Erythroid predominance in bone marrow biopsies of AML patients after decitabine treatment correlates with mutation profile and complete remission.

INTRODUCTION: Acute myeloid leukemia (AML) patients may receive hypomethylating agents (HMAs) such as decitabine (DAC) as part of their treatment. Not all patients respond to this therapy, and if they do the clinical response may occur only after 3 to 6 courses of treatment. Hence, early biomarkers predicting response would be very useful. METHODS: We retrospectively analyzed a cohort of 22 AML patients who were treated with DAC. Histology of the bone marrow biopsy, pathogenic mutations and methylation status were related to the treatment response. RESULTS: In 8/22 (36%) patients, an erythroid dominant response (EDR) pattern, defined as a ratio of myeloid cells/erythroid cells < 1, was observed. In the remaining 14 cases a myeloid predominance was preserved during treatment. No difference in the hypomethylating effect of DAC treatment was observed in patients with and without EDR, as global 5-methylcytosine levels dropped similarly in both groups. Mutational analysis by NGS using a panel of commonly mutated genes in AML, showed that patients with an early EDR harbored on average less mutations, with U2AF1 mutations occurring more frequently, whereas RUNX1 mutations were underrepresented compared to non-EDR cases. Interestingly, the development of an EDR correlated with complete remission (7/8 cases with an EDR versus only 2/14 cases without an EDR). CONCLUSION: We conclude that early histological bone marrow examination for the development of an EDR may be helpful to predict response in AML patients during treatment with DAC.

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation.

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

Mapping AML heterogeneity - multi-cohort transcriptomic analysis identifies novel clusters and divergent ex-vivo drug responses.

Subtyping of acute myeloid leukaemia (AML) is predominantly based on recurrent genetic abnormalities, but recent literature indicates that transcriptomic phenotyping holds immense potential to further refine AML classification. Here we integrated five AML transcriptomic datasets with corresponding genetic information to provide an overview (n = 1224) of the transcriptomic AML landscape. Consensus clustering identified 17 robust patient clusters which improved identification of CEBPA-mutated patients with favourable outcomes, and uncovered transcriptomic subtypes for KMT2A rearrangements (2), NPM1 mutations (5), and AML with myelodysplasia-related changes (AML-MRC) (5). Transcriptomic subtypes of KMT2A, NPM1 and AML-MRC showed distinct mutational profiles, cell type differentiation arrests and immune properties, suggesting differences in underlying disease biology. Moreover, our transcriptomic clusters show differences in ex-vivo drug responses, even when corrected for differentiation arrest and superiorly capture differences in drug response compared to genetic classification. In conclusion, our findings underscore the importance of transcriptomics in AML subtyping and offer a basis for future research and personalised treatment strategies. Our transcriptomic compendium is publicly available and we supply an R package to project clusters to new transcriptomic studies.

Improvement, Implementation, and Evaluation of the CMyLife Digital Care Platform: Participatory Action Research Approach.

BACKGROUND: The evaluation of a continuously evolving eHealth tool in terms of improvement and implementation in daily practice is unclear. The CMyLife digital care platform provides patient-centered care by empowering patients with chronic myeloid leukemia, with a focus on making medication compliance insightful, discussable, and optimal, and achieving optimal control of the biomarker BCR-ABL1. OBJECTIVE: The aim of this study was to investigate to what extent the participatory action research approach is suitable for the improvement and scientific evaluation of eHealth innovations in daily clinical practice (measured by user experiences) combined with the promotion of patient empowerment. METHODS: The study used iterative cycles of planning, action, and reflection, whereby participants' experiences (patients, health care providers, the CMyLife team, and app suppliers) with the platform determined next actions. Co-design workshops were the foundation of this cyclic process. Moreover, patients filled in 2 sets of questionnaires for assessing experiences with CMyLife, the actual use of the platform, and the influence of the platform after 3 and at least 6 months. Data collected during the workshops were analyzed using content analysis, which is often used for making a practical guide to action. Descriptive statistics were used to characterize the study population in terms of information related to chronic myeloid leukemia and sociodemographics, and to describe experiences with the CMyLife digital care platform and the actual use of this platform. RESULTS: The co-design workshops provided insights that contributed to the improvement, implementation, and evaluation of CMyLife and empowered patients with chronic myeloid leukemia (for example, simplification of language, and improvement of the user friendliness of functionalities). The results of the questionnaires indicated that (1) the platform improved information provision on chronic myeloid leukemia in 67% (33/49) of patients, (2) the use of the medication app improved medication compliance in 42% (16/38) of patients, (3) the use of the guideline app improved guideline adherence in 44% (11/25) of patients, and (4) the use of the platform caused patients to feel more empowered. CONCLUSIONS: A participatory action research approach is suited to scientifically evaluate digital care platforms in daily clinical practice in terms of improvement, implementation, and patient empowerment. Systematic iterative evaluation of users' needs and wishes is needed to keep care centered on patients and keep the innovation up-to-date and valuable for users.

