Loss of heterozygosity in childhood de novo acute myelogenous leukemia.

A genome-wide screening for loss of heterozygosity (LOH), a marker for possible involvement of tumor suppressor genes, was conducted in 53 children with de novo acute myelogenous leukemia (AML). A total of 177 highly polymorphic microsatellite repeat markers were used in locus-specific polymerase chain reactions. This comprehensive allelotyping employed flow-sorted cells from diagnostic samples and whole-genome amplification of DNA from small, highly purified samples. Nineteen regions of allelic loss in 17 patients (32%) were detected on chromosome arms 1q, 3q, 5q, 7q (n = 2), 9q (n = 4), 11p (n = 2), 12p (n = 3), 13q (n = 2), 16q, 19q, and Y. The study revealed a degree of allelic loss underestimated by routine cytogenetic analysis, which failed to detect 9 of these LOH events. There was no evidence of LOH by intragenic markers for p53, Nf1, or CBFA2/AML1. Most lymphocytes lacked the deletions, which were detected only in the leukemic myeloid blast population. Analysis of patients' clinical and biologic characteristics indicated that the presence of LOH was associated with a white blood cell count of 20 x 10(9)/L or higher but was not correlated with a shorter overall survival. The relatively low rate of LOH observed in this study compared with findings in solid tumors and in pediatric acute lymphoblastic leukemia and adult AML suggests that tumor suppressor genes are either infrequently involved in the development of pediatric de novo AML or are inactivated by such means as methylation and point mutations. Additional study is needed to determine whether these regions of LOH harbor tumor suppressor genes and whether specific regions of LOH correlate with clinical characteristics. (Blood. 2001;98:1188-1194)

Regulation of colony forming cell generation by flt-3 ligand.

The recently cloned ligand for the flt-3/flk-2 receptor was examined for its effect on colony formation by subpopulations of CD34+ cells including the least mature CD34+lin-CD38- small-medium lymphocyte-sized cell population. Flt-3 ligand (flt-3l) had little or no effect when added alone to cells. Isolated CD34+lin+ cells formed increased numbers of colony-forming cells (CFC) when flt-3l was added together with IL-3, IL-6, G-CSF, GM-CSF or c-kit ligand (KL), or with the combination of IL-3 and KL. Significant increases in CFC formation from CD34+lin- cells were consistently seen when flt-3l was added to the IL-3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34+lin- cells in liquid cultures showed that cultures containing IL-3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL-3, KL and flt-3l produced few CFC past 2 weeks of culture. Flt-3l alone or the combination of IL-3 and KL did not stimulate significant growth of CD34+lin-CD38- small-medium lymphocyte-sized cells, although these cells reproducibly generated CFC when grown in the combination of IL-1 beta, IL-3, IL-6, G-CSF, GM-CSF and KL. Addition of flt-3l to either IL-3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt-3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34+ cells.

Ontogeny of hematopoietic stem cell development: reciprocal expression of CD33 and a novel molecule by maturing myeloid and erythroid progenitors.

We have identified a molecule expressed by human marrow granulocyte/monocyte colony-forming cells (CFU-GM), erythroid colony-forming cells (CFU-E), and erythroid burst-forming units (BFU-E), but not their precursors detectable in long-term bone marrow culture. This antigen, detected by flow microfluorimetry using monoclonal antibody 7B9, is coexpressed with CD33 on many CD34+ CFCs, although only the 7B9 antigen was detected on a portion of BFU-E and CFU-E, whereas only CD33 was found on a portion of CFU-GM. Antibody 7B9 appears to be useful for isolating subsets of progenitors based on their common or selective expression of 7B9 antigen and CD33.

Expression of lymphoid-associated cell surface antigens by childhood acute myeloid leukemia cells lacks prognostic significance.

The prognostic significance of cell surface antigens associated with lymphoid and myeloid lineage differentiation on the blasts of children with acute myeloid leukemia (AML) was evaluated. Leukemic blasts from 176 patients enrolled on Childrens Cancer Study Group Protocol 213 determined to have AML by standard morphologic and cytochemical criteria were immunophenotyped. Cell surface antigens associated with myeloid differentiation were found on blasts from 88.1% of patients (CD15, 44%; CD33, 65%; CD36, 53%; glycoprotein Ib, 9.3%). However, blasts from 30.7% of patients expressed surface antigens thought to be specific for lymphoid lineage differentiation (CD2, 9.4%; CD5, 2.7%; CD19, 34.5%; CD20, 0.8%). In addition, CD34, a glycoprotein found on immature cells of both myeloid and lymphoid lineages, was expressed on the blast cells of 48.2% of patients. With the exception of the lymphoid lineage nonspecific antigen CD4, no correlation was found between white blood cell count at diagnosis, age, and French-American-British morphology, and the expression of any of the lymphoid- or myeloid-associated cell surface antigens studied. Nor was the expression of these antigens prognostically significant with respect to response to induction therapy, death during induction, survival, event-free survival, or survival/event-free survival following remission induction. Multivariate analysis showed that CD4 expression was not an independent predictor of outcome. Thus, this prospective study suggests that expression of lymphoid-associated cell surface antigens as well as myeloid-associated antigens by childhood AML blasts lacks prognostic significance.

Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia.

Acute myelogenous leukemia (AML) is a clonal disease that is heterogeneous with respect to the pattern of differentiative expression of the leukemic progenitors. In some patients, the involved stem cells manifest pluripotent differentiative expression, whereas in others, the involved progenitors manifest differentiative expression mainly restricted to the granulocytic pathway. This is in contrast to chronic myelogenous leukemia (CML) which is a clonal disease known to arise in a pluripotent stem cell. Therefore, we tested whether these leukemias could be distinguished with respect to their involvement of immature precursors by studying colony-forming cells (CFC) and their precursors from four glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML and five patients with CML. CFC were separated from their precursors by FACS for expression of CD33 and CD34 followed by growth in a long-term culture (LTC) system. The vast majority of CFC express both the CD33 and CD34 antigens, but their less mature precursors, detected by their ability to give rise to CFC in LTC, express only CD34. In three of the four patients with AML, the CD33-CD34+ cells produced CFC in LTC that appeared to be predominantly or completely normal (ie, nonclonal) in origin. In the fourth patient, a significant enrichment of nonclonal progenitors was obtained in the CD33-CD34+ population, but these cells may also have included significant numbers of clonal cells. In contrast, in four of five patients with CML, cultures of both the CD33-CD34+ and CD33+CD34+ populations produced CFC in LTC that were almost entirely clonal in origin, whereas in the fifth patient a substantial number originated from nonclonal stem cells. These data indicate that granulocyte/monocyte progenitors are predominantly clonally derived in CML and AML. In CML, their precursors are also predominantly clonal, but in some cases of AML they are not. These findings may have implications for understanding the success or failure of current therapies of AML and CML.