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Sequencing the exome is quickly becoming the preferred method for discovering disease-inducing mutations. While obtaining data sets is a straightforward procedure, the subsequent ...analysis and interpretation of the data is a limiting step for clinical applications. Thus, while the initial mutation and variant calling can be performed by a bioinformatician or trained researcher, the output from robust packages such as MuTect and GATK is not directly informative for the general life scientists. In attempt to obviate this problem we have created complementary Wolfram scripts, which enable easy downstream annotation and selection, presented here in the perspective of hematological relevance. It also provides the researcher with the opportunity to extend the analysis by having a full-fledged programming and analysis environment of Mathematica at hand. In brief, post-processing is performed by:
•Mapping of germ line and somatic variants to coding regions, and defining variant sets within Mathematica.•Processing of variants in variant effect predictor.•Extended annotation, relevance scoring and defining focus areas through the provided functions.
Summary
The concept of leukaemic stem cells (LSCs) was experimentally suggested 25 years ago through seminal data from John Dick's group, who showed that a small fraction of cells from acute myeloid ...leukaemia (AML) patients were able to be adoptively transferred into immunodeficient mice. The initial estimation of the frequency was 1:250 000 leukaemic cells, clearly indicating the difficulties ahead in translating knowledge on LSCs to the clinical setting. However, the field has steadily grown in interest, expanse and importance, concomitantly with the realisation of the molecular background for AML culminating in the sequencing of hundreds of AML genomes. The literature is now ripe with contributions describing how different molecular aberrations are more or less specific for LSCs, as well as reports showing selectivity in targeting LSCs in comparison to normal haematopoietic stem and progenitor cells. However, we argue here that these important data have not yet been fully realised within the clinical setting. In this clinically focused review, we outline the difficulties in identifying and defining LSCs at the individual patient level, with special emphasis on intraclonal heterogeneity. In addition, we suggest areas of future focus in order to realise the concept as real‐time benefit for AML patients.
Myeloproliferative neoplasms – a global view Tefferi, Ayalew; Ianotto, Jean‐Christophe; Mathews, Vikram ...
British journal of haematology,
September 2022, Letnik:
198, Številka:
6
Journal Article
5-azacytidine (azacytidine), a DNA hypomethylating agent, was recently approved as the first therapeutic agent for the treatment of myelodysplastic syndromes. The present subcutaneous dosing ...schedule, 75 mg/m
2 for 7/28 days, is based on early clinical studies and may constitute a practical problem for patients. The present in vitro study aimed at evaluating the pharmacodynamics of azacytidine, thereby providing a rationale for clinical dose-finding studies.
P39 cells were incubated with 0.1, 0.5, and 1 μM azacytidine daily for 24, 48, and 72 hours, followed by 48 hours in drug-free medium. The effects of azacytidine on cell growth, proliferation, apoptosis, cell cycle status, and promoter methylation of
E-cadherin, ER, and
HIC genes were studied.
Azacytidine decreased cell growth and proliferation, increased apoptosis, and affected cell cycle status in a dose-dependent manner. However, the exposure time, 24 to 72 hours, at doses between 0.5 and 1 μM, did not significantly affect any of these variables. Using first-order exponential pharmacokinetic model, we found that the effect of 1, 2, or 3 μM over 24 hours did not differ from that of 0.5 to 1 μM given over 48 to 72 hours. Induction of promoter hypomethylation was observed already after 24 hours of exposure with ≥0.5 μM azacytidine with no clear dose-effect relationship.
Our results indicate that optimal cellular effects of azacytidine might be achieved by shorter exposure times. The model provides information about the relation between azacytidine dose intensity and exposure time on malignant myeloid cells, which could serve as a rationale for further clinical development of practical, safe, and cost-effective dosing schedules.
Abstract We have identified a novel 7.7 Mb del(8)(q23.2q24.11) in a patient progressing to acute myeloid leukemia (AML) following a 12-year stable phase of chronic myelomonocytic leukemia (CMML). A ...surprisingly high JAK2 + allelic burden of 92% at the time of AML led us to delineate the molecular aberrations relevant for leukemogenesis. While a frameshift mutation in the TET2 gene was stably present throughout the course of disease the JAK2 mutation was acquired after initial diagnosis of CMML. At progression aCGH revealed del(8q)(q23.2q24.11) encompassing various cancer relevant genes of which RAD21 and CSMD3 are of particular interest.
Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a ...challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location.
AML in the elderly—A global view Hokland, Peter; Fernández, Isolda I.; Freeman, Sylvie D. ...
