A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia

Abstract

Relapses after initial successful treatment in acute myeloid leukemia are thought to originate from the outgrowth of leukemic stem cells. Their flow cytometrically assessed frequency is of importance for relapse prediction and is therefore assumed to be implemented in future risk group profiling. Since current detection methods are complex, time- and bone marrow consuming (multiple-tubes approach), it would be advantageous to have a broadly applicable approach that enables to quantify leukemia stem cells both at diagnosis and follow-up. We compared 15 markers in 131 patients concerning their prevalence, usefulness and stability in CD34(+)CD38(-) leukemic stem cell detection in healthy controls, acute myeloid leukemia diagnosis and follow-up samples. Ultimately, we designed a single 8-color detection tube including common markers CD45, CD34 and CD38, and specific markers CD45RA, CD123, CD33, CD44 and a marker cocktail (CLL-1/TIM-3/CD7/CD11b/CD22/CD56) in one fluorescence channel. Validation analyses in 31 patients showed that the single tube approach was as good as the multiple-tube approach. Our approach requires the least possible amounts of bone marrow, and is suitable for multi-institutional studies. Moreover, it enables detection of leukemic stem cells both at time of diagnosis and follow-up, thereby including initially low-frequency populations emerging under therapy pressure.

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

“…7,10–12,40,46 We are not aware of any published reports indicating that they should differ from AML blasts in their intrinsic susceptibility to RDL by antibodies or antibody-derived agents in combination with NK cells. For most patients with CD34-positive AML, the relapse-relevant AML-LSCs are contained predominantly in the compartments of CD34-positive and (CD34-positive CD38-negative) cells.…”

“…For most patients with CD34-positive AML, the relapse-relevant AML-LSCs are contained predominantly in the compartments of CD34-positive and (CD34-positive CD38-negative) cells. 12,40,43–46,73 Therefore, we have first enriched CD34-bearing cells from 2 patients with CD34-positive AML, for whom sufficient cell numbers were available (patients 9 & 11; Table 3), by immuno-magnetic sorting with a commercial kit. Both samples carried intermediate combined densities of CD33 plus CD123 on their blasts (MNCs; approx.…”

“…This assay was tested and validated for the detection of LSCs from a majority of patients with CD34-positive AML, which account for approximately 80% of all AML cases. 46 We intend to use this and other methods (quantitative polymerase chain reaction, qPCR, where applicable) in the future, to monitor the effect of SPM-2 plus autologous NK cells on the subset of AML-LSCs.…”

“…12 CD123 is also expressed on normal HSCs from AML patients in follow-up after induction therapy. 46 On aggregate, the published reports agree that CD123 is expressed with far greater frequency and surface densities on AML-LSCs and progenitor cells than on normal HSCs. 7 A fraction of normal HSCs may therefore survive therapies targeting CD123.…”

“…We anticipate that SPM-2 will be able to discriminate at least to a degree between AML-LSCs and remaining normal HSCs of a patient, due to its dual-targeting capacity and the greater combined surface density of CD33 and CD123 on AML-LSCs than on normal HSCs. 11,12,24,27,40,46 A preferential elimination of AML-LSCs over normal HSCs would be a distinct advantage, if it could be reached, because the surviving HSCs may be able to reconstitute the patient’s hematopoietic system after the end of therapy at least in part, possibly even without the need for a stem cell transplant. Here we have studied, whether SPM-2 in conjunction with activated human NK cells was capable of eliminating blasts from patients with a very broad range of AML subtypes, as predicted, 8 and found the prediction to be fulfilled.…”