The treatment of plasma cell myeloma

In a previous publication the ultrastructure of pleural effusions in cases of pleural mesothelioma was reported. The same method has now been applied to a study of effusions produced by pleural metastases. The findings are considered sufficiently conclusive to justify the use of electron microscopic cytology in determining the nature and sometimes the origin of such effusions.     

Busulfan and melphalan as conditioning regimen for autologous peripheral blood stem cell transplantation in multiple myeloma

Twenty-four patients with multiple myeloma (MM), three (12.5%) in complete remission (CR) and 21 (87.5%) in partial remission (PR) were treated with high-dose chemotherapy (HDCT) (busulfan 12 mg/kg+melphalan 140 mg/m2) as preparative regimen for autologous peripheral blood stem cell (PBSC) transplantation. These cells were previously collected by leukapheresis after mobilization by high-dose cyclophosphamide (HD Cy)+rhGM-CSF (18 patients) or rhG-CSF alone (six patients). Considering 23 evaluable patients following HDCT, the CR rate was 58% (14 patients) and the PR rate was 38% (nine patients). One transplant-related death occurred following this regimen (4%). With a median follow-up of 20 months (range 4-34) after transplantation, 21 patients are alive (87%). Disease progression after transplantation was observed in four patients. Overall and relapse-free actuarial survival at 24 months was 91% and 74%, respectively. 12 patients (50%) remain in CR 15 months (4-34) post transplant. The major toxicity was mucositis. Busulfan+melphalan is a safe and feasible conditioning regimen for APBSCT in MM with acceptable toxicity and a high objective response rate, which may result in prolonged survival.     

IgE plasma cell leukemia successfully treated with combination VAD (vincristine, doxorubicin, dexamethasone) and MP (melphalan, prednisolone) followed by interferon-alpha

A 69-year-old woman with IgE/kappa plasma cell leukemia (PCL) was treated with a sequential combination of VAD (vincristine, doxorubicin, dexamethasone) and MP (melphalan, prednisolone) followed by interferon-alpha (IFN alpha). A complete remission was achieved for 14 months. Maintenance therapy with IFN alpha has continued for an additional 10 months. IgE PCL is extremely rare. The biological characteristics of the myeloma cells, including surface marker, adhesion molecule, karyotype, DNA analysis, and the response to various cytokines, are presented.     

Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan

An in vitro model of acquired melphalan resistance was developed by serial incubation of an MCF-7 human breast cancer cell line in increasing concentrations of melphalan. The resulting derivative cell line, Me1R MCF-7, was 30-fold resistant to melphalan. Uptake studies demonstrated decreased initial melphalan accumulation in Me1R MCF-7 cells. Inverse-reciprocal plots of initial melphalan uptake revealed a 4-fold decrease in the apparent Vmax of Me1R MCF-7 compared with WT MCF-7 (516 amol cell-1 min-1 vs 2110 amol cell-1 min-1 respectively) as well as a decrease in the apparent Kt (36 microM vs 70 microM respectively). Two amino acid transporters have previously been identified as melphalan transporters: system L, which is sodium-independent and inhibited by 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH), and system ASC which is sodium dependent and unaffected by BCH. At low concentrations of melphalan (3-30 microM), 1mM BCH competition eliminated the differences between the two cell lines, thus implicating an alteration of the system L transporter in the transport defect in the resistant cells. Me1R MCF-7 cells were also evaluated for glutathione-mediated detoxification mechanisms associated with melphalan resistance. There was no difference between Me1R MCF-7 and WT MCF-7 in glutathione content, glutathione-S-transferase activity and expression of pi class glutathione S-transferase RNA. In addition, buthionine sulfoximine did not reverse melphalan resistance in Me1R MCF-7 cells. Therefore, Me1R MCF-7 cells provide an in vitro model of transport-mediated melphalan resistance in human breast cancer cells.     

Analysis of prednisolone in plasma by high-performance liquid chromatography

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet dector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentration in plasma following administration of a 10-mg single oral dose to a human subject is described.     