British journal of haematology,
December 2023, 2023-12-00, 20231201, Letnik:
203, Številka:
5
Journal Article
The C‐type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia‐associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its ...expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem‐ and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI 32.5;45.7) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte‐macrophage progenitors and megakaryocyte‐erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI 76.0;85.9) and 11.9% (95% CI 9.3;14.6), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/− CMPs and MEPs in the colony‐forming unit assay demonstrated CLEC12A+ subsets to favour non‐erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A‐expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A‐targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.
Introduction
In myelodysplastic syndrome (MDS), multicolor flow cytometry (FC) offers a supplement to the morphological, cytogenetic and molecular evaluation of the hematopoietic lineages in the bone ...marrow (BM). In addition to providing a robust enumeration of immature myeloid cells, FC allows for identification of aberrant antigen expression patterns. While aberrant findings are well-defined in the myelo-monocytic lineages, the erythroid and megakaryocytic cell lineages are less well characterized by conventional FC. Imaging flow cytometry (IFC) is a novel methodology that bridge multicolor FC and high-throughput capacity with collection of multispectral imagery of single cells. This allows for the simultaneous evaluation, quantification and subsequent statistical analysis of both surface-, nuclear- and cytoplasmic immunophenotypic and morphometric characteristics on a practically unlimited number of cells in suspension. In this study, we hypothesized the IFC technique to be useful in discriminating dyserythropoietic from normal BM.
Methods
Mononuclear BM cells from 6 healthy donors and 2 MDS patients were thawed and stained with monoclonal antibodies against CD235a, CD45, CD71, CD117 and CD105. DRAQ5 was included for DNA staining, and Zombie Violet was included for viability. Data acquisition was performed on an ImageStream®XMarkII (Amnis/Merck), and subsequent analysis was done using the IDEAS software package. The median number of acquired cells was 130,000 for healthy BM (range: 70,548-300,000) and for MDS samples the equivalent number was 253,255 and 260,000, respectively. Nucleated erythroid cells (NECs) were identified based on the CD45lowDRAQ5highCD71+ immunophenotype and the coexpression of either CD235 and/or CD105. Next, proerythroblasts (ProE), basophilic erythroblasts (BasoE) and poly-orthochromatic erythroblasts (Poly_OrthoE) were distinguished based on the expression of CD117 and CD105 (Fig. 1, upper panel). Morphometric features were examined within function masks covering the cellular, nuclear and cytoplasmic area. From these masks we calculated cell area, nucleus area, nucleus elongatedness, nucleus circularity score, and nuclear location (delta centroid XY) (Fig. 1, lower panel). Data from healthy controls (Fig. 1A) served as biological reference for phenotypic and morphometric measurements of MDS samples.
Results & perspectives
In one MDS patient with single lineage dysplasia (MDS-SLD) (Fig. 1B), morphological dysplastic changes were seen in less than 10% of erythropoiesis, and manifested as nuclear lobulations, cytoplasmic granules and megaloblastic changes. We examined 6470 NECs by IFC and found an increased elongatedness and a decreased circularity score of the nuclei in the ProE population, reflecting irregular nuclear shape. Moreover, for the ProE and BasoE maturation stages, we detected increased cell size as well as enlarged nuclei of BasoE. This finding corresponded to the megaloblastic changes seen by morphology. Taken together, data from IFC confirmed the morphological findings in this MDS patient.
The second patient (Fig. 1C) had MDS with excess blasts (MDS-EB-1). Morphologic examination showed mild dyserythropoiesis in less than 10% of erythropoietic cells with signs of multinuclearity, nuclear lobulations, and ringed sideroblasts. Interestingly, IFC analysis of 25,240 NECs identified aberrant delta centroid XY corresponding to decentralization of the nucleus in the ProE population, as also seen in the MDS-SLD patient. As such, with the applied IFC features, we were not able to detect changes corresponding to the morphological findings, which could be due to the low percentage of dysplastic erythroid cells. However, the deviant morphometric parameters detected by IFC in this patient identified additional dysmorphometric features of interest.
To conclude, we provide proof-of-concept of a novel experimental approach to study and quantify morphometric changes in samples from MDS patients. Certainly, the technique needs to be explored in a larger patient material and we are currently extending our study cohort. Using a different-from-normal approach, our preliminary data was sufficient to demonstrate dysmorphometric changes in two MDS patients with only mild morphological dyserythropoiesis. Thus, we propose that IFC holds great promise as a powerful tool in the challenging setting of MDS diagnostics.
No relevant conflicts of interest to declare.
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