Dose intensity analysis of melphalan and prednisone in multiple myeloma.

The average relative dose intensity (DI) of conventional oral melphalan and prednisone therapy received by 93 newly diagnosed multiple myeloma patients was correlated with survival and with percent reduction in M-protein. A survival advantage was shown with increasing average relative DI of melphalan and prednisone. Multivariate analysis showed survival to correlate with increasing DI of prednisone (P = .05) but not with the DI of melphalan (P = .93) nor with the percent decrement in M-protein (P = .10). These results suggest that the initial management of myeloma should be reassessed, with particular emphasis on more intensive therapy employing high-dose steroids.

Deoxyadenosine metabolism and toxicity in cultured L5178Y cells.

The growth of cultured L5178Y cells is inhibited by relatively low concentrations fo deoxyadenosine in the presence of deoxycoformycin, an inhibitor of adenosine deaminase. Cell viability is reduced, presumably as a consequence of the induced state of unbalanced growth which is characterized by inhibition in DNA synthesis, accumulation of cells in G1 or early S phase, a continuation in RNA synthesis, and increasing cell volume. The intracellular concentrations of purine and pyrimidine ribonucleoside phosphates remain essentially unchanged. The significant changes in the intracellular deoxynucleoside triphosphate pools are an increase in deoxyadenosine triphosphate and a decrease in deoxycytidine triphosphate.

Consequences of inhibition of guanine nucleotide synthesis by mycophenolic acid and virazole.

Mycophenolic acid and virazole are inhibitors of inosinate dehydrogenase and produce growth inhibition and loss of viability in cultured murine lymphoma L5178Y cells. Treatment with 1 muM mycophenolic acid produced the following changes in concentrations of acid-soluble nucleotides: (a) guanosine triphosphate decreased to less than 10% of control within 2 hr; (b) uridine triphosphate and cytidine triphosphate concentrations increased markedly; (c) adenosine triphosphate did not change; (d) deoxyguanosine triphosphate decreased; and (e) thymidine triphosphate increased. DNA synthesis was inhibited by 90% within 2 hr, whereas the incorporation of adenosine into RNA and of leucine into protein were much less affected. Virazole (100 muM) produces similar effects. These biochemical effects of mycophenolic acid, as well as its effects on cell growth, can be prevented by addition of guanylate to the medium. Mycophenolic acid treatment also appears to cause breakdown of high-molecular-weight DNA.

L-phenylalanine mustard (melphalan) uptake and cross-linking in the RPMI 6410 human lymphoblastoid cell line.

L-Phenylalanine mustard (melphalan) induced a time- and concentration-dependent arrest of cycling RPMI 6410 cells in the G2 phase of the cell cycle as evidenced by flow cytofluorometry. A melphalan exposure of 1 microgram/ml for 1 hr caused a temporary G2 blockage which was overcome by 48 hr. Higher concentrations or longer exposures lead to irreversible blockages. Melphalan caused DNA cross-linking which was monitored by the alkaline elution method. The cross-linking was shown to be between DNA and protein. The degree of DNA cross-linking increased for approximately 4 hr after a 1-hr drug exposure of 1 microgram/ml. At 36 to 48 hr after the drug exposure, the cells overcame the G2 block and were dividing. The DNA cross-links have apparently been repaired as they are no longer detected by alkaline elution. The extent of melphalan cross-linking was dependent on both drug dosage and exposure time. Using a culture medium lacking amino acids, it was shown that melphalan uptake into RPMI 6410 cells was inhibited by leucine, isoleucine, or glutamine. The increased uptake of melphalan and the increased cross-linking in amino acid-deficient media were reduced by readdition of the aforementioned amino acids.

DNA strand breaks induced in human T-lymphocytes by the combination of deoxyadenosine and deoxycoformycin.

There is a progressive loss of human T-lymphocyte viability upon incubation with deoxycoformycin, an adenosine deaminase inhibitor, and low concentrations of deoxyadenosine (drug concentration that reduced cell count at 48 hr after initiation to 50% of value for untreated control culture, less than 1 microM). The loss of viability was evidenced by vital staining with fluorescein diacetate and by changes in forward single light scatter measured by flow cytometry. This loss of lymphocyte viability is detectable 18 to 20 hr after the addition of deoxyadenosine and is earlier than has been reported by other investigators using trypan blue as the vital stain. Alkaline elution studies show that the incubation of T-lymphocytes with the combinations of deoxycoformycin and deoxyadenosine gives rise to DNA single-strand breaks. These DNA strand breaks are dose and time dependent and are readily detected 4 hr after the addition of deoxyadenosine. These DNA lesions are not observed with deoxycoformycin or deoxyadenosine alone. Incubations of T-lymphocytes with deoxycoformycin and deoxyadenosine (1 and 5 microM) for 7 hr result in DNA strand breaks with a frequency of 145 and 280 rad equivalents, respectively. Preliminary studies indicate that the ability of lymphocytes to repair this damage is dependent upon deoxyadenosine concentration and exposure time. The relationship of these DNA lesions to loss of lymphocyte viability in the presence of deoxycoformycin and deoxyadenosine remains to be established.

