Altered expression of growth-regulated protooncogenes in human malignant plasma cells.

The expression of three growth-regulated protooncogenes, c-myc, c-myb, and p53, and the S-phase-specific histone H3 gene, was compared in bone marrow cells from multiple myeloma patients and normal controls by measuring the amount of specific RNA by Northern blot analysis. Four samples contained at least 72% of myeloma cells, one sample 43%, and one 11%. Expression of the protooncogenes was similar in normal and myeloma bone marrow cells, whereas that of histone H3 gene was significantly reduced (between 10 and 15 times) in samples containing at least 43% of malignant plasma cells and not detectable in those containing more than 72% of neoplastic cells. Protooncogene levels of expression were compared to those of the H3 gene to distinguish the increased expression of a growth-regulated gene due to a true deregulation from overexpression reflecting solely an increase in the fraction of cycling cells. The ratios of expression of protooncogenes to histone H3 were markedly increased in multiple myeloma cells; the highest ratios were found in the patients with the highest number of malignant plasma cells. These results suggest that the expression of three growth-regulated oncogenes (c-myc, c-myb, p53) is altered in myelomatous plasma cells.

P-glycoprotein expression in plasma-cell myeloma is associated with resistance to VAD.

Tumor cell-associated expression of multidrug resistance (MDR) was quantitated in 22 patients with DNA-aneuploid myeloma using 2-parameter flow cytometry with monoclonal antibody (MoAb) C-219 for the detection of cytoplasmic p-170 and propidium iodide for nuclear DNA content. The proportion of cells expressing p-170 and the intensity of p-170-related fluorescence were determined for each patient. Among the 14 patients treated with vincristine-adriamycin-dexamethasone (VAD), the proportion of p-170-positive cells distinguished sensitive from resistant disease (P less than .01). Among a subgroup of seven patients with MDR analysis available prior to VAD therapy, two subsequent nonresponders had high proportions of C-219-reactive cells. The presence de novo of high proportions of p-170-expressing cells in another still untreated patient and in a further individual with resistance to dexamethasone and interferon (not associated with MDR) warrants systematic analysis of p-170 expression prior to therapy to determine its clinical implications for response to MDR-associated drugs as combined in the VAD regimen. Concurrent MDR expression by aneuploid tumor cells and cells in the diploid subcompartment may represent involvement of diploid cells in the myeloma disease process.

[Beta 2 microglobulin in some hematologic neoplasms]. / Beta 2 microglobulina en algunas neoplasias hematológicas.

Beta 2 microglobulin is a low molecular weight protein integrating the light chain HLA antigens. Its serum concentration is increased in different neoplasias and in renal failure. Using solid phase RIA we determined the concentration of beta 2 microglobulin in plasma and spinal fluid of 57 healthy individuals and patients with hematologic neoplasia. Serum levels were 1.34 +/- 0.34 mg/l and spinal fluid levels were 1.3 +/- 0.7 mg/l in healthy subjects. Serum levels in 29 patients with myeloma was 7.51 mg/l, significantly higher in those with renal failure (12.35 mg/l) compared to those without (4.54). In 30 patients with non-Hodgkin lymphoma the mean serum levels were 2.90 mg/l, significantly greater in those with active disease (3.18) than in those with remission (1.5). No difference was found according to the degree of malignancy. Patients with acute lymphatic leukemia had elevated values of beta 2 microglobulin while the disease was active (3.37 mg/l), decreasing to normal levels after remission (1.79 mg/l). Spinal fluid levels of beta 2 microglobulin were elevated only in patients with central nervous system involvement. Our results indicate that serum levels of beta 2 microglobulin are helpful in patients with hematologic neoplasia in assessing the activity of the disease and tumor mass, especially in multiple myeloma.

Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining.

Prior studies have shown that the P-glycoprotein is a cell membrane efflux pump that is quantitatively increased in expression in multidrug-resistant tumor cell lines. In this study, fresh tumor tissues from patients with multiple myeloma, malignant lymphoma, or metastatic breast cancer were studied immunohistochemically for P-glycoprotein expression and for in vitro sensitivity to doxorubicin. Twenty-six patients who were either previously untreated or in relapse after chemotherapy had tumor specimens submitted that could be evaluated in both assays. The testing was done independently and blindly in separate laboratories instead of our being provided relevant clinical data on the patients. Tumor cells from 12 of the 26 patients (46%) stained positively for P-glycoprotein. Fifteen of the 26 specimens (58%) exhibited drug resistance in vitro. Although only three (21%) of the 14 P-glycoprotein-negative tumors exhibited in vitro resistance to doxorubicin, all 12 fresh tumors that stained positively for P-glycoprotein were resistant to doxorubicin. The difference in frequency of intrinsic doxorubicin resistance between P-glycoprotein-negative and -positive tumors was highly significant (P less than .001). Similar trends were observed in each of the individual tumor categories and were statistically significant in myeloma and breast cancer. Four of the biopsy specimens that stained positively for P-glycoprotein and exhibited doxorubicin resistance were from patients who had not received prior cytotoxic chemotherapy. Similar conclusions were reached when results of drug sensitivity tests were ranked in relation to the median infective dose rather than by criteria based on correlations with clinical drug resistance. Our findings indicate that positive staining for P-glycoprotein associated with multidrug resistance predicts intrinsic cellular resistance of human cancers to doxorubicin. We anticipate that immunohistochemical staining for P-glycoprotein will prove useful in clinical oncology.

