Phase II evaluation of low-dose continuous 5-fluorouracil and weekly cisplatin in advanced adenocarcinoma of the stomach. A Southwest Oncology Group study.

We have conducted a Phase II trial in patients with metastatic gastric cancer utilizing low-dose continuous infusion 5-fluorouracil (5FU) and weekly cisplatin (CDDP). The 5FU was administered at a dose of 200 mg/m2 per day by 24-hour continuous infusion via a permanent central venous catheter. The CDDP was administered at a dose of 20 mg/m2/week for the first 8 weeks, then every other week thereafter. Patients were evaluated for response every 8 weeks. There were 2 complete and 2 partial responses out of 39 eligible and evaluable patients for an overall response rate of 10% (95% CI = 3-24%). The primary toxicities were nausea, hyponatremia, and anemia. Overall, treatment was well tolerated. We conclude that, although the treatment is relatively well tolerated, the regimen has minimal activity in this disease and does not deserve further study.

A phase II trial of piroxantrone in advanced ovarian carcinoma after failure of platinum-based chemotherapy: Southwest Oncology Group Study 8904.

A phase II trial of the new anthrapyrazole piroxantrone was conducted by the Southwest Oncology Group in advanced ovarian carcinoma. The objective were to evaluate its response rate and toxicity in patients who had disease persistence, progression, or recurrence either during or after platinum-containing chemotherapy. A two-stage statistical design targeted accrual to 15 eligible patients if no responses were observed. The piroxantrone starting dose was 120 mg/m2, with the provision to escalate to 150 and 180 mg/m2. There were 16 eligible patients, all of whom had received either one (12 patients) or two (4 patients) prior platinum-containing regimens; one patient had received doxorubicin. Fourteen of the 16 patients were enrolled either at the time of disease persistence/progression during initial chemotherapy or with recurrence or progression within 6 months of the previous platinum-based remain. One to 5 cycles of piroxantrone were given. Dose escalation was feasible in 7 patients but was prevented in the other 9 by neutropenia. Maximum toxicity for all cycles was none or grade 1 in 2 patients; grade 2, 5; grade 3, 8; and grade 4, 1. All but one of the grade 3 or 4 events was from myelosuppression; there were no adverse cardiac events. No responses were observed. Thus, piroxantrone appears inactive in patients with persistent, progressive, or recurrent ovarian cancer who recently had received a platinum-based regimen.

Mononuclear cell polyamine content associated with myeloid maturation in patients with leukemia during administration of polyamine inhibitors.

Fourteen patients with acute leukemia in relapse were treated with difluoromethylornithine (DFMO) alone or in combination with methylglyoxal-bis(guanylhydrazone) (MGBG) as part of Phase I studies. Five patients included in the trial exhibited morphologic evidence of cellular differentiation during the course of treatment. In one patient who exhibited no blasts and a normal white blood cell differential at the end of treatment the mononuclear cell content of all three polyamines declined after an initial increase in spermidine and spermine content. In the other patients in whom the cellular maturation was less pronounced the mononuclear cell polyamine levels remained stable or increased over the treatment time. No absolute difference was apparent between the cellular polyamine levels detected in patients at the times of the greatest increase in per cent circulating neutrophils as compared to the cellular levels present in patients whose circulating mononuclear cell number were increasing. Circulating mononuclear cell putrescine, spermidine, and spermine levels varied over two orders of magnitude from patient to patient and the range of values detected in each state completely overlapped those present in the other. It does not appear from the present study that there is a consistent human leukemic cell polyamine content at which cellular differentiation occurs.

Coexistence of myelodysplastic syndrome and untreated chronic lymphocytic leukemia with development of acute myeloid leukemia immediately after treatment of chronic lymphocytic leukemia.

A 72-year-old man originally seen for anemia and thrombocytopenia was determined to have chronic lymphocytic leukemia (CLL). Bone marrow examination at the time of CLL diagnosis revealed a small but significant population of atypical blasts. Cytogenetic analysis of the bone marrow aspirate disclosed chromosomal abnormalities (-7, +8) suggestive of a myelodysplastic syndrome. Shortly after treatment of the CLL, there was proliferation of the previously noted blast population, which cytochemical studies demonstrated to be of the myeloid series thus indicating acute myeloid leukemia superimposed on CLL. This report presents microscopic, cytogenetic, immunophenotypic, and cytochemical evidence to document the evolution of acute myeloid leukemia in the bone marrow of a patient with CLL after one course of chemotherapy.

