In order to enhance our detection of mixed chimerism we have adopted PCR methods for detection of BCR-ABL expression characteristic of MCL cells. This method requires isolation of mRNA from clinical specimens which is used to synthesize the cDNA configuration specific for the BCR-ABL fusion gene of each CML patient's Philadelphia chromosome. This BCR-ABL fusion is selectively amplified by subsequent PCR. This method increases our sensitivity of detection of CML cells to 1 cell in 10 enabling us to identify persistence or recurrence of leukemia cells lower than the levels detected hematologic, cytogenetic, or genomic Southern hybridization analysis. We have used reverse transcriptase polymerase chain reaction (RT-PCR) in collaboration with Drs Atul Bedi and Richard Jones to characterize the pluripotent stem cells of CML patients. These studies demonstrate that CML stem cells; a) contain the BCR-ABL translocation as determined by indirect immunoperoxidase staining for P210 and c) do not express BCR-ABL RNA from the rearranged gene as determined by RT-PCR (less than 0.0001% the level of BCR-ABL mRNA detected from one K562 cell). This is the first indication that BCR-ABL gene rearrangement is not sufficient for expression of the BCR-ABL fusion gene. We are prospectively preparing both DNA and RNA from specimens of the mixed chimerism study. This will provide the material for analysis of residual BCR-ABL expressing cells. A PCR approach is being tested for positive assessment of engraftment by PCR amplification of hypervariable RFLP markers. Bringing PRC technology into the lab has also provided a very sensitive means for screening and residual disease detection of lymphomas with the (14;18) chromosome translocation involving the BCL2 oncogene. We are using PCR detection of BCL2 gene rearrangement to evaluate patients with indolent lymphoma who would be eligible for our clinical protocols of BMT.
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