Marrow cytometry and prognosis in myeloma.

We have previously shown that flow cytometric analysis of acridine orange-stained bone marrow cells is useful for the objective enumeration and characterization of plasma cells from patients with myeloma, frequently exhibiting an abnormal DNA and an elevated RNA content. In this report on 77 previously untreated patients, we have investigated the biologic and prognostic implications of these quantitative tumor cell parameters. The degree of marrow involvement by tumor, both by microscopic and cytometric analysis, correlated with the clinically derived tumor mass stage. Examination of the product of relative tumor cell RNA content and marrow tumor infiltrate (as a measure of metabolic capacity for immunoglobulin production) in relationship to the myeloma protein concentration in the serum revealed differences in the efficiency of immunoglobulin production and/or catabolism. There was an inverse relationship between the degree of marrow tumor involvement and RNA index, suggesting a more aggressive behavior of myeloma in patients with a low tumor cell RNA content. Prognostically, high tumor cell RNA content identified patients with a high likelihood of response to both initial treatment (32 patients, P = 0.004) and salvage therapy (29 patients, P = 0.01). Favorable factors for survival were low clinical tumor mass stage (P = 0.07) and low marrow tumor infiltrate as determined morphologically (P = 0.04) and cytometrically (P = 0.004). Thus, the direct examination of marrow cellular DNA and RNA content permitted assessment of tumor burden and was useful in the prediction of response and survival.

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.

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.

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.

Modulation of etoposide cytotoxicity and DNA strand scission in L1210 and 8226 cells by polyamines.

The anticancer agent etoposide (VP-16) produces DNA strand scission in intact tumor cells or isolated nuclei. This activity may be mediated by topoisomerase II, an enzyme capable of producing double strand breaks in mammalian cells. Two established tumor cell lines were examined to see whether polyamines, which alter DNA conformation and topoisomerase II activities, affected the cytotoxicity, strand scission, and antitumor efficacy of VP-16. L1210 murine leukemia and 8226 human myeloma cells were treated with alpha-difluoromethylornithine (DFMO) to reduce intracellular polyamine levels via inhibition of ornithine decarboxylase. The polyamines putrescine and spermidine were markedly reduced by a 48-h incubation with 50 microM DFMO. This DFMO concentration did not inhibit colony formation in either cell line, but did reduce the growth rate of both cultures. In contrast, VP-16 produced a dose-dependent inhibition of colony formation. This was especially marked in the 8226 cell line. This correlated with DNA single strand breaks (SSBs) detected by the alkaline elution technique. When cells previously treated with DFMO were exposed to VP-16, a synergistic inhibition of colony formation (determined by isobologram analysis) was observed. However, VP-16-induced SSBs were only marginally increased by the DFMO pretreatment. When putrescine was combined concurrently with VP-16, both the in vitro cytotoxic effects and the number of DNA SSBs in L1210 cells were significantly reduced. These results demonstrate that putrescine inhibits VP-16-induced SSBs and commensurate cytotoxic effects, while DFMO, which depletes intracellular putrescine and partially reduces intracellular spermidine, acts to produce synergistic cytotoxic effects when combined with VP-16.

P31, a mammalian housekeeping protein encoded by a multigene family containing a high proportion of pseudogenes.

The primary structure of P31, a 13.300 kDa mammalian 'housekeeping' protein, was deduced from the nucleotide sequence of a rat recombinant cDNA clone. The P31 mRNA has 950 nucleotides and codes for a protein of 121 amino acids. This mRNA is present in several mouse tissues and in other mammals. Nuclei of rapidly growing cells contain mature P31 mRNA and a distinctive set of larger transcripts. P31 is encoded by a multigene family. Five members of the family were isolated from a mouse genomic library and restriction mapping and S1 nuclease analysis of four of them (P31-4, P31-12, P31-13 and P31-14) suggest that they are processed genes. The very low homology of the fifth (P31-8) with respect to the corresponding cDNA sequence indicates that it is a pseudogene. Although the features of the mRNA and the genes encoding P31 exhibit extensive similarity with those of ribosomal proteins, neither the identity nor the biological function of the protein has yet been determined.

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.

An abundant ubiquitous glycoprotein (GP100) in nucleated mammalian cells.

Two-dimensional gel electrophoresis with the 125I-Con A overlay and affinity purification with Con A-agarose revealed the presence of an abundant ubiquitous 100-kDa glycoprotein (GP100) in nucleated mammalin cells. The amount in cultured human and murine cells varies from 3 to 20 X 10(6) molecules per cell making GP100 the most abundant glycoprotein in nucleated cells. Peptide mapping shows that it is different from erythrocyte Band III protein. Several properties of GP100 suggest that it could play a structural role in nucleated cell membranes.

Intraneuronal accumulation of myeloma proteins.

We studied a patient with multiple myeloma who had immunologically identified myeloma light chain proteins within neurons projecting beyond the blood-brain barrier, but not within intrinsic neurons of the spinal cord, brain stem, and cerebral cortex. There was a possibility that these myeloma light chain proteins or their immunologically recognizable fragments were transported intra-axonally by human peripheral nerves. Myeloma light chain proteins were also identified within cerebellar Purkinje's neurons. This labeling may have been secondary to transport from cerebrospinal fluid.

Biclonal and hypodiploid multiple myeloma.

Plasma cell DNA and RNA content was measured by flow cytometry of acridine orange-stained bone marrow cells from 72 untreated patients with multiple myeloma. Biclonal or hypodiploid DNA stemlines were identified in 10 patients, nine of whom had a low RNA content and did not have response to chemotherapy. Biclonal tumors often showed atypical myeloma protein changes with chemotherapy, suggesting that one clone was reduced whereas the other remained unchanged. These findings suggest a genetic basis for the resistance of low RNA tumors to chemotherapy.

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.

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.

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.

Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C.

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.

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.

Identification and quantification of half-molecule immunoglobulins.

Abnormal IgA1 half-molecules consisting of one heavy and one light chain were found in a patient (NN) with typical multiple myeloma. Serum total protein was 9.4 g/dl, and the serum protein electrophoresis showed a monoclonal gamma-1 fraction amounting to 43.1%. The patient's serum and urine showed immunoelectrophoretically double arcs against anti-alpha antiserum having the same mobility. No double precipitin ring was demonstrated on single radial immunodiffusion analysis of the serum. The serum and the urine of this patient contained both 7.0S and 3.9S IgA-lambda type myeloma proteins. The IgA half-molecules (3.9S) were found to have a molecular weight of 59,000 daltons and were composed of one alpha-1 chain of about 40,000 daltons and one light chain of 22,000 daltons. Furthermore, enzymatic degradation suggested that the alpha chain of the half-molecules had a large deletion in its Fc portion. We suggest that its heavy and light chains were probably bound noncovalently, since the interchains connecting the heavy and light chains of these IgA half-molecules were easily dissociated with 1% SDS and 8M urea. The similar half-molecules of IgG type were also found in another patient of multiple myeloma.