We report a patient with Philadelphia (Ph)-positive, BCR-ABL rearrangement positive, chronic myeloid leukemia (CML) with a prolonged chronic phase of 24 years who was first prescribed alpha-2 interferon 22 years after initial diagnosis. This therapy was tolerated poorly on account of thrombocytopenia, but an eventual major cytogenetic response was followed soon afterwards by transformation to terminal acute myeloid leukemia (AML). Cytogenetic studies indicated that the transformed myeloblasts were karyotypically normal and Ph negative. Although polymerase chain reaction (PCR) analysis of total leukemic mRNA remained BCR-ABL positive, other molecular studies, including Southern blotting and fluorescent in situ hybridization (FISH) analyses, showed that myeloblasts were BCR-ABL rearrangement negative. PCR-based clonality studies using an X-chromosome-linked restriction fragment polymorphism within the phosphoglycerate kinase gene (PGK 1 ) further showed that the Ph-negative blast cells had a different clonal origin from the Ph-positive clone of chronic phase. We suggest that cases of underlying Ph-negative leukemic transformation in Ph-positive CML warrant further study and should be considered for trial of intensive remission induction therapy as appropriate for acute leukemia.

Generation time ( G T ) of normal human bone marrow cells cultured in the C FC-gm assay was measured by using bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. Cells were cultured in methylcellulose for 72 hr prior to the addition of BrdU and then harvested at 6-12 hr intervals for up to 72 hr. The time interval between the appearance of second and third division metaphases at the 50% level gave mean GTs which ranged from 32 to 43 hr. These values are longer than those reported for myeloblasts and promyelocytes but shorter than those previously reported for myelocytes.Human bone marrow functions as a continuously renewing cell system in which pluripotent stem cells give rise by proliferation and differentiation to functionally mature haematopoietic elements. Studies of the generation time of normal human bone marrow precursor cells in uiuo and in uifro are few, due both to ethical considerations (i.e. injecting normal individuals with [3H]TdR) and to difficulties in obtaining adequate cell numbers in uitro to apply traditional cytokinetic techniques.Bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining has proved to be a rapid means for identifying successive cell divisions in a proliferating population (Tice, Schneider & Rary, 1976; Crossen & Morgan, 1977a,b;Crossen et al., 1985). In this method, cells were cultured in medium that contained the thymidine analogue 5-bromodeoxyuridine (5-BrdU) which is readily incorporated into chromosomal DNA. Metaphases which have completed two complete cell cycles in medium containing BrdU have chromosomes in which the DNA of one chromatid has both strands substituted with BrdU while the other chromatid has only one strand substituted with BrdU. When stained by the sister chromatid exchange technique, the doubly-substituted chromosome appears pale staining while the singly substituted is dark staining. Cells that have completed one cell cycle have both chromatids dark staining while metaphases that have completed three cell cycles have three quarters of their chromatin pale staining. By harvesting the cultures at various time intervals, a detailed analysis of the proliferative characteristics of the culture can be obtained. Recent studies (Trent et af., 1986) indicate that a modification of the BrdU technique