DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

We report on investigations aimed at detecting mutated RAS genes in a variety of preleukemic disorders (1,5,6). Moreover, in all cases investigated thus far, the mutations turned out to be specific for the tumor cells and were not found in normal cells of the respective patient. Activation of RAS genes has been detected in a variety of different neoplasias with variable frequencies. By far, the highest incidence (25-50%) has been reported in acute myelocytic leukemia (AML) (6-8).To gain further information on the biological significance of RAS mutations in leukemogenesis, we investigated the occurrence of RAS gene mutations in AML compared to a broad spectrum of other preleukemic and leukemic disorders involving the myeloid lineage. For this purpose we used, in addition to DNA transfection analyses (tumorigenicity assay), a dot-blot screening procedure based on a combination of in vitro amplification of RAS-specific sequences and hybridization to mutation-specific oligonucleotide probes (9)(10)(11)

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We here summarize the analysis of 126 DNA samples from patients with hematopoietic neoplasias and solid tumors and from various tumor cell lines that were screened in the tumorigenicity assay. Thirty-eight samples were able to induce tumors after transfection in NIH/3T3 cells and injection into nude mice. Southern-blot analysis with a panel of onco-gene probes revealed human ras genes in the vast majority of cases (25 N-ras, 2 K-ras, 1 H-ras) but also activated FGF4, dbl, ret and mas genes respectively. DNA samples from the 6 remaining transfectants were cloned into EMBL-3 phages and screened with a human specific repetitive Alu probe. Direct hybridization of a transfectant cDNA library allowed cloning of the ufo oncogene. Application of the exon-trapping technique to alu-positive phage DNA from the other transfec-tants enabled us to isolate tre, cot, B-raf, p85/HUMORF8 and a novel oncogene. Int.