No abstract

To explore the possibility that deregulated HOX gene expression might commonly occur during leukemic hematopoiesis, we compared the relative levels of expression of these and related genes in phenotypically and functionally defined subpopulations of AML blasts and normal hematopoietic cells. Initially, a semi-quantitative RT-PCR technique was used to amplify total cDNA from total leukemic blast cell populations from 20 AML patients and light density cells from four normal bone marrows. Expression of HOX genes (A9, A10, B3 and B4), MEIS1 and MLL was easily detected in the majority of AML samples with the exception of two samples from patients with AML subtype M3 (which expressed only MLL). Low levels of HOXA9 and A10 but not B3 or B4 were seen in normal marrow while MLL was easily detected. PBX1a was difficult to detect in any AML sample but was seen in three of four normal marrows. Cells from nine AML patients and five normal bone marrows were FACS-sorted into CD34 ؉ CD38 ؊ , CD34 ؉ CD38 ؉ and CD34 ؊ subpopulations, analyzed for their functional properties in long-term culture (LTC) and colony assays, and for gene expression using RT-PCR. 93 ؎ 14% of AML LTC-initiating cells, 92 ± 14% AML colony-forming cells, and Ͼ99% of normal LTC-IC and CFC were CD34 ؉ . The relative level of expression of the four HOX genes in amplified cDNA from CD34 ؊ as compared to CD34 ؉ CD38 ؊ normal cells was reduced Ͼ10-fold. However, in AML samples this down-regulation in HOX expression in CD34 ؊ as compared to CD34 ؉ CD38 ؊ cells was not seen (P Ͻ 0.05 for comparison between AML and normal). A similar difference between normal and AML subpopulations was seen when the relative levels of expression of MEIS1, and to a lesser extent MLL, were compared in CD34 ؉ and CD34 ؊ cells (P Ͻ 0.05). In contrast, while some evidence of down-regulation of PBX1a was found in comparing CD34 ؊ to CD34 ؉ normal cells it was difficult to detect expression of this gene in any subpopulation from most AML samples. Thus, the down-regulation of HOX, MEIS1 and to some extent MLL which occurs with normal hematopoietic differentiation is not seen in AML cells with similar functional and phenotypic properties.

Acute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo, we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1−. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1− phenotype. However, significant engraftment was achieved using both CD34+/Thy-1− and CD34− subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients, the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1.