DETECTION OF BCR-ABL MESSENGER-RNA IN SINGLE PROGENITOR COLONIES FROMPATIENTS WITH CHRONIC MYELOID-LEUKEMIA BY PCR - COMPARISON WITH CYTOGENETICS AND PCR FROM UNCULTURED CELLS

Bone marrow and/or peripheral blood of patients with chronic myeloid l eukemia (CML) was investigated by the following three parameters: Ph' chromosome, bcr-abl expression in fresh blood and/or bone marrow and b cr-abl expression in single hematopoietic progenitor colonies generate d from blood and/or bone marrow. Expression of bcr-abl was proven by a reverse ''nested primer'' polymerase chain reaction (PCR) that is abl e to detect 1 pg of hybrid mRNA. We performed 108 investigations on 68 patients containing all three parameters: 12 on untreated patients, s even after interferon-alpha (IFN-alpha), seven after low-dose cytosine arabinoside (Ara-C), 22 after cyclic high-dose hydroxyurea (HU), 49 a fter allogeneic BMT, five before and three after stem cell mobilizatio n, and three 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 t o cytogenetics (e.g., mosaic in colonies when all metaphases were posi tive or negative). Most interesting were the results of one patient af ter IFN, one patient after ASCT, and 10 patients after BMT (14 investi gations = 13%), showing only Ph'-negative mitoses accompanied by a neg ative nested primer PCR from fresh blood/bone marrow but single bcr-ab l-positive progenitor colonies. False-positive results could be widely excluded by repeated insertion of negative controls into the experime nts. One explanation for these results could be that CML progenitors s urvive in the patient's body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectabl e by reverse PCR. When stimulated ex vivo in a colony assay by externa l growth factors, cells proliferate and produce detectable amounts of hybrid mRNA. The value of these observations is not clear. A follow-up of the patients will show if such sleeping progenitors can be activat ed in vivo. Concluding our observations, we can say that in special ca ses (therapy followup, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.