Evaluate prognostic significance of immunophenotypically and functionally defined candidate leukemia stem cell populations.
Numerous studies have demonstrated that the population of CD34+/CD38dim (low to negative) cells is highly enriched for human hematopoietic stem cells. Another marker of stem cell-like phenotype can be assessed functionally by measuring the ability of the blasts to actively export DNA binding dye, Hoechst 33342 by flow cytometry giving rise to the so called “side population”. SP has also been widely linked to stem cell potential in both hematopoietic and non-hemtatopoietic tissues. The relationship between SP and CD34+/CD38dim phenotype is not entirely clear. Analogously, most human acute myeloid leukemias contain both a CD34+/ CD38dim population and a side population. CD34+/CD38dim population has been established as a source of AML cells with high self renewal potential. Less data exists for the role of SP in self renewal in AML. The relationship between the CD34+/CD38dim population and the side populations in AML has not been fully clarified.
Twenty-two newly diagnosed AML patients were identified with CD34+ immunophenotype on the majority of leukemic blasts amongst patients enrolled on an IRB-approved protocol for analysis of blood and bone marrow samples. These patients then underwent induction chemotherapy, and response and duration of first complete remission (CR1) were determined. Wilcoxon rank sum test was used to compare stem cell populations with achievement of CR and FLT3 status; Kruskal-Wallis test to compare stem cell populations across cytogenetic categories and CR1 duration categories (more then 12 months, relapsed less than 12 months, no response), and Spearman correlation to assess correlation between age and stem cell population. For the purposes of analysis of AML prognosis fractions of blasts with CD38 expression below that of mature myeloid cells (CD38low) and those with no CD38 expression (CD38neg) expression were analyzed separately.
The median age of AML patients was 61 (range 19–84). 23% of the patients had favorable, 41% intermediate, and 36% poor risk cytogenetics 45% achieved complete remission. Median SP % was 0.04 (0-13.2), median CD38low % was 1.298 (0.02-17.4), and median CD38neg % was 0.01077 (0-0.92). We first analyzed the relationship between CD34+/CD38dim and SP phenotypes in normal and AML patients. 20 normal human bone marrow and 22 bone marrows from patients were investigated by flow cytometry. We demonstrate that normal human bone marrow SP is highly enriched for the most phenotypically immature CD34+/CD38dim small blasts likely representing the earliest identifiable hematopoietic precursors. Moreover, the highest dye efflux activity correlated with the least CD38. In contrast to normal marrow in AML, the SP did not correspond with CD38 expression levels. Most samples showed an SP phenotype that extended to include many CD38bright events more typical of lineage-committed progenitors. ABCG2 pump inhibitor Fumitremorgin C blocked dye efflux in all normal and all but one AML samples we tested. Side population did harbor the same cytogentic anbormalities as the bulk of blasts in 4/4 samples tested. SP percent did not correlate with age, cytogenetic risk category, response, CR1 duration, or Flt3 (all p>0.05). Percent of CD38low and CD38neg each negatively correlated with achievement of complete response to treatment (both p<0.01). Percent CD38neg correlated with length of CR1 (p=0.02). There was also a positive correlation with increasing age (both p=0.01) for both CD38low and CD38neg populations. Only percent CD38neg correlated with cytogenetic risk category (p=0.03).
1. Increased proportion of blasts with CD38low phenotype was strongly associated with failure of response to induction chemotherapy and was also associated with increased age.
2. Increased proportion of CD38neg blasts was strongly associated with lack of initial chemotherapy response, adverse cytogenetics, short CR1 duration and advanced age.
3. In contrast, SP frequency did not show significant association with outcome, age or cytogenetic status or CR1 duration.
Expansion of less mature (CD38 lower) compartment of blasts may explain the adverse prognosis of patients with advanced age and poor cytogenetic predictors.
Becker:Genzyme: Research Funding.
Asterisk with author names denotes non-ASH members.