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 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.

Therapy-related myeloid neoplasms (tMN) develop after exposure to cytotoxic and radiation therapy, and due to their adverse prognosis, it is of paramount interest to identify patients at high risk. The presence of clonal hematopoiesis has been shown to increase the risk of developing tMN. The value of analyzing hematopoietic stem cells harvested at leukapheresis before autologous stem cell transplantation (ASCT) with next-generation sequencing and immunophenotyping represents potentially informative parameters that have yet to be discovered. We performed a nested case-control study to elucidate the association between clonal hematopoiesis, mobilization potential, and aberrant immunophenotype in leukapheresis products with the development of tMN after ASCT. A total of 36 patients with nonmyeloid disease who were diagnosed with tMN after treatment with ASCT were included as case subjects. Case subjects were identified from a cohort of 1130 patients treated with ASCT and matched with 36 control subjects who did not develop tMN after ASCT. Case subjects were significantly poorer mobilizers of CD34+ cells at leukapheresis (P = .016), indicating that these patients possess inferior bone marrow function. Both clonal hematopoiesis (odds ratio, 5.9; 95% confidence interval, 1.8-19.1; P = .003) and aberrant expression of CD7 (odds ratio, 6.6; 95% confidence interval, 1.6-26.2; P = .004) at the time of ASCT were associated with an increased risk of developing tMN after ASCT. In conclusion, clonal hematopoiesis, present at low variant allele frequencies, and aberrant CD7 expression on stem cells in leukapheresis products from patients with nonmyeloid hematologic cancer hold potential for the early identification of patients at high risk of developing tMN after ASCT.

Background The hallmark of myelodysplastic syndrome (MDS) remains dysplasia in the bone marrow (BM). However, diagnosing MDS may be challenging and subject to inter‐observer variability. Thus, there is an unmet need for novel objective, standardized and reproducible methods for evaluating dysplasia. Imaging flow cytometry (IFC) offers combined analyses of phenotypic and image‐based morphometric parameters, for example, cell size and nuclearity. Hence, we hypothesized IFC to be a useful tool in MDS diagnostics. Methods Using a different‐from‐normal approach, we investigated dyserythropoiesis by quantifying morphometric features in a median of 5953 erythroblasts (range: 489–68,503) from 14 MDS patients, 11 healthy donors, 6 non‐MDS controls with increased erythropoiesis, and 6 patients with cytopenia. Results First, we morphometrically confirmed normal erythroid maturation, as immunophenotypically defined erythroid precursors could be sequenced by significantly decreasing cell‐, nuclear‐ and cytoplasm area. In MDS samples, we demonstrated cell size enlargement and increased fractions of macronormoblasts in late‐stage erythroblasts (both p < .0001). Interestingly, cytopenic controls with high‐risk mutational patterns displayed highly aberrant cell size morphometrics. Furthermore, assisted by machine learning algorithms, we reliably identified and enumerated true binucleated erythroblasts at a significantly higher frequency in two out of three erythroblast maturation stages in MDS patients compared to normal BM (both p = .0001). Conclusion We demonstrate proof‐of‐concept results of the applicability of automated IFC‐based techniques to study and quantify morphometric changes in dyserythropoietic BM cells. We propose that IFC holds great promise as a powerful and objective tool in the complex setting of MDS diagnostics with the potential for minimizing inter‐observer variability.

Summary Targeted therapy directed against rare disease‐propagating leukaemic stem cells (LSCs) is a promising prospect for improving the outcome of acute myeloid leukaemia (AML) patients. Thus, distinguishing LSCs from normal haematopoietic stem and progenitor cells (HSPCs) is essential. The CLEC12A receptor has been proposed as a specific marker of LSCs, and consequently as an appealing treatment target. To explore the role of CLEC12A in further detail, we investigated whether a sorting strategy based on the activity of aldehyde dehydrogenase and CLEC12A expression could separate residual normal HSPCs from LSCs in bone marrow from 5 AML patients. We demonstrate that this distinction was possible in 2/5 cases, however with evidence of pre‐leukaemic mutations in the CLEC12A‐ stem cells in one case. In contrast, cytogenetic and/or molecular aberrations were detected in both the CLEC12A+/− cell subsets in 3/5 AML cases studied. Furthermore, targeted next generation sequencing (NGS) of the sorted cell subsets revealed a pronounced clonal heterogeneity in the CLEC12A‐ cells suggestive of the leukaemia often originating in this immature cell subset. In conclusion, we provide proof‐of‐concept that precision diagnostics employing targeted cytogenetic/NGS‐based analyses on highly purified cell subsets could be a powerful tool for selecting patients eligible for LSC‐directed therapy.

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.