Evolutionary landscape of clonal hematopoiesis in 3,359 individuals from the general population.

Knowledge about evolution of clonal hematopoiesis, which may drive malignant progression, is crucial for clinical decision-making. We investigated the landscape of clonal evolution by error-corrected sequencing on 7,045 sequential samples from 3,359 individuals in the prospective population-based Lifelines cohort, with a special focus on cytosis and cytopenia. Spliceosome (SRSF2/U2AF1/SF3B1) and JAK2 mutated clones show highest growth rates over a median 3.6-year period, while clone sizes for DNMT3A and TP53 increase only marginally, independent of cytosis or cytopenia. Nevertheless, large differences are observed between individuals carrying the same mutation, indicative of modulation by non-mutation-related factors. Clonal expansion is not dependent on classical cancer risk factors (e.g., smoking). Risk for incident myeloid malignancy diagnosis is highest for JAK2, spliceosome, or TP53 mutations and absent for DNMT3A, and it is mostly preceded by cytosis or cytopenia. The results provide important insight into high-risk evolutionary patterns to guide monitoring of "CHIP" and "CCUS."

Inducible MLL-AF9 Expression Drives an AML Program during Human Pluripotent Stem Cell-Derived Hematopoietic Differentiation.

A t(9;11)(p22;q23) translocation produces the MLL-AF9 fusion protein, which is found in up to 25% of de novo AML cases in children. Despite major advances, obtaining a comprehensive understanding of context-dependent MLL-AF9-mediated gene programs during early hematopoiesis is challenging. Here, we generated a human inducible pluripotent stem cell (hiPSC) model with a doxycycline dose-dependent MLL-AF9 expression. We exploited MLL-AF9 expression as an oncogenic hit to uncover epigenetic and transcriptomic effects on iPSC-derived hematopoietic development and the transformation into (pre-)leukemic states. In doing so, we observed a disruption in early myelomonocytic development. Accordingly, we identified gene profiles that were consistent with primary MLL-AF9 AML and uncovered high-confidence MLL-AF9-associated core genes that are faithfully represented in primary MLL-AF9 AML, including known and presently unknown factors. Using single-cell RNA-sequencing, we identified an increase of CD34 expressing early hematopoietic progenitor-like cell states as well as granulocyte-monocyte progenitor-like cells upon MLL-AF9 activation. Our system allows for careful chemically controlled and stepwise in vitro hiPSC-derived differentiation under serum-free and feeder-free conditions. For a disease that currently lacks effective precision medicine, our system provides a novel entry-point into exploring potential novel targets for personalized therapeutic strategies.

Effectiveness of digital care platform CMyLife for patients with chronic myeloid leukemia: results of a patient-preference trial.

BACKGROUND: Two most important factors determining treatment success in chronic myeloid leukemia (CML) are adequate medication compliance and molecular monitoring albeit still being suboptimal. The CMyLife platform is an eHealth innovation, co-created with and for CML patients, aiming to improve their care, leading to an increased quality of life and the opportunity of hospital-free care. OBJECTIVE: To explore the effectiveness of CMyLife in terms of information provision, patient empowerment, medication compliance, molecular monitoring, and quality of life. METHODS: Effectiveness of CMyLife was explored using a patient-preference trial. Upon completion of the baseline questionnaire, participants actively used (intervention group) or did not actively use (questionnaire group) the CMyLife platform for at least 6 months, after which they completed the post-intervention questionnaire. Scores between the intervention group and the questionnaire group were compared with regard to the within-subject change between baseline and post-measurement using Generalized Estimating Equation models. RESULTS: At baseline, 33 patients were enrolled in the questionnaire group and 75 in the intervention group. Online health information knowledge improved significantly when actively using CMyLife and patients felt more empowered. No significant improvements were found regarding medication compliance and molecular monitoring, which were already outstanding. Self-reported effectiveness showed that patients experienced that using CMyLife improved their medication compliance and helped them to oversee their molecular monitoring. Patients using CMyLife reported more symptoms but were better able to manage these. CONCLUSIONS: Since hospital-free care has shown to be feasible in time of the COVID-19 pandemic, eHealth-based innovations such as CMyLife could be a solution to maintain the quality of care and make current oncological health care services more sustainable. TRIAL REGISTRATION: ClinicalTrials.gov NCT04595955 , 22/10/2020.

