DETECTION OF RAS ONCOGENE POINT MUTATIONS IN HL-60 PROMYELOCYTIC LEUKEMIA-CELLS USING A 2-STEP POLYMERASE CHAIN-REACTION - RELEVANCE TO MONITORING OF MINIMAL RESIDUAL DISEASE IN ACUTE MYELOID-LEUKEMIA

In this paper we demonstrate the use of a Ras oncogene specific polyme rase chain reaction (PCR) for the detection of minimal residual diseas e in leukemia. Using a two-step procedure and mutation specific amplim ers, we were able to detect one HL-60 cell in 10(5) peripheral blood m ononuclear cells (PBMC) bearing a mutation in N-ras codon 61. We then applied this method to the evaluation of immunologic and chemical purg ing procedures that are used for autologous bone marrow transplantatio n (ABMT). We routinely found that a positive PCR signal was still obta inable after both immunologic and chemical purging despite the observa tion that no viable clonogenic cells remained after the purging proced ure. Since we also found that prolonged exposure of HL-60 cells to sod ium azide did not eliminate a positive PCR signal, we conclude that in tact deoxyribonucleic acid in nonviable cells fan contribute to a posi tive PCR result and give misleading information regarding the efficacy of tumor cell removal. Thus, while this method may have utility far i n vivo monitoring of minimal residual disease, the mutation-specific P CR using tumor cell DNA is not as reliable as expected for the assessm ent of short term in vitro therapies.