Response patterns of purified myeloma cells to hematopoietic growth factors.

Tumor cells were isolated from the bone marrow of seven patients with multiple myeloma and from the peripheral blood of three patients with plasma cell leukemia using Ficoll-Hypaque (FH) density sedimentation followed by immune rosette depletion of T, myeloid, monocytoid, and natural killer (NK) cells. Enrichment to greater than or equal to 93% plasma cells was confirmed with Wright's-Giemsa staining, with intracytoplasmic immunoglobulin staining, and with staining using monoclonal antibodies (MoAbs) directed at B, T, myeloid, monocytoid, and myeloma antigens in indirect immunofluorescence assays. Myeloma cells neither proliferated nor secreted Ig in response to G/M-CSF, G-CSF, M-CSF, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2), or interleukin-4 (IL-4). Significant proliferation (SI greater than or equal to 3.0) was induced by interleukin-6 (IL-6) in six of ten patients (SI of 31 and 43 in two cases); and to interleukin-3 (IL-3) and interleukin-5 (IL-5), independently, in two patients each. Peak proliferation to IL-5 or IL-6 and to IL-3 occurred in cells pulsed with 3[H] thymidine at 24 and 48 hours, respectively; and proliferation to combinations of factors did not exceed that noted to IL-6 alone; Ig secretion was not documented under any culture conditions. Three myeloma-derived cell lines similarly studied demonstrated variable responses. The heterogeneity in the in vitro responses of myeloma cells and derived cell lines to exogenous growth factors enhances our understanding of abnormal plasma cell growth and may yield insight into the pathophysiology of plasma cell dyscrasias.

Tumor cell heterogeneity in multiple myeloma: antigenic, morphologic, and functional studies of cells from blood and bone marrow.

Tumor cells from six patients with immunoglobulin G (IgG) multiple myeloma were analyzed for surface antigens, cytoplasmic paraprotein, morphology, and response to various culture conditions. The tumor marker was the paraprotein idiotype. Low numbers of tumor cells were found in the blood of most of the patients. In some patients, the circulating tumor cells were solely B lymphocytes, whereas in other patients, they were lymphoid, lymphoplasmacytoid, and plasmacytoid. Dual surface antigen analysis of blood and bone marrow cells confirmed that the tumor may be composed of a spectrum of cell types. Thus, cells may range from surface-idiotype+,CD19+,CD20+, PCA-1-,cytoplasmic-idiotype- lymphocytes, to CD19-,PCA-1+,cytoplasmic-idiotype+ plasma cells that are surface-idiotype- or weakly surface-idiotype+. In one patient, some of the tumor cells co-expressed surface idiotype and CD10. The tumor B lymphocytes were activated in vitro to synthesize paraprotein by pokeweed mitogen (PWM), and by low molecular weight B cell growth factor (BCGF). In contrast, spontaneous synthesis of paraprotein by more mature tumor cells was inhibited by agents that also inhibit nonmyeloma plasma cells. These agents included PWM, gamma interferon, and phorbol ester. The results demonstrate that in multiple myeloma there exist different tumor cell types that are similar, by a variety of criteria, to normal B lineage cells at different stages of differentiation. Thus, further evidence is provided for the hypothesis of myeloma cell differentiation.

Phenotypic analysis of human myeloma cell lines.

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation, primarily in bone marrow, of a clone of plasma cells. The nature of the stem cells feeding the tumoral compartment is still unknown. To investigate this special point, we have studied the phenotypes of nine well-known human myeloma cell lines (HMCLs) and compared them with those of normal lymphoblastoid cell lines (LCLs). Twenty-four clusters of differentiation involved in B lymphopoiesis were investigated using a panel of 65 monoclonal antibodies (MoAbs). For each cluster, the percentage of positive cells and the antigen density were determined, giving rise to a "quantitative phenotype". We thus classified the HMCLs into two different groups: those with cytoplasmic mu chains (c mu+) and those without (c mu-). In the first (c mu+) group, comprising seven cell lines, the HMCLs had a phenotype of pre-B/B cells close to that of Burkitt's lymphoma cell lines. They expressed low densities of surface mu chains, without detectable cytoplasmic or surface light chains. Three of them were infected with the Epstein Barr virus (EBV). These c mu+ HMCLs bore most of the B-cell antigens except CD23. They expressed the CALLA antigen (CD10) and lacked the plasma-cell antigen PCA1. In contrast, LCLs expressed surface light chains, high densities of CD23, low densities of PCA1 antigen, and no CD10 antigen. The c mu- HMCLs had a plasma-cell phenotype, lacking most of the B-cell antigens and expressing high densities of PCA1 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual parameter analysis of myeloma cells by flow cytometry. DNA content of cells containing monotypic cytoplasmic immunoglobulin.