5-bromodeoxyuridine (BUdR) quenching of acridine orange fluorescence distinguishes cycling and non-cycling normal and malignant bone marrow cells in vitro.

Suppression of the green fluorescence of acridine orange by 5-bromodeoxyuridine incorporation into cellular DNA was measured by flow cytometry. Bone marrow cells from normal volunteers and patients with chronic myelogenous leukemia acute lymphocytic leukemia acute myelogenous leukemia and multiple myeloma were incubated with BUdR in vitro. By 24 h acridine orange stained cycling cells that had synthesized DNA in the presence of BUdR were differentiated from quiescent cells as a second peak with quenched green fluorescence (DNA). After 72 h in culture 11-65% of the G0/G1 cells from normal bone marrows and bone marrows with myeloid leukemia were identified as cycling in culture by the presence of a second peak with quenched green fluorescence. A greater percentage of cells with BUdR quenched AO fluorescence was associated of acridine orange was higher in the cycling cells that had synthesized DNA in the presence of BUdR than in the non-cycling G0/G1 cells. In one patient with AML there was quenching of the DNA fluorescence of the aneuploid population but not the diploid population indicating that the aneuploid leukemia cells were proliferating. In contrast in patients with multiple myeloma, the quenched fluorescence of the diploid cell population and not the aneuploid cells, indicated that the diploid cells were proliferating. The cells from patients with untreated ALL failed to proliferation prohibiting an in vivo assessment of growth. Although measurements of proliferation obtained by this method are clearly influenced by the cell's adaptation to culture, measurement of BUdR quenching of acridine orange fluorescence is technically feasible and can identify and allow characterization of the cycling population of cells.

Alterations in human circulating and bone marrow mononuclear cell polyamine levels in hematologic malignancies as a consequence of difluoromethylornithine administration.

The effect of administering increasing intravenous doses of difluoromethylornithine on human tumor cell polyamine levels was determined in patients with hematologic malignancies. Difluoromethylornithine from 5.5. to 64 gm/m2 per day was administered to nine patients with refractory acute leukemia or multiple myeloma. Putrescine, spermidine, and spermine levels were determined on a daily basis in the circulating mononuclear cells and on a weekly basis in the mononuclear cells of the bone marrow. Tumor cell putrescine levels declined in 5 patients, spermidine levels declined in 4 patients, and spermine levels declined in 3 patients. Alterations in the polyamine levels of the bone marrow mononuclear cells paralleled those occurring in the peripheral blood mononuclear cells in the patients with leukemia. Seven to ten days of DFMO treatment were required for mononuclear cell polyamine levels to decrease. The higher drug doses were not significantly more effective than the lower doses in bringing about a decline in tumor cell polyamine levels, either with respect to treatment time required for onset of response or with respect to the ultimate extent of response.

Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guanylhydrazone) levels: phase I evaluation of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) treatment of human hematological malignancies.

Nine patients with hematological malignancies were treated with difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The number of circulating blast cells decreased in all of the patients treated with DFMO and MGBG for longer than 1 wk. Morphological evidence of myeloid maturation was evident in four patients with leukemia and the circulating M Protein decreased in one patient with multiple myeloma. The polyamine content of the mononuclear cells in both the peripheral blood and bone marrow was transiently increased after the initial MGBG dose. During administration of DFMO decreases were achieved in the peripheral blood mononuclear cell putrescine levels in 7 patients, spermidine levels in 5 patients, and spermine levels in 4 patients. Alterations in bone marrow mononuclear cell polyamine levels were similar to those which occurred in the peripheral cells. An average of 9 days of DFMO treatment was required to lower mononuclear cell polyamine levels. Three of the 4 evaluable patients receiving multiple MGBG doses had an increased mononuclear cell content of MGBG after DFMO pretreatment. Enhancement of cellular MGBG levels was not directly correlated to the degree of cellular polyamine depletion.

Species specificity of antibodies to regulatory subunits of cyclic AMP-dependent protein kinases.

