Angele Kelder

High Stem Cell Frequency in Acute Myeloid Leukemia at Diagnosis Predicts High Minimal Residual Disease and Poor Survival

Purpose: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34+CD38− stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. Experimental Design: First, the leukemogenic potential of unpurified CD34+CD38− cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34+CD38− compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. Results:In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34+CD38− stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34+ percentage showed no such correlations. Conclusions: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34+CD38− population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.

High Prognostic Impact of Flow Cytometric Minimal Residual Disease Detection in Acute Myeloid Leukemia: Data From the HOVON/SAKK AML 42A Study

PURPOSE Half the patients with acute myeloid leukemia (AML) who achieve complete remission (CR), ultimately relapse. Residual treatment-surviving leukemia is considered responsible for the outgrowth of AML. In many retrospective studies, detection of minimal residual disease (MRD) has been shown to enable identification of these poor-outcome patients by showing its independent prognostic impact. Most studies focus on molecular markers or analyze data in retrospect. This study establishes the value of immunophenotypically assessed MRD in the context of a multicenter clinical trial in adult AML with sample collection and analysis performed in a few specialized centers. PATIENTS AND METHODS In adults (younger than age 60 years) with AML enrolled onto the Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research Acute Myeloid Leukemia 42A study, MRD was evaluated in bone marrow samples in CR (164 after induction cycle 1, 183 after cycle 2, 124 after consolidation therapy). RESULTS After all courses of therapy, low MRD values distinguished patients with relatively favorable outcome from those with high relapse rate and adverse relapse-free and overall survival. In the whole patient group and in the subgroup with intermediate-risk cytogenetics, MRD was an independent prognostic factor. Multivariate analysis after cycle 2, when decisions about consolidation treatment have to be made, confirmed that high MRD values (> 0.1% of WBC) were associated with a higher risk of relapse after adjustment for consolidation treatment time-dependent covariate risk score and early or later CR. CONCLUSION In future treatment studies, risk stratification should be based not only on risk estimation assessed at diagnosis but also on MRD as a therapy-dependent prognostic factor.

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30–40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34+ AML patients. At diagnosis, subfractions of the CD45dimCD34+CD38dim/− compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45dimCD34+ blast compartment in 6 out of 7 patients. Moreover, within CD45dimCD34+CD38dim/− fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.

Residual normal stem cells can be detected in newly diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and predict for optimal response to imatinib

Insensitivity of chronic myeloid leukemia (CML) hematopoietic stem cells to tyrosine kinase inhibitors (TKIs) prevents eradication of the disease and may be involved in clinical resistance. For improved treatment results more knowledge about CML stem cells is needed. We here present a new flow cytometric approach enabling prospective discrimination of CML stem cells from their normal counterparts within single-patient samples. In 24 of 40 newly diagnosed CML patients residual normal CD34+CD38− stem cells could be identified by lower CD34 and CD45 expression, lower forward/sideward light scatter and by differences of lineage marker expression (CD7, CD11b and CD56) and of CD90. fluorescent in situ hybridization (FISH) analysis on Fluorescence-activated cell sorting sorted cells proved that populations were BCR–ABL positive or negative and long-term liquid culture assays with subsequent colony forming unit assays and FISH analysis proved their stem cell character. Patients with residual non-leukemic stem cells had lower clinical risk scores (Sokal, Euro), lower hematological toxicity of imatinib (IM) and better molecular responses to IM than patients without. This new approach will expand our possibilities to separate CML and normal stem cells, present in a single bone marrow or peripheral blood sample, thereby offering opportunities to better identify new CML stem-cell-specific targets. Moreover, it may guide optimal clinical CML management.

Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor

AIM To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells. MATERIAL AND METHODS PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP(+)) multinucleated cells (MNCs) and bone resorption were assessed. RESULTS TRACP(+) MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP(+) MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL. CONCLUSION Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity.

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.

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Abstract A29: High aldehyde dehydrogenase activity is general marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia

Only a minority of cells, the leukemic stem cells (LSCs), within Acute Myeloid Leukemia (AML) are typically responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which originates mainly from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure leukemia. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukaemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high aldehyde dehydrogenase (ALDH) activity compartment contains the LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. High ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry. Here we have analyzed ALDH activity in normal HSCs and LSCs, both present within the same BM, in 23 AML patient samples. In 7 AML cases, a high SSCloALDHbr cell population was identified (>5%). We show that in 9 BM AML samples, defined as CD34min (ie In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as mafosfamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Citation Information : Clin Cancer Res 2010;16(7 Suppl):A29