Five year survival rates for childhood AML in children are currently 55–65%. AML is an extremely heterogeneous disease, and while prognostic significance of some karyotypic abnormalities has become evident, the biology of the disease remains largely unknown. Full characterisation of leukaemia initiating cells which may be responsible for relapse has not yet been undertaken.
We investigated the leukaemic stem cell populations from 10 childhood primary AML samples by comparing expression of CD34, CD38 and CD45RA, a marker of a committed granulocyte-macrophage progenitor (GMP)-like population frequent in adult AML, in vitro daunorubicin sensitivity and engraftment in immuno-compromised NOD/Shi-scid/IL-2Rgnull (NOG) mice. Consequently, we were able to classify AML samples into 4 subgroups. These comprised Group 1, CD34+CD38- AML (n=1); Group 2, CD34-CD38+CD45RA- (<10% CD34+ blasts) AML (n=4); Group 3, CD34+CD38+CD45RA- (>10% CD34+ blasts) AML (n=4); and Group 4, CD34-CD38+CD45RA+ (<10% CD34+ and >10% CD45RA+ blasts) (n=1). There was no apparent enrichment for high risk prognostic karyotypes in any of the groups. The Group 1 AML presented at 3y with t(16;21); In Group 2 AMLs, the mean presentation age was 11y, 2 carried good prognostic t(8;21), while 1 had MLL involvement and 1 had FLT3-ITD with chromosome 13 isodisomy, both higher risk indicators; The Group 3 AMLs presented with a mean age of 11.9y and 2 carried good prognostic inv(16) whereas 2 had FLT3-ITD one with additional chromosome 13 isodisomy, t(5;11) and TP53 loss. Finally, the Group 4 AML presented at 1.5y with a normal karyotype. When we compared the 2 most frequent subgroups, Group 2 had a much shorter mean EFS of 122d (n=2) compared with a 275d (n=4) for Group 3 (the mean follow up was 282d and 1013.5d, respectively).
We next sorted four cell subpopulations based on CD34 and CD38 expression (CD34+CD38-, CD34+CD38+, CD34-CD38- and CD34-CD38+ blasts) and compared in vitro sensitivity to daunorubicin. In Group 1, CD34- and CD34+ cells were equally sensitive at nanomolar IC50 doses. In 2 of the Group 2 AMLs,CD34-CD38- cells were the most resistant to daunorubicin at micromolar IC50 doses (2.5-10mM) whereas the CD34-CD38+ cells (also the dominant subpopulation in this group) were the most sensitive cells exhibiting nanomolar IC50 doses (750–800nM). In contrast, the Group 3 AMLs were overall more sensitive to daunorubicin exhibiting lower nanomolar IC50 doses. Again in this group, the CD34-CD38- cells were typically the most resistant (this time being the dominant subpopulation) whereas the CD34+CD38+ were the most sensitive cells. Finally, in the Group 4 AML, while CD45RA+ cells rapidly underwent spontaneous apoptosis, CD45RA- cells exhibited extreme resistance to daunorubicin (IC50 >10mM) and CD38 expression had no impact on sensitivity. The reduced sensitivity of Group 2 AMLs to daunorubicin compared with Group 3 could, therefore, be an underlying factor contributing to shorter EFS.
Finally, we initiated comparison of the stem cell qualities of the different subpopulations from representative samples from each of the two major subgroups, first by assessment of differentiation potential in vitro, and second by engraftment in vivo using NOG mice. In on-going experiments, the time to leukaemia will be compared between mice injected with unsorted and sorted cells and, at terminal cull, cells harvested from organs will be characterised by karyotype and immunophenotype and tested for clonogenic potential via subsequent serial transplantations. Peripheral blood sampling currently suggests higher human CD45+ engraftment in mice injected with sorted versus unsorted cells, and these are CD34+CD33+CD3- recapitulating the AML phenotype. We anticipate that particular subpopulations will be enriched for AML stem cells with the ability to repopulate the leukaemia.
Overall, we have shown that childhood AML is diverse with respect to stem cell characteristics. AMLs with low CD34 (Groups 2 and 4) exhibit the greatest overall resistance to daunorubicin as well as shorter EFS. Furthermore, in the majority of AMLs, CD34-CD38- blasts exhibit the least sensitivity to daunorubicin. Novel therapies which can target these resistant subpopulations with leukaemia initiating activity could significantly improve the treatment responses in this clinically challenging disease.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.