Polyclonal antibodies were generated against regulatory subunits (RI and RII) of type-I and type-II cAMP-dependent protein kinases from rat skeletal muscle. Western immunoblot analyses showed specific cross-reactivity of rat and bovine RI with anti-RI. Similarly, RII from both species was specifically recognized by anti-RII. Quantitative immunoassays, using antisera against proteins from either species, indicated selectivity towards regulatory subunits from the same species. Molecular basis for this selectivity was examined by comparison of peptide maps of 32P-8-azido-cAMP-labelled or autophosphorylated peptides. Detailed analysis of two-dimensional peptide fingerprints demonstrated extensive homology between either RI or RII from the two species. The data suggests that the overall protein-chemical and functional determinants characterizing type-I and type-II regulatory subunits of cyclic AMP dependent protein kinase from different species are substantially similar. However, minor differences in structure, also predicted by amino-acid sequences for RI and RII obtained by molecular cloning, may account for the distinct immunological properties of the proteins from rat and bovine tissues.

Characteristics of cyclic AMP enhancement of retinoic acid induction of increased transglutaminase activity in HL60 cells.

When the human myeloid leukemia cell line (HL60) is induced to differentiate with retinoic acid (RA), there is a concentration-dependent increase in transglutaminase (TGase) activity which peaks on day 5. While dibutyryl 3',5'-cyclic adenosine monophosphate (db-cAMP) alone produced only a slight increase in TGase activity in HL60 cells, the concomitant addition of db-cAMP (100 microM) with RA (10(-12)-10(-4) M) potentiates RA induction of TGase activity. Maximal increases in TGase activity (2- to 10-fold) were observed with 10(-4)-10(-7) M RA and when db-cAMP was present from 24 to 48 h after the addition of RA. The cyclic nucleotide enhancement was dose-dependent from 10 to 100 microM of cAMP. Less marked increases were observed with 8-bromo-cAMP and with the phosphodiesterase inhibitor theophylline. Although the simultaneous addition of PGE1 or PGE2 (10(-8)-10(-6) M) produced no enhancement of RA-induced TGase activity, adding PGE1 or PGE2 24 or 48 h following RA treatments produced an enhancement of TGase activity. The phosphodiesterase inhibitor potentiated the increases produced by db-cAMP and the prostaglandins. Dibutyryl cAMP enhanced the ability of RA to induce the cells to reduce nitroblue tetrazolium (NBT), a functional measure of differentiation, at lower concentrations of RA and with shorter treatment durations. cAMP potentiates RA-induced TGase activity in HL60 cells and the combination appears to be associated with enhanced RA-induced differentiation.

Polyamines increase in human peripheral blood and bone marrow mononuclear cells following administration of methylglyoxal bis(guanylhydrazone).

Eight patients who had refractory leukemia and 1 patient with refractory multiple myeloma were treated with the polyamine biosynthesis inhibitors methylgloxal bis(guanylhydrazone) (MGBG) and difluoromethylornithine (DFMO). After the first dose of MGBG there was an increase in polyamine content in the mononuclear cells of both the peripheral blood and the bone marrow despite the administration of DFMO in all patients with leukemia. Putrescine levels increased in the mononuclear cells of all patients, cellular spermidine levels increased in 4 and cellular spermine levels increased in 5 patients. The cellular polyamine levels remained elevated above the pretreatment levels for up to 1 week in some patients. Subsequent treatment with MGBG, administered after 1-2 weeks of DFMO treatment, also promoted increases in mononuclear cell polyamine concentrations. Since enhanced tumor cell uptake of MGBG after DFMO priming is hypothesized to be dependent on a decrease in cellular polyamine levels, the increase in cellular polyamines after MGBG has important implications for the scheduling of this drug combination. From these observations, withholding MGBG until DFMO treatment has produced a decrease in tumor cell polyamine concentrations would be the schedule most likely to enhance the uptake of MGBG.

Difluoromethylornithine enhances the uptake of methylglyoxal-bis(guanylhydrazone) prior to inhibiting leukemic cell proliferation.

