Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection

Measurable residual disease in acute myeloid leukemia using flow cytometry: approaches for harmonization/standardization

Introduction: Measurable residual disease (MRD) in acute myeloid leukemia (AML) is a rapidly evolving area with many institutes embarking on it, both in academic and pharmaceutical settings. However, there is a multitude of approaches to design, perform, and report flow cytometric MRD. Together with the long-term experience needed, this makes flow cytometric MRD in AML nonstandardized and time-consuming. Areas covered: This paper briefly summarizes critical issues, like sample preparation and transport, markers and fluorochromes of choice, but in particular focuses on the main issues, which includes specificity and sensitivity, hereby providing a new model that may circumvent the main disadvantages of the present approaches. New approaches that may add to the value of flow cytometric MRD includes assessment of leukemia stem cells, MRD in peripheral blood, and approaches to use multidimensional image analysis. Expert commentary: MRD in AML requires standardization/harmonization on many aspects, for which the present paper offers possible guidelines

MRD Tailored Therapy in AML: What We Have Learned So Far

Acute myeloid leukemia (AML) is a heterogeneous clonal disease associated with a dismal survival, partly due to the frequent occurrence of relapse. Many patient- and leukemia-specific characteristics, such as age, cytogenetics, mutations, and measurable residual disease (MRD) after intensive chemotherapy, have shown to be valuable prognostic factors. MRD has become a rich field of research where many advances have been made regarding technical, biological, and clinical aspects, which will be the topic of this review. Since many laboratories involved in AML diagnostics have experience in immunophenotyping, multiparameter flow cytometry (MFC) based MRD is currently the most commonly used method. Although molecular, quantitative PCR based techniques may be more sensitive, their disadvantage is that they can only be applied in a subset of patients harboring the genetic aberration. Next-generation sequencing can assess and quantify mutations in many genes but currently does not offer highly sensitive MRD measurements on a routine basis. In order to provide reliable MRD results, MRD assay optimization and standardization is essential. Different techniques for MRD assessment are being evaluated, and combinations of the methods have shown promising results for improving its prognostic value. In this regard, the load of leukemic stem cells (LSC) has also been shown to add to the prognostic value of MFC-MRD. At this moment, MRD after intensive chemotherapy is most often used as a prognostic factor to help stratify patients, but also to select the most appropriate consolidation therapy. For example, to guide post-remission treatment for intermediate-risk patients where MRD positive patients receive allogeneic stem cell transplantation and MRD negative receive autologous stem cell transplantation. Other upcoming uses of MRD that are being investigated include: selecting the type of allogeneic stem cell transplantation therapy (donor, conditioning), monitoring after stem cell transplantation (to allow intervention), and determining drug efficacy for the use of a surrogate endpoint in clinical trials

AML/normal progenitor balance instead of total tumor load (MRD) accounts for prognostic impact of flowcytometric residual disease in AML

Measurable residual disease (MRD) in AML, assessed by multicolor flow cytometry, is an important prognostic factor. Progenitors are key populations in defining MRD, and cases of MRD involving these progenitors are calculated as percentage of WBC and referred to as white blood cell MRD (WBC-MRD). Two main compartments of WBC-MRD can be defined: (1) the AML part of the total primitive/progenitor (CD34+, CD117+, C

Activin-A induces fewer, but larger osteoclasts from monocytes in both healthy controls and fibrodysplasia ossificans progressiva patients

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease characterized by heterotopic ossification (HO) that occurs in muscle tissue, tendons, and ligaments. The disease is caused by mutations in the Activin receptor type I (ACVR1) gene resulting in enhanced responsiveness to Activin-A. Binding of this molecule to the mutated receptor induces HO. Bone metabolism normally requires the coupled action of osteoblasts and osteoclasts, which seems to be disturbed during HO. We hypothesize that Activin-A may also counteract the formation of osteoclasts in FOP patients. In this study we investigated the effect of Activin-A on osteoclast differentiation of CD14+ monocytes from FOP patients and healthy controls. The lymphocytic and monocytic cell populations were determined by FACS analysis. Expression of the mutated R206H receptor was assessed and confirmed by allele specific PCR. The effect of Activin-A on osteoclastogenesis was assessed by counting the number and size of multinucleated cells. Osteoclast activity was determined by culturing the cells on Osteo Assay plates. The influence of Activin-A on expression of various osteoclast related genes was studied with QPCR. Blood from FOP patients contained similar percentages of classical, intermediate, or non-classical monocytes as healthy controls. Addition of Activin-A to the osteoclastogenesis cultures resulted in fewer osteoclasts in both control and FOP cultures. The osteoclasts formed in the presence of Activin-A were, however, much larger and more active compared to the cultures without Activin-A. This effect was tempered when the Activin-A inhibitor follistatin was added to the Activin-A containing cultures. Expression of osteoclast specific genes Cathepsin K and TRAcP was upregulated, gene expression of osteoclastogenesis related genes M-CSF and DC-STAMP was downregulated by Activin-A. Since Activin-A is a promising target for inhibiting the formation of HO in FOP, it is important to know its effects on both osteoblasts and osteoclasts. Our study shows that Activin-A induces fewer, but larger and more active osteoclasts independent of the presence of the mutated ACVR1 receptor. When considering FOP as an Activin-A driven disease that acts locally, our findings suggest that Activin-A could cause a more pronounced local resorption by larger osteoclasts. Thus, when targeting Activin-A in patients with neutralizing antibodies, HO formation could potentially be inhibited, and osteoclastic activity could be slightly reduced, but then performed dispersedly by more and smaller osteoclasts