Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.

Chemotherapy resistant leukaemic stem cells (LSC) are thought to be responsible for relapses after therapy in acute myeloid leukaemia (AML). Flow cytometry can discriminate CD34(+) CD38(-) LSC and normal haematopoietic stem cells (HSC) by using aberrant expression of markers and scatter properties. However, not all LSC can be identified using currently available markers, so new markers are needed. CD45RA is expressed on leukaemic cells in the majority of AML patients. We investigated the potency of CD45RA to specifically identify LSC and HSC and improve LSC quantification. Compared to our best other markers (CLL-1, also termed CLEC12A, CD33 and CD123), CD45RA was the most reliable marker. Patients with high percentages (>90%) of CD45RA on CD34(+) CD38(-) LSC have 1·69-fold higher scatter values compared to HSC (P < 0·001), indicating a more mature CD34(+) CD38(-) phenotype. Patients with low (<10%) or intermediate (10-90%) CD45RA expression on LSC showed no significant differences to HSC (1·12- and 1·15-fold higher, P = 0·31 and P = 0·44, respectively). CD45RA-positive LSC tended to represent more favourable cytogenetic/molecular markers. In conclusion, CD45RA contributes to more accurate LSC detection and is recommended for inclusion in stem cell tracking panels. CD45RA may contribute to define new LSC-specific therapies and to monitor effects of anti-LSC treatment.

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered.

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric-based CD34-definition, without use of cut-off values. We discriminated CD34-positive (n = 548) and CD34-negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34-status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut-offs (5-20%) can now entirely be explained by considering the number of CD34-negative cases. In the total patient group, the absence of neoplastic CD34-positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.

Relevance of leukemic stem cells in acute myeloid leukemia: heterogeneity and influence on disease monitoring, prognosis and treatment design.

Acute myeloid leukemia is a bone marrow disease characterized by a block in differentiation of the myeloid lineage with a concomitant uncontrolled high proliferation rate. Development of acute myeloid leukemia from stem cells with specific founder mutations, leads to an oligoclonal disease that progresses into a very heterogeneous leukemia at diagnosis. Measurement of leukemic stem cell load and characterization of these cells are essential for prediction of relapse and target identification, respectively. Prediction of relapse by monitoring the disease during minimal residual disease detection is challenged by clonal shifts during therapy. To overcome this, characterization of the potential relapse-initiating cells is required using both flow cytometry and molecular analysis since leukemic stem cells can be targeted both on extracellular features and on stem-cell specific signal transduction pathways.