AML is a highly heterogeneous disease from the biological and clinical standpoint, for which prognostic factors have become increasingly important in the choice and planning of therapeutic procedures. Currently, cytogenetic and molecular aberrations are the best prognostic indicators for AML patients . However, these factors predict primarily for groups of patients and cannot prognosticate well for individual patients within any given risk strata. For example, CBF cytogenetic abnormalities are generally considered favorable; yet approximately half of these patients relapse [18–20]. So, new prognostic tools based on biological analyses need to be developed.
AML is regarded as a stem cell disease, and the failure of chemotherapy in eradicating primitive leukemia initiating cells likely contributes to the relapse of AML. Numerous studies have emphasized the correlation between the enrichment of the CD34+CD38- phenotype in AML at diagnosis and a high level of residual disease after treatment [12, 13]. And it is generally accepted that CD34+CD38- cells are enriched for LICs, however, this population is heterogeneous and includes both normal and leukemic stem cells. In this regard, measure of “real” LICs promises to serve as a powerful, individualized prognostic tool, enhancing delivery of risk-adapted therapies. Recent data have generated important advances in the field, including the identification of novel leukemia initiating cells-specific cell surface antigens. Several studies have showed that the aberrant expression of specific markers on the cell surface of leukemia progenitors is characteristic for LIC, differentiating them from their normal counterparts, including CD90, CD123, CLL-1, CD96, CD47, Tim3 and so on [8–11]. However, aberrant marker-positive CD34+CD38- cells at diagnosis are partly of malignant origin, while the corresponding marker-negative population is not totally of normal origin. For example, Rhenen et al. reported that CD123 seems not very useful in leukemic stem cell detection due to the high expression of CD123 in normal control samples .
In the present study, we established the Flow-FISH protocol as an effective method to distinguish normal and leukemic cells within the CD34+CD38- cell subpopulation. As leukemic cytogenetic marker was never present in normal counterparts, we could get the reliable result of “real” LIC load in leukemic burden through Flow-FISH analysis for patients with recurrent cytogenetic abnormalities. We showed that this leukemia initiating cells subpopulation was not so rare in patients, and we found that FISH+CD34+CD38- cells ranged from 0.01% to 52.8% in the bulk of blasts. Although the results of the present finding need to be confirmed in a larger cohort, our data do show that a high LIC load at diagnosis determines a high relapse rate of leukemia and a poor clinical outcome. A percentage of FISH+CD34+CD38- cells of more than 1% is strongly associated with reduced OS and EFS suggesting that residual disease is likely to be high in these patients. Hence, the assessment of the level of FISH+CD34+CD38- cells at diagnosis could help clinicians to identify patients as an effective prognostic indicator in AML with recurrent chromosomal abnormalities, eg. CBF-AML (Data not shown), and thereby refining the selection of therapeutic strategies and, possibly, long-term clinical outcome.
This ability to distinguish these cell populations offers the potential to simultaneously observe the efficacy of chemotherapy drugs against putative LICs and their toxicity against normal HSCs. It may also facilitate better identification of therapeutic targets. Recently, Minderman et al. reported a unique technology to perform FISH in suspension . The ability to visualize and perform photometric/morphometric analysis of imagery from tens of thousands of cells thus combines quantitative image analysis with the statistical power of flow cytometry, may further enhance the sensitivity and specificity of LIC detection.
To the best of our knowledge, Flow-FISH is used, for the first time, to detect LIC subpopulation in human bone marrow samples. In accordance with our results, high levels of “real” LIC load detected at diagnosis in AML patients treated with standard chemotherapy, was significantly correlated with increased risk of poor clinical outcome. However, it is still not clear if the prognostic value of the LIC load detected with Flow-FISH is associated with the risk stratification of the associated cytogenetic abnormality and other known gene mutations such as FLT3-ITD because, most probably, of the limited cases in this cohort. Future larger cohort studies should solve this issue. In conclusion, Flow-FISH discrimination of leukemic and normal candidate stem cells could be used as a powerful tool to predict clinical outcome and help physicians to evaluate criteria for treatment in AML with recurrent cytogenetic abnormalities.