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. Publisher9s Note: There is a Blood Commentary on this article in this issue.

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⁻ stem- cell 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 AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34⁺CLL-1⁺ cells indicate that anti–CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

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. 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 patient's 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. 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.