Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells?

Abstract

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALP high cells appear to sustain leukemogenesis over time.

Flow cytometric significance of cellular alkaline phosphatase activity in acute myeloid leukemia

“…Aside from antigen expression properties on AML bulk cells, and that antigen coexpression on leukemic stem cells (LSC) makes it difficult to specifically target this compartment, LSCs have unique features based on metabolic response to drugs, cell stress control, and other key aspects of stemness and chemotherapy resistance. Based on the clonal nature of tumors, and after the demonstration that leukemic stem cells from patients with AML reconstitute the full spectrum of phenotypes in the malignant populations that they regenerate in transplanted mice, we hypothesized that ALP could help to identify primitive leukemic cells [14]. Moreover, it has been demonstrated that ALP activity may be useful for the diagnosis of acute myelomonocytic and monocytic leukemia in dogs [24].…”

“…Additionally, assays dealing with live kinetic analysis, like calcium flux and enzyme rate, or assays that depend on continued active drug transport just prior to analysis, like side population analysis must be done with live cells, preferably with as little sample manipulation as possible. Recent work by the authors [14] showed elevated ALP activity in CD34+ cells in highly refractory cancers. This study was made possible by using this new non-toxic cell-permeant fluorescent ALP for live cells without need to add multidrug resistant transporter inhibitors, taking advantage of one of the most-widely used enzyme-based standards, known as the aldehyde dehydrogenase (ALDH) assay [28].…”

“…Previously, we demonstrated measurement of ALP activity with ABCG2 efflux pump activity using this reagent in combination with side population analysis, making its use feasible for primitive stem cells expressing ABC transporters. This can only be done with live healthy cells at the point of analysis [14].…”

“…All EDTA-anticoagulated bone marrow ( n = 38) and blood ( n = 5) samples were prepared using a modified previously developed method [27] aimed at avoiding the lysis of erythrocytes during sample preparation, which can result in unwanted damage to leukocytes, and conceivably to leukemic cells. As most enzyme functions are performed at 37°C, we first established an optimal Alkaline Phosphatase Live Stain (APLS, Thermo Fisher Scientific) stable loading time (t = 20 min) and temperature (T = 37°C) to measure ALP enzymatic activity [14]. This dye is a cell-permeable fluorescent substrate for ALP that is non-toxic to cells.…”

“…Because stem cells lack specific cell surface markers, the identification of this compartment can be difficult, making cancer stem cells especially elusive [13]. Thus, we undertook a prospective cohort study based on our previous work [14], with the objective to investigate whether there might be differences in ALP activity within primitive leukemic cells, and its association with the potential risk of recurrence and mortality in newly diagnosed patients with AML.…”