal cord compression and hypercalcaemia. MBD is the result of MM plasma cells-mediated activation of osteoclast activity and suppression of osteoblast activity. Bisphosphonates (BP), pyrophosphate analogues with high bone affinity, are the only pharmacological agents currently recommended for the treatment and prevention of MBD and remain the standard of care. Pamidronate and zoledronic acid are the most commonly used BP to treat MBD. Although generally safe, frequent high doses of BP are associated with adverse events such as renal toxicity and osteonecrosis of the jaw. As such, optimal duration and dosing of BP therapy is required in order to minimise BP-associated adverse events. The following guidelines provide currently available evidence for the adoption of a tailored approach when using BP for the management of MBD.

Multiple myeloma (MM) is a haematological malignancy characterised by the clonal proliferation of plasma cells in the bone marrow. More than 80% of patients with MM display evidence of myeloma bone disease (MBD), characterised by the formation of osteolytic lesions throughout the axial and appendicular skeleton. MBD significantly increases the risk of skeletal-related events such as pathologic fracture, spinal cord compression and hypercalcaemia. MBD is the result of MM plasma cells-mediated activation of osteoclast activity and suppression of osteoblast activity. Bisphosphonates (BP), pyrophosphate analogues with high bone affinity, are the only pharmacological agents currently recommended for the treatment and prevention of MBD and remain the standard of care. Pamidronate and zoledronic acid are the most commonly used BP to treat MBD. Although generally safe, frequent high doses of BP are associated with adverse events such as renal toxicity and osteonecrosis of the jaw. As such, optimal duration and dosing of BP therapy is required in order to minimise BP-associated adverse events. The following guidelines provide currently available evidence for the adoption of a tailored approach when using BP for the management of MBD.

Understanding the interactions of the immune system and B-cell tumours has great therapeutic potential. Both in myeloma and Waldenstrom’s macroglobulinaemia (WM), there are measurable changes in the immune system with abnormalities of T-cells, NK cells and the ability to respond to external immunological stimuli. The homeostatic balance between the activation and suppression of immune antitumour effector cells in vivo is controlled by a network of cytokines and regulatory cells in a defined micro-environment. In myeloma, malignant plasma cells appear to have multiple mechanisms of inhibiting the antitumour immune response. These include inactivation of clonal cytotoxic T-cell, depression of NK and dendritic cell function and altering the regulatory T and pro-inflammatory Th17 cell balance in favour of a suppressive state. In addition, in myeloma, trogocytosis (demonstrated by malignant membrane transfer to T-cells) of plasma cells by T-lymphocytes results in the development of inhibitory Treg cells. In addition myeloid-derived suppressor cells (MDSC) are numerically increased, localised to the bone marrow micro-environment and functionally more active in patients with myeloma as compared to normal. The state of the immune homeostatic balance in WM has not yet been so clearly defined but results in the development of clonal cytotoxic T-cells, with possible abnormalities in Treg and Th17 populations and defects in NK cell function under active investigation.

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.

Deficiencies in B7:CD28 costimulation are considered to be one of the major causes of the failure to generate a tumor-specific immune response. Up-regulating the expression of the B7 molecules on malignant B cells has been shown to stimulate cytotoxic T cells. Plasma cells from patients with myeloma express a tumor-specific idiotype but lack CD80 (B7-1) and have a variable expression of CD86 (B7-2). This study has identified the incidence and clinical significance of high CD86 expression on plasma cells at diagnosis and studied the ability of trimeric human CD40 ligand (huCD40LT) to up-regulate the expression of the B7 family on malignant plasma cells. CD86 expression on plasma cells was increased in 54% of the patients studied at diagnosis (n = 35) and was associated with a significantly shorter survival (median, 28 versus 57 months; χ2 = 4.6;P = .03) and a higher tumor load (patients with more than 50% bone marrow plasma cells, 47% versus 6%; χ2 = 7.2; P = .005). CD86 expression was highest on immature and primitive plasma cells (CD38++, CD45+) of both patients and controls and was associated with a CD40+, CD20+, CD19−, CD138+ phenotype. The shortened survival was associated with high CD86 only on mature (CD38++, CD45−) plasma cells (χ2 = 7.6; P = .006). There was no significant correlation between high CD86 and other known prognostic markers, including serum β2-microglobulin, serum thymidine kinase, and labeling index. The addition of huCD40LT to short-term cultures up-regulated both CD80 and CD86 expression on B cells (CD19+) and CD80 on plasma cells (CD38++), but did not up-regulate CD86 expression on plasma cells. Thus, B7-2–positive myeloma consists of a subgroup of patients with a relatively poor prognosis, and CD40LT may be useful in immunotherapy protocols because it up-regulates CD80 expression on malignant plasma cells without inducing B7-2–positive myeloma.

