Alexander N. Snel

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.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

Introduction Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. Results For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patients bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n = 91), which reflect the total neoplastic burden, revealed four patient groups with different survival. Conclusion and Perspective Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Normal hematopoietic stem cells within the AML bone marrow have a distinct and higher ALDH activity level than co-existing leukemic stem cells.

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDHbright). The ALDHbright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDHlow. In CD34-positive AML, LSC are also ALDHlow but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.

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 LB-45: High aldehyde dehydrogenase activity is a marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia: novel therapeutic implications.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Only a minority of cells, the leukemic stem cells (LSC), within AML are responsible for tumor growth and maintenance. Many patients experience relapse after therapy which originates from outgrowth of therapy resistant LSC. Therefore, eradication of LSC is necessary to cure AML. Both the normal hematopoietic stem cells (HSC) and LSC co-exist in the bone marrow (BM) of AML patients and success of anti-LSC strategies relies on specific elimination of LSC while sparing HSC. LSC are contained within the CD34+CD38-, the side population (SP) and the high aldehyde dehydrogenase (ALDH) activity compartments. ALDH is a detoxifying enzyme responsible for oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as cyclophosphamide. It has been shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blasts. In view of applicability of LSC specific therapies the detoxification by ALDH is clinically very important. A difference in ALDH activity between HSC and LSC might be used to preferentially kill LSC while sparing HSC. To establish ALDH activity differences between HSC and LSC it should be possible to discriminate between them. We have shown that LSC can be identified and discriminated from HSC using stem cell-associated cell surface markers, such as CLL-1, lineage markers (CD7, CD19, CD56) and recently CD34/CD45 expression and cell size characteristics (Terwijn, Blood 111: 487, 2008). This offers the opportunity to identify co-existing LSC and HSC in the AML BM. We now show that, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that HSC that co-exist with LSC in BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. We have analyzed ALDH activity in HSC and LSC, both present in the BM from 18 AML patients. In nine BM AML samples, defined as CD34negative (<1%CD34+ blasts), the CD34+ compartment contained only normal CD34+CD38− HSC. The ALDH activity in these CD34+ HSC, is a factor 4,4 (range 1,7–18,9) higher than in LSC. In nine BM AML samples, defined as CD34positive AML, the CD34+CD38- HSC have a 7,7 fold (range 1,73–29,2 fold) higher ALDH activity as compared to putative LSC. In both CD34-positive and CD34-negative AML, we confirmed the identity of HSC and LSC by screening for molecular aberrancies present in AML blasts. The level of the ALDH activity of HSC within the AML BM is similar to that of HSC in NBM of healthy donors. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype) at diagnosis. Consequently, AML patients with high ALDH activity in HSC might benefit from treatment with agents that will be converted by ALDH enzymes, such as cyclophosphamide, whereby the difference between the activity in LSC and HSC will define the therapeutic window. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2011-LB-45