Difluoromethylornithine (DFMO) is a nonreversible inhibitor of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in the polyamine biosynthetic pathway. When HL60 leukemic cells were incubated in the presence of concentrations of DFMO from 0.05 mM to 5 mM, there was a concentration-dependent inhibition of ODC activity apparent within 24 h. Likewise, cellular polyamine levels were reduced by the presence of DFMO in a concentration-dependent manner after 4 days. The growth of cells incubated with 0.5 mM or greater was inhibited after 3-4 cell doublings. When the concentration of DFMO was less than 0.5 mM, growth was not inhibited. Methylglyoxal-bis(guanylhydrazone) (MGBG) uptake was enhanced in cells treated with concentrations of 0.05-0.5 mM DFMO, but not enhanced in cells treated with DFMO concentrations of 1 mM or greater. DFMO-induced cellular polyamine depletion does enhance MGBG uptake into HL60 cells, but treatment with high concentrations of DFMO, which deplete polyamines to the extent that growth is inhibited, negate this effect.

Prognostic factors for survival of 194 patients with low infiltrate leukemia.

Prognostic factors were identified from the histories of 194 patients diagnosed as having low infiltrate leukemia (LIL) between 1973 and 1981, when the policy was to delay treatment until there was evidence of progressive disease or life-threatening morbidity. Their median age was 59 yr; 63% were male; 30 had had a malignant disease previously. Presenting symptoms included anemia, 82%; infections, 15%; and hemorrhage, 16%. The group's median survival was 42 weeks, with high marrow cellularity and percentage of blasts, impairment of bone marrow, liver or renal function, age over 65 yr and performance status less than 80% associated with poorer survival. Cytogenetic changes associated with poor survival included loss of chromosome 5 or 7, additional chromosome 8, karyotypic instability, and presence of 100% abnormal metaphases. A regression model including terms for blood and bone marrow features, and age at hospital admission was able to stratify patients into prognostic groups in the same population and in an independent population admitted prior to 1973. Further prospective testing of the model is required. Twenty-six of the 78 patients who were eventually treated with combination chemotherapy achieved complete remission. The presence of Auer rods, diagnosis of acute leukemia or refractory anemia with excess blasts, rapid increase in marrow infiltrate and favorable cytogenetic karyotype were associated with response. Delaying treatment in all patients was found to have improved only modestly the survival of patients with LIL.

Purification of cyclic AMP- and cyclic GMP-dependent protein kinases from rat skeletal muscle.

Cyclic AMP-dependent protein kinase (cAMP-PrK) regulatory subunits, RI and RII, and cyclic GMP-dependent protein kinase (cGMP-PrK) have been simultaneously purified from skeletal muscle, utilizing sequential affinity chromatography on cyclic AMP-Sepharose. Rat skeletal muscle extract was chromatographed over DEAE-cellulose. Appropriate fractions, enriched in RI, RII or cGMP-PrK were further purified by affinity chromatography on cAMP-Sepharose. The protein kinase units were specifically eluted with cAMP or cGMP. A novel procedure, using two affinity columns, differing in their linkage of cAMP via either N6 or C-8 bonds, was developed to obtain RII free of other cyclic nucleotide binding proteins. In all cases, affinity chromatography was followed by HPLC gel exclusion chromatography to remove residual contaminating proteins. Proteins were purified to essential homogeneity as judged by silver stained SDS polyacrylamide gels. This procedure yields protein kinase subunits of high purity, and may be applicable to the isolation of these proteins from other sources.

De novo hemolytic uremic syndrome in renal transplant recipients immunosuppressed with cyclosporine.

Three renal transplant recipients were studied who developed the hemolytic uremic syndrome (HUS) during cyclosporine (CsA) immunosuppression. All patients displayed characteristic findings including normocytic, normochromic anemia, thrombocytopenia, erythrocyte morphologic abnormalities, increased numbers of reticulocytes, bone marrow hyperplasia, and renal failure. The clinical studies support the diagnosis of an extrinsic hemolytic process. Renal biopsy specimens demonstrated fibrin deposition and glomerular thrombosis. The renal failure was not responsive to antirejection therapy. The first patient was converted to azathioprine and consequently had a gradual improvement in renal function. Failure to convert the second patient resulted in adequate immunosuppression and subsequent graft loss. The third patient's improvement corresponded with a moderate CsA dose reduction. The occurrence of HUS in these patients represents a de novo presentation after CsA immunosuppression. An analysis of these cases indicates that CsA should be discontinued if HUS persists after moderate dose reduction.