Individual myeloma cells in the peripheral blood of 7 patients with multiple myeloma were detected by mRNA in situ hybridization (ISH) using biotinylated antisense oligonucleotide probes to non-germline sequences of the CDR3 region of the immunoglobulin heavy chain gene. Peripheral blood samples from these patients were studied over a period of 2-3 years. The number of circulating myeloma cells varied from 0.1-23% of the mononuclear cell population. Longitudinal studies showed that the highest number of circulating myeloma cells were present during escape phase and thus the percentage of mRNA ISH+ cells correlated with the clinical state of the patient. This technique is the most accurate means of monitoring and quantitating individual myeloma cells in the peripheral blood of patients with myeloma and provides insight into the relevance of circulating myeloma cells.

The dysregulation of specific oncogenes due to either mutation or activation has previously been reported in a small number of patients with myeloma but the extent of oncogene dysregulation during the course of the disease is not known. The oncoprotein phenotype of plasma cells in 146 bone marrow samples from 81 patients with multiple myeloma was determined by dual colour flow cytometry using a predetermined panel of 8 monoclonal antibodies. High intensity CD38 expression was used to distinguish the plasma cell population and the cells were permeabilised to detect intracellular antigen expression. In situ hybridization using biotinylated cDNA probes for c-myc and bcl-2 was used to determine mRNA expression and to validate the flow cytometric assay. The normal range of expression for each of 6 oncoproteins (c-myc, c-fos, c-neu, bcl-2, p-ras, p53 mutant) and 2 tumour suppressor gene products (p53 wild and Rb) was determined in plasma cells from 33 normal bone marrows. Disease progression was associated with the concurrent abnormal expression of at least one oncogene and one tumour suppressor gene where as stable disease was associated with a normal expression of at least one or both (chi2 = 34.1; p0.001). At diagnosis there was a correlation between serum beta2 microglobulin and the concurrent overexpression of both an oncoprotein and a tumour suppressor gene product. Longitudinal studies of 33 different patients over 4 years, suggests that the progressive evolution of myeloma is a multistep process of genomic instability producing ongoing alterations in the expression of both oncogenes and tumour suppressor genes.

For patients with multiple myeloma the most important laboratory correlate of prognosis and disease activity is the bromodeoxyuridine (BrdUrd) plasma cell labelling index (LI). However, the traditional immunofluorescent microscope LI technique, like other manual enumeration assays, can suffer from poor precision and accuracy. In this study the LI of different subpopulations of plasma cells (CD38++) as determined by flow cytometry was correlated with disease state. The mean LI of the total CD38++ population was significantly higher (2.7 +/- 0.4%) than the LI determined by the traditional slide technique (0.6 +/- 0.1%) for 65 samples tested. Primitive plasma cells (CD38++, CD45++) had a higher labelling index than mature plasma cells (CD38++, CD45-) (7.0 +/- 1.3% v 1.8% +/- 0.3%) and in one patient the LI of the primitive plasma cells was 46%. In addition, the LI of the mature plasma cells was lower than the total plasma cell population. As expected, there was a significant difference between the LI of patients in plateau phase and progressive disease but this difference was greatest when the LI of the primitive plasma cells was studied (9.2 +/- 2.9% v 2.2 +/- 0.7%; z = 19.9, P < 0.001). This study has raised some concerns about the sensitivity and accuracy of the traditional labelling index and has shown that the increased LI associated with progressive disease is almost entirely attributable to an increase in the LI of the primitive plasma cell subpopulation and that the LI of primitive plasma cells provides a more clinically significant correlation with disease status than the traditional assay.

Therapy for patients with multiple myeloma (MM) is currently unsatisfactory and most patients eventually succumb to relapsed disease. DCs are a subset of leukocytes with the capacity to initiate and control the adaptive immune response against many cancers, including MM. In MM patients, in vivo DC function is often abnormal, however, it appears that it can be restored by in vitro manipulation. This has led to the development of DC immunotherapy for MM patients. We review the background research leading to the recognition of an anti-MM immune response, and discuss abnormalities in DC function, potential tumor-associated Ags, and the results of clinical trials of DC immunotherapy in MM patients.