Characterization of p16(INK4A) expression in multiple myeloma and plasma cell leukemia

Loss of p16(INK4A) (p16) expression is frequently associated with the development of epithelial and lymphoid malignancies. However, the frequency and significance of p16 abnormalities in multiple myeloma (MM) and the more aggressive phase of plasma cell leukemia (PCL) have not been well defined. Accordingly, the goal of this study was to define the expression and function of p16 in fresh samples of MM and PCL. We found that p16 protein was highly expressed in primary MM cells, although it was undetectable in fresh samples of PCL. Additionally, p16 protein was also absent in four of four MM-derived cell lines. To determine the mechanism for p16 underexpression in PCL and MM-derived cell lines, we performed PCR analysis to evaluate both gene deletion and the presence of methylation. Interestingly, the p16 gene was present and methylated in all patient PCL cells and MM cell lines, whereas it was unmethylated in patient MM cells and normal B cells. Furthermore, treatment with the demethylating agent 5-deoxyazacytidine or p16 retrofection restored p16 protein expression and induced G1 growth arrest in patient PCL cells and MM cell lines. These results suggest that inactivation of the p16 gene by methylation may be associated with decreased growth control and the development of PCL in a subset of patients with MM.     

Intermittent etidronate therapy to prevent corticosteroid-induced osteoporosis

BACKGROUND AND METHODS: Osteoporosis is a recognized complication of corticosteroid therapy. Whether it can be prevented is not known. We conducted a 12-month, randomized, placebo-controlled study of intermittent etidronate (400 mg per day for 14 days) followed by calcium (500 mg per day for 76 days), given for four cycles, in 141 men and women (age, 19 to 87 years) who had recently begun high-dose corticosteroid therapy. The primary outcome measure was the difference in the change in the bone density of the lumbar spine between the groups from base line to week 52. Secondary measures included changes in the bone density of the femoral neck, trochanter, and radius and the rate of new vertebral fractures. RESULTS: The mean (+/-SE) bone density of the lumbar spine and trochanter in the etidronate group increased 0.61 +/- 0.54 and 1.46 +/- 0.67 percent, respectively, as compared with decreases of 3.23 +/- 0.60 and 2.74 +/- 0.66 percent, respectively, in the placebo group. The mean differences between the groups after one year were 3.72 +/- 0.88 percentage points for the lumbar spine (P = 0.02) and 4.14 +/- 0.94 percentage points for the trochanter (P = 0.02). The changes in the femoral neck and the radius were not significantly different between the groups. There was an 85 percent reduction in the proportion of postmenopausal woman with new vertebral fractures in the etidronate group as compared with the placebo group (1 of 31 patients vs. 7 of 32 patients, P = 0.05), and the etidronate-treated postmenopausal women also had significantly fewer vertebral fractures per patient (P = 0.04). CONCLUSIONS: Intermittent etidronate therapy prevents the loss of vertebral and trochanteric bone in corticosteroid-treated patients.     

B-lymphocyte suppression in multiple myeloma is a reversible phenomenon specific to normal B-cell progenitors and plasma cell precursors