DNA-RNA measurements in patients with acute leukemia undergoing remission induction therapy.

Prior to therapy, 111 newly diagnosed adult patients with acute leukemia had DNA content measured (cell-cycle distributions) and 91 had RNA content measured using flow cytometry of acridine orange-stained bone marrow biopsies. The RNA index (RI) (ratio of mean RNA in G0/G1 of the sample to the median RNA in G0/G1 of normal lymphocytes) distinguished acute myelogenous leukemia (AML) from acute lymphocytic leukemia (ALL). The mean RI in AML was 2.2 and in ALL 1.5. RI did not predict for achievement of complete remission (CR). In AML the mean S-phase percent was 11.2 in patients who achieved CR and 13.1 in those who failed to respond to therapy, whereas in ALL it was 14.5 in those responding and 8.0 in those not responding (P = .02). In the 77 patients with either AML or ALL who achieved CR, a low pretreatment S-phase percent and a high RI correlated with a long duration of CR. S-phase percent and RI were also measured during remission induction and during maintenance therapy. Between days 8 and 18, the RI of responders decreased. In both AML and ALL an increase in S-phase percent between days 18 and 22, prior to morphological CR, was observed in responders but not in nonresponders. The mean S-phase percent at the time of morphological remission was 17.3. A high S-phase percent at this time correlated with a longer duration of CR. Although neither pretreatment S-phase percent nor RI was found to predict for achievement of CR in AML, both predicted for length of CR. Increases in S-phase percent during therapy indicated recovery of normal hematopoiesis.

Remission in skin infiltrate of a patient with mycosis fungoides treated with cyclosporine.

A 57-year-old woman with mycosis fungoides that had failed to respond to cytotoxic chemotherapy was treated with cyclosporine. Mycosis fungoides and Sézary syndrome are disorders of helper T cells. Cyclosporine is a fungal endecapeptide of novel chemical structure that causes preferential inhibition of T helper cells. Because of this in vitro inhibition of T helper cells, we used cyclosporine to treat a patient who had mycosis fungoides that was refractory to cytotoxic combination chemotherapy. With cyclosporine administered initially as an intravenous infusion and orally after 10 days, there was immediate improvement in the patient's symptoms. This subjective improvement was accompanied by a decrease in her skin infiltration, noted on physical examination and microscopically. Despite continued administration of the cyclosporine, symptoms recurred after 3 1/2 months of therapy.

Transglutaminase activity increases in HL60 cells induced to differentiate with retinoic acid and TPA but not with DMSO.

HL60 cells induced to differentiate into myeloid cells by retinoic acid exhibited a 300-fold increase in transglutaminase (TGase) activity which peaked on day 5. HL60 cells induced to differentiate into monocytes by a phorbol ester tetradecanoylphorbol-12-myristate-13-acetate (TPA) had a greater than 840-fold increase in TGase activity on day 7. In contrast, cells induced to differentiate along the myeloid pathway by dimethyl sulfoxide (DMSO) exhibited no increase in TGase activity. Elevation of TGase activity appears to be characteristic of monocyte differentiation and retinoic acid-induced myeloid differentiation but not of myeloid differentiation in response to DMSO.

Phase I evaluation of intravenous difluoromethylornithine--a polyamine inhibitor.

Difluoromethylornithine (DFMO), a non-competitive inhibitor of ornithine decarboxylase (ODC), the rate limiting enzyme of the polyamine synthetic pathway was evaluated in a Phase I trial. Intravenous DFMO was given to twenty patients with refractory leukemia by continuous infusion in doses from 5.5 to 64 g/m2. Toxicity clearly attributable to the drug was not severe and other than nausea and vomiting did not increase with dose. The previously reported ototoxicity which occurred with the oral form appeared to be less frequent. Loss of hearing which improved when the drug was stopped was seen in four patients, three of whom were simultaneously receiving aminoglycosides. Anorexia occurred in some patients at all doses. Vomiting, necessitating dosage reduction, was a significant problem at the highest dose administered. No patient achieved a remission but there was stabilization or decrease in circulating blast cells in several patients. This growth inhibition did not appear to be dosage related.