Dual-parameter flow cytometric analysis of monotypic cytoplasmic immunoglobulin (CIg) and DNA content on 15 myeloma marrows allowed S-phase determination of the CIg(+) tumor separately from the CIg(-) hematopoietic cell pool. The median percentage of the CIg(+) cells in S-phase was 2% compared with 5% for the CIg(-) cells. The median survival of patients with more than 2%, and those with 2% or less CIg(+) S-phase cells was 2 months and more than 13 months, respectively, from the time of study. Ploidy analysis identified four patterns of plasma cell DNA content: entirely hyperdiploid, entirely diploid, combined diploid and tetraploid stemlines, and tumors containing diploid and aneuploid CIg(+) cells. A monotypic CIg(+) double stemline myeloma was distinguished from two aneuploid tumors containing admixed normal, diploid polyclonal plasma cells. This technique provides an improved and expedient means for determining the proliferating fractions of myeloma cells and enhances recognition of double stemline tumors and clonal evolution in myeloma.

Sialic acid content in mouse myeloma cells and derived B-cell hybridomas with different metastatic potentials.

In a previous work we have reported on the metastatic capacities of mouse myeloma cells (NSO) and of two hybridomas derived from them. The present study was designed to examine the sialic acid content of these cell types, their attachment to glass and their capping in the presence of wheat germ agglutinin (WGA). The results showed no significant differences in sialic acid content between NSO and Hybridoma A (Hy A) cells which are non and low metastatic, respectively, whereas Hybridoma B (Hy B), which is highly metastatic, contained 1.4-1.6-fold higher sialic acid compared to the other two cell types. NSO cells exhibited a high, Hy A a moderate and Hy B a negligible attachment to glass. Capping experiments showed a low response of NSO cells, a moderate response of Hy A and a high response of Hy B in the presence of WGA. The results are discussed with regard to the metastatic potential of these cell types taking into consideration the properties cell surface sialic acid is known to endow to cells, determining various aspects of their behaviour.

Restrictive cardiomyopathy with kappa light chain deposits in myocardium as a complication of multiple myeloma. Histochemical and electron microscopic observations.

kappa Light chain deposits occurring in myocardium as a complication of multiple myeloma were identified ultrastructurally and immunohistochemically in a right ventricular endomyocardial biopsy specimen from a patient who presented with clinical and hemodynamic findings of restrictive cardiomyopathy. These deposits were not evident on routine histopathologic examination; they were Congo red-negative and gave a positive immunoperoxidase reaction for kappa light chains and a negative reaction for lambda chains. They consisted of amorphous, electron-dense granules that formed discontinuous layers adjacent to the plasma membranes of cardiac myocytes, arteriolar endothelial and smooth-muscle cells, and neural elements. These observations underscore the need for critical study of endomyocardial biopsy specimens, using electron microscopy and immunohistochemical reagents, for the precise identification of protein components in tissue deposits in patients suspected of having cardiac amyloidosis or related disorders.

Expression of an antibody Fv fragment in myeloma cells.

The antigen binding site on antibodies is fashioned by loops at the tips of the beta-sheet framework of both heavy and light chain variable domains. A heterodimer of both variable domains (Fv fragment), incorporating loops from an anti-lysozyme antibody, was expressed and secreted from myeloma cells in good yield (8 mg/l in supernatant from roller bottles), and shown to bind lysozyme. The two subunits were found to be in dynamic equilibrium but are overwhelmingly associated at neutral pH. The small size of Fv fragments (25 x 10(3) Mr) make them attractive for structural studies, in vivo imaging, and therapy.

Identification of a second transforming gene, rasn, in a human multiple myeloma line with a rearranged c-myc allele.

