Detection of bcr-abl m-RNA in Single Progenitor Colonies by PCR
Bone marrow and/or peripheral blood of patients with CML was investigated by the following 3 parameters: Ph’ chromosome, bcr-abl expression in fresh blood and/or bone marrow and bcr-abl expression in single hemopoietic progenitor colonies generated from blood and/or bone marrow. Expression of bcr-abl was proved by a reverse “nested primer” PCR that is able to detect 1pg of hybrid m-RNA. We performed 108 investigations containing all 3 parameters: 12 on untreated patients, 7 after IFNα, 7 after low-dose Ara-C, 22 after cyclic high-dose hydroxyurea, 49 after allogeneic BMT, 5 before and 3 after stem cell mobilization and 3 after autologous stem cell transplantation (ASCT). In 53 cases (49%) cytogenetics and PCR gave identical results. In 40 cases (37%) PCR from single colonies gave additional information compared to cytogenetics (i.e. mosaic in colonies when all mitoses where positive or negative). Most interesting were the results of one patient after IFN, one patient after ASCT and 10 patients after BMT (14 investigations = 13%) showing only Ph’negative mitoses accompanied by a negative “nested primer” PCR from fresh blood/ bone marrow but single positive progenitor colonies. False positive results could be widely excluded by repeated insertion of negative controls into the experiments. One explanation for these results could be that CML progenitors survive in the patients’ body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectable by reverse PCR. When stimulated ex vivo in a colony assay by external growth factors, cells proliferate and produce detectable amounts of hybrid-m-RNA. The value of these observations is not clear. A follow up of the patients will show if such sleeping progenitors can be activated in vivo. Concluding our observations we can say that in special cases (therapy follow up, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.
KeywordsPolymerase Chain Reaction Chronic Myeloid Leukemia Chronic Myelogenous Leukemia Single Coloni Positive Coloni
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