The reduced levels of normal immunoglobulin in patients with myeloma may be due to suppression of normal B-cell differentiation. However, reports on the numbers of B cells vary, with some finding decreases consistent with immunoparesis, and others reporting expansions of phenotypically aberrant cells. We have therefore assessed the phenotype and levels of B lymphocytes in patients at presentation (n = 23), in plateau or complete remission (PB n = 42, BM n = 18), and in relapse (PB n = 17, BM n = 14), in comparison to normal individuals (n = 10). Phenotypic analysis was performed using five-parameter flow cytometry, with CD14 used to exclude monocytes where necessary. We found no evidence of a phenotypically distinctive blood or marrow B-cell population in patients with myeloma, nor of an increase in the levels of any B-cell subset. Numbers of blood CD19+ 38+ normal plasma cell precursors were significantly reduced in presentation/relapse patients, but not in patients in plateau/remission. Total CD19+ cells were significantly reduced only in patients with circulating myeloma cells, detected by IgH-PCR. In the marrow, CD19+ B cells expressing CD5, CD10, CD34, CD38, CD45(low) and Syndecan-1 were significantly decreased at presentation/relapse, but not in patients in plateau/remission. The majority of these antigens are expressed by normal B-cell progenitors, indicating that myeloma also affects the early stages of B-cell development. The suppression of progenitor cells was not restricted to B-lymphoid differentiation, as total CD34+ cells were also significantly reduced in the marrow of myeloma patients at presentation. These results indicate that, if neoplastic B cells are present in myeloma, they are low in number and have a phenotype similar to their normal counterparts. Furthermore, there is a reversible suppression of CD19+ B lymphocytes that correlates inversely with disease stage, and specifically affects the early and late stages of normal B-cell differentiation.     

Oligoclonal protein bands and Ig isotype switching in multiple myeloma treated with high-dose therapy and hematopoietic cell transplantation

Multiple myeloma (MM) is usually characterized by production of a single serum monoclonal protein of constant isotype and light-chain restriction. Multiple Ig isotypes and isotype switches, which are rare in untreated patients, are reported to be more common in patients undergoing myeloablative therapy. These additional protein bands, detected by immunofixation electrophoresis (IFE), could be due to altered paraprotein production by the malignant plasma cell clone or oligoclonal Ig production during recovery of B-cell function after myeloablative therapy. We analyzed abnormal protein bands (APB), distinct from the presenting paraprotein, in 550 patients receiving high-dose therapy with autologous hematopoietic cell transplantation at a single institution. Fifty-five patients (10%) had APB, 48 had oligoclonal bands (OB), and 23 had an apparent isotype switch (IS) on IFE (16 had both OB and IS). Morphologic and flow cytometric examination of bone marrow in 17 patients with IS showed no evidence of a clonal plasma cell isotype switch. Patients with APB had significantly higher complete response to therapy (67% v 37%, P = .001). To assess the independent prognostic relevance of APB, a multivariate analysis was performed among 471 patients surviving at least 12 months from first transplant (all patients developing APB had done so by 12 months from first transplant). APB (in 50 patients) was a favorable feature for both event-free (rank 3, P = .004) and overall survival (rank 3, P = .0005). We propose that OB and IS are likely to be due to recovery of Ig production rather than alterations in the biology of the malignant plasma cell clone.     

Renal replacement therapy in multiple myeloma and systemic amyloidosis

Renal failure frequently complicates both multiple myeloma and systemic amyloidosis. Renal replacement therapy (RRT) may be poorly tolerated and its role in such patients is not clearly defined. Of fifty patients (26 males and 24 females) referred to a single centre because of renal failure associated with multiple myeloma or systemic amyloidosis 37 progressed to end-stage renal failure and 30 of these patients received RRT. Nine patients have been treated by CAPD, 13 by haemodialysis, and 8 patients have required both forms of dialysis. Overall one year and two year survival rates were 66% and 57% respectively. The median duration on RRT was 7.5 months (range 1-96 months) with a 51% one year, and a 46% two year survival rate. Of 7 patients with amyloidosis who underwent renal transplantation, 3 died within 6 months of transplantation. Undiagnosed cardiac involvement contributed to this early mortality. We conclude that renal replacement therapy is appropriate for some patients with multiple myeloma and systemic amyloidosis who develop endstage renal failure. Careful assessment and selection of patients is necessary prior to renal transplantation.     

