Chronic myelogenous leukemia (CML) is a pluripotent stem cell disease, which is associated with an abnormal chromosome, termed the Philadelphia chromosome (Ph). The Ph fuses parts of the BCR and ABL genes, and the resulting Bcr-Abl oncoprotein is expressed in blood and bone marrow cells of CML patients. Two modes of therapy have been unusually effective at inducing long term remission in chronic phase CML patients: bone marrow transplantation from a matched donor and interferon-alpha treatment. Several tests are currently used to monitor treatment of CML but only the reverse transcriptase (RT) polymerase chain reaction (PCR) method is sufficiently sensitive to detect minimal residual disease. RT-PCR is known to detect as few as one cell expressing BCR-ABL in a total of about 100,000 cells. However, the standard RT-PCR method is widely used as not quantitative. Competitive quantitative PCR methods have been developed in recent years. We are adapting an approach developed by Cross and Goldman. This method involves adding known levels of BCR-ABL junction sequences, containing an insert to distinguish it from the native junction sequences, to an unknown sample and comparing the intensity of the amplimers. The reliability of the method is increased by analyzing serial patient samples from a period of time to determine whether the level of BCR-ABL-RNA is decreasing or increasing. For example, a pattern of decreasing level of BCR-ABL RNA would indicate a deepening remission.
The aims of this grant are to adapt the Cross and Goldman method and use the test to monitor bone marrow transplant CML patients and CML patients being treated with interferon alpha. This approach will be also used in Ph+ ALL patients. The method will be established using known Bcr-Abl positive and negative cell lines; next studies will be done on cells from blood and marrow with emphasis on minimal residual disease; and the utility will be evaluated by performing a blind study with patient samples. Lastly, a core laboratory will be established for analysis blood and marrow samples of CML patient samples under study within the P01 grant by competitive PCR.
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