Abnormal Platelet Counts and Clonal Hematopoiesis in the General Population.

Clonal hematopoiesis (CH) is defined by the presence of somatic mutations that may cause clonal expansion of hematopoietic cells. Here, we investigated the association between platelet count abnormalities, CH and consequences on overall survival and the development of hematological malignancies. Individuals with thrombocytopenia (n = 631) or thrombocytosis (n = 178) ≥60 years, and their age- and sex-matched controls, were selected within the population-based Lifelines cohort (n = 167,729). Although the prevalence of CH was not increased in thrombocytopenia cases compared with their controls (37.9% vs 39.3%; P = 0.639), mutations in spliceosome genes (SF3B1, SRSF2, U2AF1) were significantly enriched in thrombocytopenia cases (P = 0.007). Overall, CH in combination with thrombocytopenia did not impact on survival, but thrombocytopenia in combination with multiple mutated genes (hazard ratio [HR] = 2.08, 95% confidence interval [CI], 1.24-3.50; P = 0.006), mutations in TP53 (HR = 5.83, 95% CI, 2.49-13.64; P < 0.001) or spliceosome genes (HR = 2.69, 95% CI, 1.29-5.63; P = 0.009) increased the risk of death. The prevalence of CH in thrombocytosis cases was higher compared with controls (55.8% vs 37.7%; P < 0.001). Especially mutations in JAK2 (P < 0.001) and CALR (P = 0.003) were enriched in individuals with thrombocytosis. The presence of CH in individuals with thrombocytosis did not impact on overall survival. However, during follow-up of 11 years 23% of the individuals with thrombocytosis and CH were diagnosed with hematological malignancies. From these, 81% were diagnosed with myeloproliferative disease and 76% carried driver mutations JAK2, CALR, or MPL.

Monocytosis and its association with clonal hematopoiesis in community-dwelling individuals.

Monocytosis may occur in numerous inflammatory conditions but is also the defining feature of chronic myelomonocytic leukemia (CMML). Clonal somatic mutations detectable in CMML may occur with aging in otherwise healthy individuals, so-called "clonal hematopoiesis" (CH). We investigated whether the combination of CH and monocytosis would represent an early developmental stage of CMML. We studied community-dwelling individuals with monocytosis (≥1 × 109/L and ≥10% of leukocytes) in the population-based Lifelines cohort (n = 144 676 adults). The prevalence and spectrum of CH were evaluated for individuals ≥60 years with monocytosis (n = 167 [0.8%]), and control subjects 1:3 matched for age and sex (n = 501). Diagnoses of hematological malignancies were retrieved by linkage to the Netherlands Cancer Registry (NCR). Monocyte counts and the prevalence of monocytosis increased with advancing age. Older individuals with monocytosis more frequently carried CH (50.9% vs 35.5%; P < .001). Monocytosis is associated with enrichment of multiple gene mutations (P = .006) and spliceosome mutations (P = .007) but not isolated mutated DNMT3A, TET2, or ASXL1. Persistent monocytosis over 4 years was observed in 30/102 evaluable individuals and associated with a higher prevalence of CH (63%). Myeloid malignancies, including 1 case of CMML, developed in 4 individuals with monocytosis who all carried CH. In conclusion, monocytosis and CH both occur at an older age and do not necessarily reflect clonal monocytic proliferation. In a fraction of older subjects with monocytosis, CH might constitute early clonal dominance in developing malignant myelomonocytic disease. Mutational spectra deviating from age-related CH require attention.

2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms.

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

Peripheral blood cytopenias in the aging general population and risk of incident hematological disease and mortality.