Multiple myeloma is a disease characterized by a long, slowly progressive phase and a final, more aggressive one. Little is known about the mechanism of transformation of myeloma cells, although the clinical characteristics of the disease suggest a multi-step process. Recently, a myeloma cell line, NCI-H929, was isolated from a patient with aggressive preterminal disease and found to have a rearranged myc allele. This myeloma cell line has been further characterized in a focus formation assay to determine whether its unusual growth characteristics were associated with a second activated transforming gene. We now report that the NCI-H929 myeloma cell line has an activated rasn allele in addition to a rearranged myc allele. This is the first identification of an activated transforming gene in a multiple myeloma cell line; furthermore, the characterization of two independently activated oncogenes in this B cell malignancy has implications for both the pathogenesis and evolution of the disease.

Establishment and characterization of an amylase-producing human myeloma cell line.

Two stable lines of IgA lambda-producing plasma cells (KHM-1A and KHM-1B) that were free of the Epstein-Barr virus were established from a patient with multiple myeloma complicated by hyperamylasemia. Surface marker studies of the two cell lines showed that the cells had no surface immunoglobulins but were positive for cytoplasmic immunoglobulins (IgA lambda) and for HLA-DR and PCA-1. Secretion of IgA monoclonal immunoglobulin by the two lines was detected by a plaque-forming cell assay and by an enzyme-linked immunosorbent assay of culture media. KHM-1B cells also secreted alpha-amylase, but no such activity was detected in the culture-conditioned supernatant fluid of KHM-1A.

Correlated flow cytometric analysis of H-ras p21 and nuclear DNA in multiple myeloma.

Correlated analysis of the H-ras oncogenes product (p21) and of nuclear DNA content was performed by flow cytometry (FCM) in patients with DNA-aneuploid multiple myeloma (MM). Bone marrow cells from normal donors and MM patients in remission served as controls. Seventy-four percent of 23 patients with active MM had higher p21 fluorescence in aneuploid tumor cells than were observed in normal donor or myeloma remission bone marrows; 39% of the 23 patients also showed high H-ras p21 expression in diploid cells. There was an inverse relationship between p21 levels and the presence of trisomy 11; especially high p21 levels were noted in patient without trisomy 11. The frequent elevation of p21 protein in aneuploid plasma cells suggests the involvement of the H-ras oncogene in the pathophysiology of MM, which is further supported by a shorter survival among patients with high p21 levels.

Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement.

The expression of early and mature B cell markers, surface beta 2-microglobulin (B2M) and cytoplasmic immunoglobulin (clg) by aneuploid tumor cells in bone marrow aspirates from 44 patients with multiple myeloma was evaluated by correlated DNA immunofluorescence flow cytometry. Myeloma tumor cells of almost 90% of the patients contained monoclonal clg and expressed the mature plasma cell antigen R1-3 as well as surface B2M; common acute lymphoblastic leukemia antigen (CALLA) was present in 55%, B2 in 17%, and B4 in 23% of samples studied. Coexpression of CALLA and clg in 46% of all patients identified a novel myeloma phenotype without known counterpart in the normal differentiation of B cells. CALLA and clg were independently expressed and gave rise to CALLA+/clg-, CALLA+/clg+, and CALLA-/clg+ cells. The association of CALLA and mature plasma cell markers may define discrete stages of neoplastic plasma cell differentiation.

Phenotypic and functional analysis of 1,25-dihydroxyvitamin D3 receptor mediated modulation of the human myeloma cell line RPMI 8226.

Several recent studies have demonstrated the presence of specific receptors for the 1,25-dihydroxyvitamin D3 (calcitriol) in activated normal lymphocytes. By DNA cellulose chromatography, we show evidence of such specific receptors in the human myeloma cell line RPMI 8226. Nanomolar concentrations of 1,25-dihydroxyvitamin D3 reduce the proliferation of RPMI 8226 cells significantly and simultaneously induce the appearance of both new properties and phenotype expression, such as butyrate esterase, enhanced expression of CD20 (B1), CD15 (Leu-M1) antigens and lambda chains, and decreased expression of the PC1 antigen using microfluorometric analysis. But such an increased expression of membrane lambda chains was not associated with an enhanced secretion of lambda chains. Furthermore, the bone resorbing activity produced normally by RPMI 8226 cells was reduced significantly after 1,25-dihydroxyvitamin D3 treatment. The possible mechanisms and significance of these new functional and phenotypic properties are discussed with respect to the B-cell lineage.

Protein Rou. A human IgA hybrid.

Protein Rou is a human IgA2 myeloma protein that carries the isoallotype marker n A2m(2). Partial amino acid sequence of its H chain (alpha) shows that the hinge region and the CH2 domain are homologous to alpha 2-chain and the CH1 and the CH3 domains homologous to alpha 1. Moreover, the CH1 domain contains the H-L disulfide bond identical to alpha 1. It is concluded that Rou H chain is a hybrid molecule caused by a recombination between alpha 1 and alpha 2 genes. The recombination event occurred between alpha 1-exon 1 and alpha 2-exon hinge and corresponds to position 222-223 of the alpha-chain.