Expression of CD47 in de novo acute myelogenous leukemia with normal karyotype and its clinical significance

Abstract

Objective To investigate the CD47 expression in de novo acute myelogenous leukemia (AML) patients with normal karyotype and its clinical significance. Methods One hundred thirty-seven cases of de novo AML with normal karyotype and 3 healthy volunteers were selected. Relative CD47 expressions in normal bone marrow hematopoietic stem cells (HSC) and multipotent progenitor (MPP) from healthy volunteers, as well as bone marrow mononuclear cells (MNC) and leukemia stem cells (LSC, Lin- CD34+ CD38- CD90-) from AML patients were determined by flow cytometry. CD47 expression on the Lin- CD34+ CD38- LSC-enriched fraction of specimen was determined by flow cytometry. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected by using the Genome Analyzer platform. CD34+ CD38- CD47hi and CD34+ CD38- CD47lo expressing cells were identified and purified using FACS. Two groups of cells were inoculated with MethoCult H4445 medium on agarose-containing methylcellulose plates. After 12 days, MPP colony forming units (CFU) were counted, and 1×105 CD34+ CD38- CD47lo and CD34+ CD38- CD47hi cells were transplanted into NSG (NOD-SCID IL-2R γ null) mice irradiated by 280 cGy, and mice were sacrificed after 8 weeks. The ratio of human CD45+ cells was detected by flow cytometry. Results The expression of CD47 in AML patients was higher than that in the healthy control. CD47 was expressed in all FAB (French-American-British) subtypes of AML. No significant difference in CD47 expression among different FAB subtypes was found (F= 0.545, P > 0.05). Among the 37 patients with CD34+ CD38- CD47hi, 17 (46%) were FLT3-ITD negative, and 20 (54%) were FLT3-ITD positive. Among the 100 patients with CD34+ CD38- CD47hi, 63 (63%) cases were FLT3-ITD negative, 37 (37%) cases were FLT3-ITD positive. The rate of FLT3-ITD positive in patients with CD34+ CD38- CD47lo had no statistical difference compared with patients with CD34+ CD38- CD47hi (χ 2= 3.79, P > 3.79). The CD34+ CD38- CD47lo or CD34+ CD38- CD47hi which was selected by FACS, was inoculated with the methylcellulose plate containing agarose for 12 days, and CD34+ CD38- CD47lo cells could form CFU. The NSG mouse transplantation experiment showed that CD34+ CD38- CD47lo cells could be reconstructed hematopoiesis, and CD34+ CD38- CD47hi implantation failed. Conclusion CD34+ CD38- CD47hi could enrich LSC, which may be a potential marker to detect minimal residual disease. Key words: Leukemia, myelogenous, acute; Antigens, CD47; Colony-forming units assay