Consequences of methotrexate inhibition of purine biosynthesis in L5178Y cells.

Addition of 1 muM methotrexate to cultures of L5178Y cells results in an initial inhibition of thymidine, uridine, and leucine incorporation into acid-insoluble material followed, after about 10 hr, by a partial recovery in the extent of incorporation of these precursors. Acid-soluble adenosine triphosphate and guanosine triphosphate concentrations are greatly reduced initially, but guanosine triphosphate concentrations appear to recover partially by 10 hr. Acid-soluble uridine triphosphate and cytidine triphosphate concentrations initially increase after methotrexate treatment but then, with time, they too decline. Hypoxanthine and guanine are more effective than is adenine in overcoming the methotrexate-induced inhibition of thymidine incorporation. These results suggest that, in the presence of methotrexate, guanine nucleotides become limiting for nucleic acid synthesis before adenine nucleotides do. The block of purine de novo synthesis in L5178Y cells by methotrexate is almost complete and is not reversed with time. This suggests that the additional purine nucleotides that are available for nucleic acid synthesis 8 to 10 hr after addition of methotrexate are being derived from nucleic acid breakdown. Consistent with this is the observed reduction in the number of polyribosomes and hence, presumably in messenger RNA levels.

Are the current criteria for response useful in the management of multiple myeloma?

One hundred seventy-three patients with multiple myeloma were treated from the time of diagnosis with standard oral melphalan and prednisone at 28-day intervals until they became refractory to treatment. Response to treatment was determined according to the Chronic Leukemia-Myeloma Task Force (TF) criteria, and independently according to the Southwest Oncology Group (SWOG) criteria. Survival by disease stage and response according to the two sets of criteria were analyzed for patients living longer than 3 months. The median survival of responding and nonresponding (TF criteria) stage II patients was 43.8 and 40.3 months, respectively (P = .29). By SWOG criteria, median survival for responding and nonresponding stage II patients was 48.3 and 39.0 months, respectively (P = .12). In stage III patients, median survival for responders and nonresponders (TF criteria) was 34.0 and 21.7 months, respectively (P = .01), compared with 35.5 and 24.4 months (P = .04) by SWOG criteria. These data would suggest that the TF criteria predicts a survival disadvantage only in very advanced myeloma and that applying the stricter limits for the definition of response of the SWOG does not further aid in selecting a subgroup of myeloma patients with poorer survival.

Elevated c-myc messenger RNA in multiple myeloma cell lines.

Oncogene analyses of four human myeloma cell lines provided no indication of gene amplification or rearrangement using DNA probes for the met, raf, abl, mos, erb B, Her-2-neu, fos, myb-7, fms, L-myc, sis, and myb-1 genes. However, a consistent elevation of up to 23-fold in the level of c-myc mRNA was observed in all of the cell lines studied. No restriction fragment length polymorphism (in exons one, two, or three) or c-myc gene amplification has as yet been demonstrated to account for the c-myc mRNA elevation. The c-myc mRNA has a half-life of 25 min which is comparable to that observed in other systems. The elevation in c-myc mRNA is further evidence for the role of the c-myc proto-oncogene in the pathogenesis of myeloma.

Cytogenetic and biological characterization of two new human plasma cell lines.

Two new human plasma cell lines designated as ACB-885 and ACB-1085 have been established from a 39-year-old patient with multiple myeloma. These cell lines have definitive plasma cell features by morphologic examination, and essentially all of the cells are positive for cytoplasmic IgG kappa immunoglobulin. These cells are negative for standard T-cell surface markers and mature B-cell markers, such as B1, B2, and HLA-DR, but are strongly positive for the antigen defined by OKT-10. The cells are negative for Epstein-Barr virus. The cell lines have a doubling time of 30-35 hours and a growth fraction approaching 100%. Cytogenetic analysis showed a 2n chromosome number of 45-46 with very similar karyotypic abnormalities in both the plasma cell lines and the original tumor material. One of the chromosomes in each of the pairs of chromosomes number #1, #2, #6, #7, #8, #10, #12, #13, and #22 were consistently missing. These were replaced by eight marker chromosomes that resulted from chromosomal rearrangements involving mainly these missing chromosomes. Almost all of the breakpoints occurring in the marker chromosomes were identified, and eight of these breakpoints have been reported in other studies of myeloma plasma cells. Homogeneously staining regions were observed in two marker chromosomes suggesting gene amplification in these chromosomal regions.

Clinical pharmacology of oral thioguanine in acute myelogenous leukemia.

