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Using synthetic oligomers we investigated fresh samples of acute myeloid leukemia (AML) for the presence of mutated ras oncogenes. Our original results showed that five of eight samples contained a mutation in codon 13 of the N-ras gene. In a subsequent study involving 37 samples, we found only one N-ras-13 mutation, and, in addition, mutations in codon 61 of the N-ras gene in four cases and a mutation in codon 12 of the Ki-ras gene in two cases. Amplification of ras genes was not observed. We conclude that in approximately 20% to 25% of AML cases, a mutated ras oncogene is present, predominantly the N-ras gene. The occurrence of mutations does not correlate with the cytological features of the leukemia.

A deficiency of protein C (PC), antithrombin, or protein S is strongly associated with deep-vein thrombosis in selected patients and their families. However, the strength of the association with venous thrombosis in the general population is unknown. This study was a population-based, patient-control study of 474 consecutive outpatients, aged less than 70 years, with a first, objectively diagnosed, episode of venous thrombosis and without an underlying malignant disease, and 474 healthy controls who matched for age and sex. Relative risks were estimated as matched odds ratios. Based on a single measurement, there were 22 (4.6%) patients with a PC deficiency (PC activity, less than 0.67 U/mL or PC antigen, less than 0.33 U/mL when using coumarins). Among the controls, the frequency was 1.5% (seven subjects). Thus, there is a threefold increase in risk of thrombosis in subjects with PC levels below 0.67 or 0.33 U/mL [matched odds ratio, 3.1; 95% confidence interval (CI), 1.4 to 7.0]. When a PC deficiency was based on two repeated measurements, the relative risk for thrombosis increased to 3.8 (95% CI, 1.3 to 10); when it was based on DNA-confirmation, the relative risk increased further to 6.5 (95% CI, 1.8 to 24). In addition, there was a gradient in thrombosis risk, according to PC levels. The results for antithrombin are similar to those for PC, although less pronounced (relative risk, 2.2; 95% CI, 1.0 to 4.7). We could not find an association between reduced total protein S (relative risk, 0.7; 95% CI, 0.3 to 1.8) or free protein S levels (relative risk, 1.6; 95% CI, 0.6 to 4.0) and thrombosis risk. Although not very frequent, PC and antithrombin deficiency are clearly associated with an increase in thrombosis risk.

Using a Brown Norway rat leukaemia model (BNML), which is a realistic model of human myelocytic leukaemia, we compared the antileukaemic activity, influence on cell cycle kinetics and effect on normal haematopoiesis of 5 aza-2-deoxycytidine (aza-dC) and arabinofuranosyl-cytosine (ara-C). The antileukaemic activity was evaluated by means of a survival study. For aza-dC a dose-response relationship was demonstrated for doses up to 50 mg kg-1 (3 times q 12 h); a higher dose resulted in only a slight increase in median survival time (MST). For ara-C a weak dose-response relationship was observed. At the maximum dose of aza-dC and ara-C tested, aza-dC induced a 10-day longer survival time than ara-C, which means 2 logs more of leukaemic cell kill for aza-dC. By means of flow cytometric analysis and a 3HTdR uptake study it was shown that aza-dC does not influence the cell cycle kinetics in the first 24 h after exposure, in contrast to ara-C which caused the characteristic G1/S blockage and synchronization. The influence of aza-dC and ara-C on normal haematopoiesis was evaluated with the CFU-S assay. The dose-response curve for CFU-S did not show a significant difference in stem cell cytotoxicity between aza-dC and ara-C. In the BNML model aza-dC is a much more effective antileukaemic agent than ara-C, while the toxic effect on normal haematopoiesis is comparable to that of ara-C.

In the present study we demonstrate that Aza-dC in combination with Amsacrine has major antileukaemic properties in patients who have not already received extensive Ara-C therapy. Eight out of 11 patients in their first relapse of acute leukaemia achieved complete remission. Cross resistance between Ara-C and Aza-dC was revealed by the lack of antileukaemic activity in five patients with with Ara-C resistant leukaemia. Combination therapy with Aza-dC/Ams-acrine induced a considerable period of a granulocytopenia (28-35 days), while the toxic effect on erythro- and megakaryopoiesis was comparable to that reported for high dose Ara-C/Amsacrine chemotherapy. Remarkable is the long disappearance time for leukaemic blast cells in bone marrow, i.e. 3-5 weeks in some cases. Analysis of cell membrane markers showed a loss of the early differentiation antigens CD34 and CD33 from leukaemic bone marrow cells after 7 days of Aza-dC treatment, which is suggestive of leukaemic cell differentiation. In the small group of patients tested for DNA hypomethylation no association existed between the degree of hypomethylation and clinical response. Non-haematologic side effects were considerable in patients receiving the highest dosages of Aza-dC and consisted of severe, although usually reversible, gastrointestinal and neurological complications. In comparison with Ara-C, Aza-dC causes less nausea and vomiting and is therefore better tolerated.