The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg^®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34⁺/CD38−/CD123⁺ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33⁺ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33⁺ subpopulation of leukemic cells. Moreover, AML stem cells (CD34⁺/CD38⁻/CD123⁺ cells) highly purified (>98% purity) from patients with (CD33⁺) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34⁺/CD38⁻ cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33⁺ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg^®) in acute myeloid leukemia.