The use of intravenous intermediate dose melphalan and dexamethasone as induction treatment in the management of de novo multiple myeloma

The variable absorption of melphalan from the gastrointestinal tract results in response rates between 40 and 60%. High dose melphalan increases response rates but at the cost of increased morbidity and mortality. We have investigated intravenous intermediate dose melphalan and dexamethasone in the treatment of patients presenting with de novo multiple myeloma with the object of reducing toxicity while preserving an improved response rate compared to oral melphalan and prednisolone. The results show that this treatment can be delivered safely on an outpatient basis in patients up to the age of 78 yr; 82% of patients achieved an objective response and 30% a complete haematological and clinical remission. Median overall survival for the whole group is 37 months.     

High-dose busulphan/melphalan with autologous stem cell rescue in Ewing's sarcoma

Eighteen patients with poor risk Ewing's sarcoma (including 11 patients with metastatic disease at presentation) received consolidation therapy of busulphan and melphalan with autologous stem cell rescue. There were nine females. The median age at diagnosis was 14.2 years (range 2.75-30 years). There was one early death due to cytomegalovirus pneumonitis. One patient developed a single generalised convulsion during busulphan therapy. Severe renal toxicity was not encountered. One patient developed veno-occlusive disease of the liver (VOD) which eventually resolved. With a median follow up of 2 years, 13 patients survive including six with initial metastatic disease. We conclude that high-dose busulphan/melphalan is well-tolerated and should be evaluated for efficacy in a larger series of patients with high risk Ewing's sarcoma.     

Spontaneous spinal epidural hematoma with spinal cord compression complicating plasma cell myeloma. A case report

STUDY DESIGN: A case is reported in which a patient had acute paraplegia with sensory loss caused by a spontaneous epidural hematoma that was ascribed to bleeding of pre-existing myeloma lesions of the thoracic vertebrae. OBJECTIVES: To highlight the causes of secondary epidural hematomas with special attention to pre-existing vertebral or epidural lesions. SUMMARY OF BACKGROUND DATA: There are no apparent previous reports of epidural spinal hematomas ascribed to underlying malignant diseases. Benign dysplasia, such as hemangioma or Paget's disease, has been implicated in a few cases. METHODS: A case of spontaneous dorsal epidural hematoma is reported in a patient followed up for plasma cell myeloma with osteolytic lesions in the lower thoracic spine. There was no history of major trauma or coagulation disorders. Complete loss of motor and sensory function in both lower limbs was noted, with sphincter dysfunction. Magnetic resonance imaging of the thoracic spine showed a large posterolateral epidural hematoma responsible for spinal cord compression. RESULTS: The patient failed to improve despite surgical decompression within 6 hours of symptom onset. He died 13 days later of refractory bacterial pneumonia. A large epidural hematoma adjacent to myelomatous lesions of the thoracic vertebrae was found at autopsy. CONCLUSIONS: This is the first reported case of spontaneous epidural hematoma ascribed to underlying malignant disease, with confirmation of the diagnosis by postmortem examination. Possible mechanisms include tumor-related epidural inflammation and fragility of epidural venous plexuses.     

The functional phenotype of the primitive plasma cell in patients with multiple myeloma correlates with the clinical state

The malignant plasma cells from patients with multiple myeloma display considerable phenotypic heterogeneity. All plasma cells express high intensity CD38 (CD38++), cytoplasmic immunoglobulin and either kappa or lambda light chains. Subpopulations of mature (CD45-), immature (CD45+) and primitive (CD45++, CD19+) plasma cells can be defined but little is known about the functional differences and clinical significance of these subpopulations. Three colour flow cytometry and permeabilisation was used to determine the expression of functionally important antigens in plasma cell subpopulations. These antigens included the labelling index (LI, bromodeoxyuridine), number of nucleoside transporter per cell, p-glycoprotein (JSB-1), and oncoprotein expression (c-myc, c-fos, c-neu, bcl-2, p-ras, p53m, p-53w, and Rb). In progressive disease there was an increase in the absolute number but not the percentage of CD45++ plasma cells. There was a significant difference in the mean LI of the CD38++, CD45++ population in progressive disease compared with stable disease (9.2% vs 2.2%; z = 19.9, p < 0.001). The LI of CD45++ cells ranged up to 45% and provided a better correlation with disease status than the LI of the total cell population. Any increase in nucleoside transporters or p-glycoprotein expression was almost entirely attributable to an increase in the primitive plasma cell population. In 96% (n = 28) of samples from patients in progressive disease there was at least one abnormality in the functional phenotype of the primitive plasma cells. This is in contrast with 44% of samples from patients in stable disease (n = 58). These studies suggest that the functional phenotype of the primitive plasma cell determines the clinical phenotype of patients with myeloma.     

