Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States.

Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame. (Funded by the Queen Wilhelmina Fund Foundation of the Dutch Cancer Society and others.).

| The cancer stem cell (CSC) concept has important therapeutic implications, but its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined. Evidence of their heterogeneous origins, frequencies and their genomic, as well as their phenotypic and functional, properties has added to the confusion and has fuelled new ideas and controversies. Participants in The Year 2011 Working Conference on CSCs met to review these issues and to propose a conceptual and practical framework for CSC terminology. More precise reporting of the parameters that are used to identify CSCs and to attribute responses to them is also recommended as key to accelerating an understanding of their biology and developing more effective methods for their eradication in patients. PERSPECTIVES NATURE REVIEWS | CANCER VOLUME 12 | NOVEMBER 2012 | 767

A B S T R A C T PurposeHalf 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 MethodsIn 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). ResultsAfter 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 (Ͼ 0.1% of WBC) were associated with a higher risk of relapse after adjustment for consolidation treatment timedependent covariate risk score and early or later CR. ConclusionIn 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.

In CD34 ؉ acute myeloid leukemia (AML), the malignant stem cells reside in the CD38 ؊ compartment. We have shown before that the frequency of such CD34 ؉ CD38 ؊ cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival.Specific targeting of CD34 ؉ CD38 ؊ cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34 ؉ CD38 ؊ stemcell compartment in AML (77/89 patients). The CD34 ؉ CLL-1 ؉ population, containing the CD34 ؉ CD38 ؊ CLL-1 ؉ cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1 ؉ blasts. CLL-1 expression was not different between diagnosis and relapse (n ‫؍‬ 9). In remission, both CLL-1 ؊ normal and CLL-1 ؉ malignant CD34 ؉ CD38 ؊ cells were present. A high CLL-1 ؉ fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34 ؉ CD38 ؊ cells in normal (n ‫؍‬ 11) and in regenerating bone marrow controls (n ‫؍‬ 6). This IntroductionDespite high-dose chemotherapy, only 30% to 40% of patients with acute myeloid leukemia (AML) survive, which is due mainly to relapse of the disease. 1 AML is generally regarded as a stem-cell disease. However, there is debate whether normal stem cells undergoing leukemogenic mutations is the explanation for leukemogenesis. Alternatively, leukemogenic mutations occurring at a later developmental stage, resulting in stem cell-like behavior, might be an alternative or additional option. [2][3][4] For CD34 ϩ AML, several authors have shown that leukemic stem cells are present in the CD34 ϩ CD38 Ϫ compartment. 5,6 It has been proven in vitro that these stem cells are more resistant to chemotherapy, compared with the progenitor CD34 ϩ CD38 ϩ cells. 7 In vivo, after chemotherapy, the residual malignant CD34 ϩ CD38 Ϫ cells are thought to differentiate to a limited extent, producing leukemic cells with an immunophenotype, which usually reflects that at diagnosis. Sensitive techniques allow early detection of small numbers of these differentiated leukemic cells, called minimal residual disease (MRD), which eventually causes relapse of the disease. 8 Since in this concept the stem cell is the origin of MRD and relapse, stem cell-targeted therapy would be of potentially high benefit for AML patients. Moreover, early detection of leukemic stem cells after chemotherapeutic treatment might offer prognostic value in predicting relapse of the disease. Different options for stem-cell identification and/or targeted therapy have been described such as anti-CD123, anti-CD44, and anti-CD33, but all have some (potential) disadvantages, including expression on normal stem cells and/or nonhematologic tissues. [9][10][11] Since the bone marrow of a (chemotherapy-) treated patient cannot be considered normal, it is extremely important to study whether after treatment nor...

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The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34 + precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future. Cancer and Immunology, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. E-mail: a.vandeloosdrecht@vumc.nl © F e r r a t a S t o r t i F o u n d a t i o n

Purpose: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34 + CD38À 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 À cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34 + CD38À 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 + CD38À 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 + CD38À population at diagnosis reflects a higher percentage of chemotherapyresistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.