Although thioguanine has been in clinical use for over 20 years, few data are yet available on the clinical pharmacology of thioguanine administered orally. We have studied the plasma thioguanine levels in acute myelogenous leukemia patients during remission induction (daunomycin 60 mg/m2 on day 1, arabinosylcytosine 200 mg/m 2. day for 7 days by infusion, thioguanine 100 mg/m2 PO every 12 h for 7 days) and remission maintenance (arabinosylcytosine 200 mg/m2 . day for 4 days by infusion, thioguanine 100 mg/m2 PO every 12 h for 4 days). Hourly blood samples were taken after thioguanine administration, and plasma thioguanine levels were measured by high-performance liquid chromatography with an anion-exchange column. Prior to the chromatography the thioguanine was oxidized by alkaline potassium permanganate to the corresponding 6-sulfonate, which was monitored by means of fluorescence detection. Peak plasma levels of thioguanine were observed 2-4 h after administration and varied from 0.03-0.94 microM. Plasma levels of thioguanine were markedly lower in patients with severe nausea and emesis. Food intake at the same time as thioguanine administration also tended to lower plasma drug levels. The 30-fold range in thioguanine plasma levels observed in this study suggests that intermittent IV administration may provide a better means of standardizing the dosage of thioguanine.

Adenosine and deoxyadenosine toxicity in colony assay systems for human T-lymphocytes, B-lymphocytes, and granulocytes.

Adenosine and deoxyadenosine toxicity was examined in colony assay systems for human T lymphocytes, B lymphocytes, and granulocytes. In the absence of deoxycoformycin, an adenosine deaminase inhibitor, no growth inhibition was observed in the three systems with concentrations of adenosine or deoxyadenosine of at least 200 microM. Deoxycoformycin itself had no growth-inhibitory effect at concentrations of at least 10 micrograms/ml. Combinations of deoxycoformycin (1 microgram/ml) and either adenosine or deoxyadenosine gave growth inhibition in all three systems. Deoxyadenosine was the most toxic in all the systems, the LD50 values being 20-25 microM. The LD50 values for adenosine were 45-55 microM. There was no evidence of selective toxicity by adenosine or deoxyadenosine with these three colony assay systems. In the T-lymphocyte colony system deoxyadenosine appeared to be toxic to both the inducer/helper and the suppressor/cytotoxic T-lymphocyte subpopulations.

Loss of viability and induction of DNA damage in human leukemic myeloblasts and lymphocytes by m-AMSA.

The effects of m-AMSA on in vitro viability and on the induction of DNA damage were examined in low-growth-fraction cell populations of human leukemic myeloblasts and normal lymphocytes. A significant individual variation in the drug-induced reduction of in vitro viability was observed in studies with five selected leukemic patients. The concentration of m-AMSA required to reduce viability by 50% within 48 h ranged from 0.25 microM to in excess of 5.0 microM for the leukemic myeloblasts as against about 2.0 microM for the samples of normal lymphocytes. Alkaline elution studies showed that m-AMSA induced protein-associated DNA strand breaks (PADB) in both myeloblasts and lymphocytes. Depending upon the m-AMSA concentration, there was a 4- to 9-fold difference in the level of PADBs induced by a given drug concentration in the myeloblasts of eight patients studied. The level of PADBs was saturable with respect to both drug concentration (5-10 microM) and exposure time (45-10 microM). The PADBs were repaired rapidly in all the lymphocyte and myeloblast samples studied, with over 90% of this DNA damage being repaired within 45 min after resuspension of the cells in drug-free medium. These studies of m-AMSA in low-growth-fraction samples of human lymphocytes and myeloblasts show both similarities and differences in the action of this drug compared with previously published studies using the high-growth-fraction mouse L1210 system.

Treatment of multiple myeloma with deoxycoformycin.

A comparison of adenosine deaminase activity in intact human plasma cells and lymphocytes in vitro showed that plasma cells had at least as much activity of this enzyme as did T or non-T lymphocytes. This observation led us to examine the effectiveness of deoxycoformycin in the treatment of multiple myeloma. Thirteen patients with advanced refractory myeloma were treated with deoxycoformycin at 5 mg/m2 daily for 3 days every 2 weeks until response or progression. Of the seven evaluable patients who received more than one cycle of therapy, two had a greater than 50% reduction in the level of myeloma protein and two had a demonstrable reduction in soft tissue disease. Toxicity consisted of marked nausea, anorexia lasting several days, and mild transient confusion in some patients. Plasma levels of deoxyadenosine and adenosine peaked on day 4 or 5 with average values of 1.9 and 0.6 microM, respectively. Red cell levels of dATP reached approximately 40% of ATP levels. The viability of plasma cells was shown to be greatly reduced in in vitro incubations with deoxycoformycin and low levels of deoxyadenosine (ID50 of 6 microM).

Individual variation in inosine triphosphate accumulation in human erythrocytes.

Erythrocytes from 5% of a normal population accumulated relatively high amounts of radioactive inosine triphosphate (i.e., greater than 70 nmoles/10(10) cells in 2 hr) when they were incubated with [14C]hypoxanthine. The incidence of this characteristic in a mentally retarded population was 16%. Inosine triphosphate was synthesized from [14C]hypoxanthine, but not from [14C]adenine or [14C]guanine. The metabolism of [14C]adenine and [14C]guanine was the same in erythrocytes that accumulated "normal" and "high" amounts of inosine triphosphate. Inosine triphosphate did not accumulate in leukocytes.