Figure 1.
AML can be divided in two subtypes based on frequency and pattern of CD34 expression.
Representative flow cytometric staining patterns of CD34 expression (CD34 versus SCC) are shown for (A) a CD34-negative AML case (0.03% CD34+ cells within the blast compartment) and (B and C) a CD34-positive AML case (72% CD34+ cells within the blast compartment). The CD34-negative AML cases contain a discrete, usually less than 1%, CD34+ cell population. This CD34+ population is completely devoid of molecular aberrancies (in this case mutated NPM1)(A). The CD34-positive AML case contains a large CD34+ cell population, usually more than 1%, which contains the leukemia-associated mutated NPM1 protein (B). The CD34+CD38– and CD34+CD38– fractions from a CD34-positive case contain the FLT3-ITD and NPM1 mutation (C).
Figure 2.
CD34+CD38– HSC have higher ALDH activity than co-existing CD34+CD38– LSC.
(A–C) CD34+CD38– HSC within normal bone marrow are ALDHbright and the level of ALDH activity decreases upon differentiation to CD38+ progenitors. Representative flow cytometric ALDH activity patterns (ALDH versus SSC) are shown for (A) total CD45dim normal bone marrow cells treated with or without DEAB and (B) CD45dimCD34+ cells (middle panel) and both CD45dimCD34+ cells (red) and CD45dimCD34– cells (blue)(right panel). In C, the ALDH activity versus SCC of CD34+CD38– stem cells (panel 1 in green and panel 3 in bleu) and CD34+CD38+ progenitor cells (red in panel 2 and panel 3) from the normal BM is shown. (D–I) In CD34-positive AML, the ALDH activity of CD34+CD38– HSC is higher than that of the CD34+CD38– LSC. The aldefluor assay was performed on cells of a CD34-positive AML case (AML-951) and cells were subsequently labeled with anti-CD45 PERCP, anti-CD34 PC7, anti-CD38 APC and anti-CLL1 PE. The CD34+CD38– stem cells (D, purple) showed to be partly CLL-1+ and partly CLL-1– (E). The CLL-1+ stem cells (green) are FSC/SSChigh as compared to the CLL-1– cells (purple)(F). Investigation of the ALDH activity of the CD34+CD38– compartment showed that the CD34+CD38– stem cell population (D) segregates into an ALDHbright (red) and an ALDHlow (blue) population (G). The ALDHbright cells (red) are CLL-1 negative (H) and contain only wild type FLT3 kinase (I, upper panel). The ALDHlow cells (blue) are largely CLL-1 positive (H) and contain FLT3-ITD+ cells (I, lower panel). The arrow indicates the FLT3-ITD.
Table 1.
CD34+CD38–stem cell frequencies and their marker expression in the ALDHbright and ALDHlow compartment in CD34-positive AML.
Table 2.
Presence of FLT3-ITD and mutated NPM1 in ALDHbright and ALDHlow compartments in CD34-positive AML.
Figure 3.
ALDHbright CD34+CD38– cells are devoid of both the FLT3 and NPM1 mutation.
AML-808 contains both FLT3-ITD and mutated NPM1 and has no immunophenotypic aberrancy present. However, AML-808 can be segregated into a normal and a leukemic CD34+CD38– compartment by measuring ALDH activity (A–C). The CD34+CD38– stem cells (A, orange) segregate into two separate ALDH compartments (B). The CD34+CD38–ALDHbright cells of AML-808 do neither contain FLT3-ITD nor mutated NPM1 (C, upper panels), while the CD34+CD38-ALDHlow cells (blue) do (C, middle panel). The ALDHlow cells have both mutations (C, lower panel). AML-575 contains both FLT3-ITD and mutated NPM1. The CD34+CD38– cells (red) segregate into two separate ALDH compartments (D, middle panel). The ALDHbright (red) cells within AML-575 lack the immunophenotypic aberrancy CD33 and are low in SSC compared to the ALDHlow cells (blue)(F). The CD34+CD38-ALDHbright cells do neither contain FLT3-ITD nor mutated NPM1 (G, upper panels), while the CD34+CD38-ALDHlow cells do (G, middle panel). The ALDHlow cells have both mutations (G, lower panel). (H) CD34+CD38– HSC, CD34+CD38– LSC (purification based on CLL1+, CD34 expression and scatter properties) from AML-598, and CD34+CD38– HSC from normal BM were purified. RNA was isolated and RNA sequencing was performed. The most abundant ALDH mRNA expressed within both normal HSC fractions (AML and normal BM) was the ALDH1A1 enzyme. LSC had hardly any expression of the ALDH1A1 form. Numbers indicate the amount of ALDH1A1 reads within that fraction.
Figure 4.
The ALDH activity of CD34+CD38– HSC is higher than the CD34– bulk in CD34-negative AML.
The aldefluor assay was performed on the bulk of cells (AML-464). Cells were subsequently labeled with anti-CD45 PERCP, anti-CD34 PC7, anti-CD38 APC and anti-CD56 PE. (A) The CD34+CD38– stem cells (A, left panel, orange) were negative for CD56 (A, middle panel, CD34+CD38– in orange) and low in FSC/SCC (A, right panel). (B) The ALDH activity of these CD34+CD38– cells is high and shows a separate SCClowALDHbright population of cells (B, CD34+CD38– cells in orange). (C) Detection of FLT3-ITD by length fragment analysis showed that the ALDHbright compartment was devoid of FLT3-ITD (top) while the ALDHlow compartment has the FLT-ITD (bottom); arrow indicate the mutation.
Table 3.
CD34+CD38– frequency and aberrant marker expression in ALDHbright and ALDHlow compartments in CD34-negative AML.
Table 4.
The presence of FLT3-ITD and mutated NPM1 in ALDHbright and ALDHlow compartments in CD34-negative AML.
Figure 5.
The presence of FLT3-ITD and mutated NPM1 in ALDHbright CD34+CD38− progenitor populations.
(A) In CD34-negative AML cases, the ALDHbright population of AML cells consists, besides CD34+CD38− stem cells of CD38dim and CD38+ progenitors (A, middle panel, AML-464) (Table S3). The complete ALDHbright compartment in CD34-negative AML has a normal phenotype as proven by the absence of FLT3-ITD in the CD34+CD38− (A, right upper panel) as well as the CD38dim (A, right middle panel) and CD38+ (data not shown) population of cells. The ALDHlow compartment contains the FLT3-ITD (arrow in right lower panel), indicating leukemic cells. (B) In CD34-positive AML cases, the ALDHbright population contains, apart from normal CD34+CD38− HSC, CD34+CD38+ progenitors and CD34− cells (Figure 5B middle panel)(Table S3). The ALDHbright CD34+CD38− stem cells lack molecular aberrancies (B, right upper two panels) and are therefore normal. The CD34+CD38+ progenitor compartment (B, right middle two panels) in this case has very small FLT3-ITD and NPM1 mutant peaks likely originating from purification of ALDHlow cells within the ALDHbright compartment. The ALDHlow compartment is largely neoplastic (B, right lower 2 panels).