Abstract

In acute myeloid leukemia (AML), apart from the CD34(+)CD38(-) compartment, the side population (SP) compartment contains leukemic stem cells (LSC). We have previously shown that CD34(+)CD38(-) LSC can be identified using stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. A similar study was performed for AML SP in order to further characterize the SP cells with the aim of narrowing down the putatively very low stem cell fraction. FACS analysis of 48 bone marrow and peripheral blood samples at diagnosis showed SP cells in 41 of 48 cases which were all partly or completely positive for the markers, including CD123. SP cells in normal bone marrow (NBM) were completely negative for markers, except CD123. Further analysis revealed that the SP fraction contains different subpopulations: 1. Three small lymphoid subpopulations (with T-, B- or NK- cell markers); 2. A differentiated myeloid population with high forward and sideward scatter (FSC(high)/SSC(high)), high CD38 expression and usually with aberrant marker expression; 3. A more primitive FSC(low)/SSC(low), CD38(low), marker negative myeloid fraction; 4. A more primitive FSC(low)/SSC(low), CD38(low), marker positive myeloid fraction. NBM contained the first three populations, although the aberrant markers were absent in the population 2. Suspension culture assay showed that FSC(low)/SSC(low) SP cells were highly enriched for primitive cells. FISH analyses showed that cytogenetically abnormal colonies originated from sorted marker positive cells, while the cytogenetically normal colonies originated from sorted marker negative cells. In conclusion, AML SP cells could be discriminated from normal SP cells at diagnosis based on expression of CLL-1 and lineage markers. This reveals the presence of a low frequency (median 0.0016 %) SP subfraction as a likely candidate to be enriched for leukemia stem cells. ______________________________________________________________________________ Author contributions: B.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; A.van Rhenen: collection and/or assembly of data; A.K.: collection and/or assembly of data; M.A.van der Pol: collection and/or assembly of data; M.T.: collection and/or assembly of data; C.B.: collection and/or assembly of data; A.H.W.: collection and/or assembly of data; G.J.O.: provision of study material or patients, final approval of manuscript; S.Z.: provision of study material or patients, manuscript writing, final approval of manuscript, G.J.S.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.