PROGENITOR COLONY RT-PCR ANALYSIS IN CML TREATMENT

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DESCRIPTION: (Applicant's Abstract) Chronic myelogenous leukemia (CML) is usually associated with a reciprocal translocation between the BCR and ABL genes that gives rise to synthesis of the hybrid BCR/ABL oncoprotein. A detailed study will be conducted of CML patients undergoing a new treatment protocol involving an induction regimen, in vivo purging, stem cell mobilization into the peripheral blood, leukapheresis, autologous peripheral blood stem cell transplantation and post-transplant treatment with alpha-interferon. The planned research will be a correlative laboratory and clinical study. It will monitor the presence of leukemic cells in bone marrow at four critical stages in the treatment procedure by reverse transcription followed by the polymerase chain reaction (RT-PCR) for the BCR/ABL translocation product. The most important RT-PCR assays to be done in this study will be those that use individual colonies derived in vitro from myeloid progenitor cells for analysis of the BCR/ABL product. In studies on CML patients in which only uncultured material is used for this RT-PCR or in which cytogenetic analysis or fluorescence in situ hybridization is done to detect the BCR/ABL translocation, the majority of the assayed cells are not the leukemic progenitors. In the proposed study, RT-PCR will be done on RNA from individual colonies cultured from progenitor cells in bone marrow and peripheral blood samples from CML patients and, by both qualitative and competitive quantitative RT-PCR, on RNA from the corresponding uncultured samples. The results of RT-PCR analysis of the frequency of leukemic progenitor cells will also be compared to those from conventional cytogenetic analyses. This study will permit a direct comparison of the level of contamination of peripheral blood samples and bone marrow samples with leukemic cells after in vivo purging. Also, the proposed analysis will provide needed preliminary data for a larger study to test the relationship between the levels of the BCR/ABL-positive myeloid progenitor cells in the autologous peripheral blood stem cells used for engraftment and long-term disease-free survival.