Peripheral blood cytopenias may precede the development of hematological malignancies and frequently pose clinical challenges in the older population. The natural course of (mild) cytopenias during aging and their association with hematological disorders in community-dwelling individuals are not well studied. Within the population-based Lifelines cohort (n = 167729), we studied changes in peripheral blood counts, occurrence of cytopenias, and associated hematological outcomes in the context of aging. Development of hematological malignancies and (cause-specific) mortality were evaluated by linkage to nationwide registries. Anemia and thrombocytopenia emerged with older age, in line with a general age-related decline in these blood counts. For neutropenia, no increase in prevalence with older age was observed. Using standard reference limits to define cytopenias, anemia (hazard ratio [HR], 1.84; 95% confidence interval [CI], 1.59-2.12), thrombocytopenia (HR, 1.58; 95% CI, 1.32-1.89), and, especially the concomitant presence of anemia and thrombocytopenia (HR, 4.75; 95% CI, 2.98-7.55) were associated with inferior overall survival. Only a minor proportion of deaths was explained by diagnosed hematological malignancies, with the majority attributable to other causes. Neutropenia, either isolated (HR, 0.88; 95% CI, 0.73-1.06) or combined with another cytopenia, did not affect overall survival. For individuals aged ≥60 years, 5-year cumulative incidence of hematological malignancies was 0.60% (95% CI, 0.50-0.70), with higher incidences among those with anemia (P < .001) or thrombocytopenia (P < .001) but not neutropenia (P = .201). Highest cumulative incidences of diagnoses and mortality from hematological malignancies were observed in individuals with >1 cytopenia. We conclude that anemia and thrombocytopenia, but not neutropenia, are associated with inferior overall survival of community-dwelling individuals. Hematological malignancies develop in a small fraction of these cases.

Prevalence, predictors, and outcomes of clonal hematopoiesis in individuals aged ≥80 years.

Clonal hematopoiesis (CH), characterized by a fraction of peripheral blood cells carrying an acquired genetic variant, emerges with age. Although in general CH is associated with increased mortality and morbidity, no higher risk of death was observed for individuals ≥80 years. Here, we investigated CH in 621 individuals aged ≥80 years from the population-based LifeLines cohort. Sensitive error-corrected sequencing of 27 driver genes at a variant allele frequency ≥1% revealed CH in the majority (62%) of individuals, independent of gender. The observed mutational spectrum was dominated by DNMT3A and TET2 variants, which frequently (29%) displayed multiple mutations per gene. In line with previous results in individuals ≥80 years, the overall presence of CH did not associate with a higher risk of death (hazard ratio, 0.91; 95% confidence interval, 0.70-1.18; P = .48). Being able to assess the causes of death, we observed no difference between individuals with or without CH, except for deaths related to hematological malignancies. Interestingly, comparison of mutational spectra confined to DNMT3A and TET2 vs spectra containing other mutated genes, showed a higher risk of death when mutations other than DNMT3A or TET2 were present (hazard ratio, 1.48; 95% confidence interval, 1.06-2.08; P = .025). Surprisingly, no association of CH with cardiovascular morbidity was found, irrespective of clone size. Further, CH associated with chronic obstructive pulmonary disease. Data on estimated exposure to DNA damaging toxicities (ie, smoking, a history of cancer [as a proxy for previous genotoxic therapy], and job-related pesticide exposure) showed an association with spliceosome and ASXL1 variants, but not with DNMT3A and TET2 variants.

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

Multicenter Next-Generation Sequencing Studies between Theory and Practice: Harmonization of Data Analysis Using Real-World Myelodysplastic Syndrome Data.

In the age of personalized medicine, genetic testing by means of targeted sequencing has taken a key role. However, when comparing different sets of targeted sequencing data, these are often characterized by a considerable lack of harmonization. Laboratories follow their own best practices, analyzing their own target regions. The question on how to best integrate data from different sites remains unanswered. Studying the example of myelodysplastic syndrome (MDS), we analyzed 11 targeted sequencing sets, collected from six different centers (n = 831). An intersecting target region of 43,076 bp (30 genes) was identified; whereas, the original target regions covered up to 499,097 bp (117 genes). Considering a region of interest in the context of MDS, a target region of 55,969 bp (31 genes) was identified. For each gene, coverage and sequencing data quality was evaluated, calculating a sequencing score. Analyses revealed huge differences between different data sets as well as between different genes. Analysis of the relation between sequencing score and mutation frequency in MDS revealed that most genes with high frequency in MDS could be sequenced without expecting low coverage or quality. Still, no gene appeared consistently unproblematic for all data sets. To allow for comparable results in a multicenter setting analyzing MDS, we propose to use a predefined target region of interest and to perform centralized data analysis using harmonized criteria.