Mechanisms of multiple myeloma cell growth

The mechanism of human multiple myeloma cell growth was studied utilizing eleven myeloma cell lines established in vitro or in vivo (Scid mouse). Four bone marrow derived cell lines grew dependently on IL-6 or bone marrow stromal cells. Seven extramedullary lesion derived cell lines grew spontaneously and additively proliferated in response to IL-6. All cell lines expressed the IL-6 receptor (IL-6R) and IL-6RmRNA, but none expressed IL-6mRNA. No IL-6 activity was detected in the myeloma cell culture supernatant. Both the anti-IL-6 antibody and anti-IL-6R antibody neutralized IL-6-induced proliferation, but did not inhibit spontaneous proliferation of extramedullary lesion derived cell lines. While establishing cell lines, it was found that the proliferating fraction was primarily included in a fraction which was non-adherent to stromal cells and composed of undifferentiated plasmablasts. Undifferentiated plasmablasts proliferated in response to IL-6, in contrast to the adherent, mature form of myeloma cells which did not proliferate in response to IL-6. Innoculation of myeloma cells into Scid mice induced subcutaneous tumor formation. These tumors were composed of undifferentiated plasmablasts, which also proliferated in response to IL-6. These results imply that the growth of bone marrow derived myeloma cell lines are dependent on the IL-6 paracrine mechanism and that the growth of extramedullary lesion derived cell lines primarily autonomous and additively dependent on the IL-6 paracrine mechanism.     

Intermittent calcitriol therapy in secondary hyperparathyroidism: a comparison between oral and intraperitoneal administration

BACKGROUND: Intermittent oral or intravenous doses of calcitriol given two or three times per week are commonly used to treat secondary hyperparathyroidism (secondary HPT). This study was undertaken to compare the biochemical and skeletal responses to thrice weekly intraperitoneal (i.p.) versus oral doses of calcitriol in children with secondary HPT undergoing peritoneal dialysis (CCPD). METHODS: Forty-six patients aged 12.5+/-4.8 years on CCPD for 22+/-25 months were randomly assigned to treatment with oral (p.o.) or i.p. calcitriol for 12 months; 17 subjects given p.o. calcitriol and 16 subjects given i.p. calcitriol completed the study. Bone biopsies were performed at the beginning and at the end of the study, while determinations of serum and total ionized calcium, phosphorus, alkaline phosphatase, parathyroid hormone (PTH) and calcitriol levels were done monthly. RESULTS: Serum total and ionized calcium levels were higher in subjects treated with i.p. calcitriol, P < 0.0001, whereas serum phosphorus levels were higher in those given p.o. calcitriol, P < 0.0001. For the i.p. group, serum PTH levels decreased from pre-treatment values of 648+/-125 pg/ml to a nadir of 169+/-57 pg/ml after nine months. In contrast, serum PTH levels did not change from baseline values of 670+/-97 pg/ml in subjects given p.o. calcitriol, P < 0.0001 by multiple regression analysis. Serum alkaline phosphatase levels were also lower in patients treated with i.p. calcitriol, P < 0.0001, but there was no difference between groups in the average dose of calcitriol given thrice weekly. The skeletal lesions of secondary HPT improved in both groups, 33% of patients developed adynamic bone lesion. CONCLUSION: Differences in the bioavailability of calcitriol and/or in phosphorus metabolism may account for the divergent biochemical response to p.o. and i.p. calcitriol.     

The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7;14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.