Limited response to idiotype vaccination in patients with myeloma suggests that there is a need to develop better immunotherapy strategies. It has been determined that the number of high-potency CMRF44(+)CD14(-)CD19(-) dendritic cells (DCs) in the blood of patients with myeloma (range, 0.03%-0.8% of mononuclear cells [MNCs]; n = 26) was not significantly different from that in controls (range, 0.05%-0.8% of MNCs; n = 13). Expression of the costimulatory molecules CD80 and CD86 on DCs from these patients (mean, 29% +/- 17% of MNCs and 85% +/- 10% of MNCs, respectively) was also normal (mean, 29% +/- 17% and 86% +/- 16% of MNCs, respectively). Up-regulation of CD80 expression in response to stimulation by human (hu)CD40LT + interleukin (IL)-2 was significantly reduced on the DCs of patients with myeloma during stable disease (n = 9) and was absent during progressive stages (n = 7) of disease. Similar effects were seen on B cells but not on monocytes of the same group of patients. CD86 expression on DCs was high before (86%) and after (89%) stimulation. Inhibition of CD80 up-regulation was neutralized by either anti-transforming growth factor (TGF)-beta (1) or anti-IL-10. Upregulation of CD80 on DCs of controls was inhibited by rTGF-beta (1) in a dose-dependent manner. Serum TGF-beta (1) and IL-10 levels were normal in most patients studied. Cytoplasmic TGF-beta (1) was increased in plasma cells during progressive disease. Thus patients With myeloma have normal numbers of DCs, but CD80 expression may fail to be up-regulated in the presence of huCD40LT because of tumor-derived TGF-P, or IL-10. Autologous high-potency DCs may have to be tested for CD80 up-regulation and biologically modified ex vivo before idiotype priming for immunotherapy. (C) 2001 by The American Society of Hematology.

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients with multiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscope technique which is time consuming and requires a degree of subjectivity in its interpretation. We have assessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has been compared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been compared with CD38-FITC + CD138-FITC and CD38-biotin + streptavidin FITC to identify plasma cells. The mean channel fluorescent intensity of the plasma cell peaks for each of these markers was 12. 7, 17.4 and 35.3 respectively demonstrating the superiority of CD38-biotin + streptavidin FITC. Analysis after propidium iodide staining provided a good correlation with the slide technique (r = 0. 71; P0.0001) but the bromodeoxyuridine method did not correlate with the slide method (r = 0.09; P = NS). The labeling index values obtained from either of the flow methods were greater than the microscopic method. Thus a labeling index of4% will replace the traditional1% threshold for identifying patients with a significantly increased labeling index. The advantages of the new method are that it takes less time to perform, is more objective and provides additional data on ploidy and cell cycle status.

Summary A 32 year old Caucasian male presented with fever, massive hepatosplenomegaly and lymphadenopathy. Peripheral blood and bone marrow findings were consistent, both morphologically and immunophenotypically, with a natural killer cell leukemia/lymphoma. The disease was rapidly progressive and was fatal within five days after presentation.

The expression of 6 different oncoproteins and 2 tumour suppressor gene products in the plasma cells of 63 bone marrow samples was used to determine a profile of the oncogenic phenotype of patients with multiple myeloma. Dual label flow cytometry after periodatelysine paraformaldehyde fixation was used to detect cell surface phenotype and intracellular protein expression simultaneously. The normal range for both the incidence and intensity of expression was determined for each protein by analysing plasma cells (high CD38 intensity) in 22 normal bone marrow samples. The percentage of myeloma patients with a greater than normal incidence of plasma cells expressing these proteins was 53% for c-myc, 28% for Rb, 28% for bcl-2, 27% for c-fos, 24% for p53 wild, 22% for p53 mutant, 13% for c-neu and 13% for pan-ras. When a panel of 8 antibodies was used, 82% of the samples (n = 28) had an increased incidence of expression by at least one oncoprotein or tumour suppressor gene product. The 5 patients with a normal incidence of expression of all 8 proteins were in plateau stage and 4 had not received chemotherapy for more than 12 months. The number of patients with an increased incidence of expression by 2 or more oncoproteins was significantly greater (X2 = 9.0; p0.005) in progressive disease (55%) than in stable disease (14%) but there was no specific phenotype pattern associated with progressive disease. All 6 oncoproteins and both tumour suppressor gene products had a greater incidence and intensity of expression in progressive than in stable disease. The expression of c-myc oncoprotein correlated with c-myc mRNA expression in the same samples (n = 10) but c-myc did not correlate with either the plasma cell labelling index (r = -0.15) nor serum thymidine kinase (r = 0.10). Our results suggest that there is a heterogeneous, non-systematic but almost universal presence of activated oncogenes and tumour suppressor genes in the plasma cells of patients with multiple myeloma and that disease progression is associated with the accumulation of a variety of secondary genetic changes which confer increased malignant behaviour.

An mRNA in situ hybridization (ISH) method which used non-radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (18-22mer) was prepared from non-germline sequences of the rear-ranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal individuals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used were from the same patient. Using simultaneous fluorescent immunocytochemistry it was shown that more than 95% of the ISH-positive cells expressed the malignant light chain in their cytoplasm. ISH positive cells were found in 1-4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour-specific therapeutic procedures using antisense technology for patients with myeloma.