[The cause of anemia is underdiagnosed in primary care]. / Onderdiagnostiek naar anemie in de eerste lijn.

OBJECTIVE: To determine if general practitioners can diagnose the cause of anemia, based on the requested laboratory tests. DESIGN: Retrospective observational study. METHOD: The research population consisted of 20.004 adult patients with established anemia, who had blood samples analyzed by Atalmedial in 2019. The cause of anemia was found when criteria based on the NHG-standard were met. We considered the NHG-guideline to be followed when hemoglobin was requested in the first diagnostic request and when the correct combination of blood tests was requested in the second diagnostic request. Descriptive statistics and multilevel regression analysis were performed. RESULTS: A possible cause of anemia has been found in 38,7% of the patients within two diagnostic requests, regardless of the adherence to the NHG-guideline. The chance of finding a cause of anemia was smaller in men than women of the same age, whereas the chance was highest in women over the age of 80 and between 18 and 44. The NHG-guideline for anemia was followed in 11.794 (59%) of the patients in the first diagnostic request. 19,3% (11,4% of total) of these patients also had a second diagnostic request. In 10,4% (1,2% of total) of these patients, the NHG-guideline was adhered to in the second diagnostic request. CONCLUSION: A cause of anemia is, based on laboratory tests, often not diagnosed in daily practice in the primary care. The reason for this is insufficient laboratory follow-up after initial testing when no cause of anemia is found. The NHG-guideline for anemia is poorly adhered to.

The effectiveness of an automated algorithm as a tool for investigating the cause of anaemia in undiagnosed patients from general practitioners.

BACKGROUND: The Dutch guideline algorithm for the analysis of anaemia in patients of general practitioners (GPs) was programmed in a Clinical Decision Support system (CDS-anaemia) to support the process of diagnosing the cause of anaemia in the laboratory. This study investigates the diagnostic yield of the automated anaemia algorithm compared to that of the manual work up by the GP. METHODS: This retrospective population-based study consisted of 2697 people ≥18 years. Anaemia was defined according to the Dutch College of General Practitioners (DCGP) guideline. Causes of anaemia were based on the DCGP guidelines with the corresponding blood tests. The number of blood tests and causes of anaemia were measured in two separate periods in both the (CDS-anaemia) pilot group and a control group in which routine care was provided. RESULTS: Patients from GPs supported by CDS-anaemia had higher chances of having more anaemia-related blood tests being performed. This resulted in finding significantly more causes of anaemia in the pilot group compared to the control group with respect to iron deficiency (resp. 31.3% vs 14.5%), possible iron deficiency (resp. 11.4% vs 2.8%), iron deficiency in acute phase (2.6% vs 0.5%), chronic disease/infection/inflammation (23.5% vs 1.9%), vitamin B12 deficiency (4.5% vs 1.9%), possible vitamin B12 deficiency (16.8% vs 8.7%), folate deficiency (3.3% vs 0.9%) and possible bone marrow disorder (3.8% vs 0.0%); p < 0.05. CONCLUSIONS: This study suggests that an automated-algorithm support can effectively aid in the diagnostic work-up of anaemia in primary care to find more causes of anaemia.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

The Glycolytic Gatekeeper PDK1 defines different metabolic states between genetically distinct subtypes of human acute myeloid leukemia.

Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1low AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1high AMLs however are OXPHOSlow, wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment.

CombiFlow: combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones.

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can coexist within patients. Differences in signaling and drug sensitivity of such subclones complicate treatment and warrant tools to identify them and track disease progression. We previously identified >50 AML-specific plasma membrane (PM) proteins, and 7 of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25, and CD123) were implemented in routine diagnostics in patients with AML (n = 256) and myelodysplastic syndrome (n = 33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a principal component-based analysis to identify distinctive marker expression profiles and to generate single-cell t-distributed stochastic neighbor embedding landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival, supporting a role for these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples. Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and understanding of clonal heterogeneity in AML.

The IL1-IL1RAP axis plays an important role in the inflammatory leukemic niche that favors acute myeloid leukemia proliferation over normal hematopoiesis

Upregulation of the plasma membrane receptor IL1RAP in Acute Myeloid Leukemia (AML) has been reported but its role in the context of the leukemic bone marrow niche is unclear. Here, we studied the signaling events downstream of IL1RAP in relation to leukemogenesis and normal hematopoiesis. High IL1RAP expression was associated with a leukemic GMP-like state, and knockdown of IL1RAP in AML reduced colony-forming capacity. Stimulation with IL1ß resulted in the induction of multiple chemokines and an inflammatory secretome via the p38 MAPK and NFκB signaling pathways in IL1RAP-expressing AML cells, but IL1ß-induced signaling was dispensable for AML cell proliferation and NFκB-driven survival. IL1RAP was also expressed in stromal cells where IL1ß induced expression of inflammatory chemokines and cytokines as well. Intriguingly, the IL1ß-induced inflammatory secretome of IL1RAPexpressing AML cells grown on a stromal layer of mesenchymal stem cells affected normal hematopoiesis including hematopoietic stem/progenitor cells while AML cell proliferation was not affected. The addition of Anakinra, an FDA-approved IL1 receptor antagonist, could reverse this effect. Therefore, blocking the IL1-IL1RAP signaling axis might be a good therapeutic approach to reduce inflammation in the bone marrow niche and thereby promote normal hematopoietic recovery over AML proliferation after chemotherapy.

Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock.

Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.

Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones.

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models.

Recently, NOD-SCID IL2Rγ-/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34+ cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34+ cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis.

Establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches.

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.

Dynamics in N-terminal pro-brain natriuretic peptide concentration in patients with non-ST-elevation acute coronary syndrome.

BACKGROUND: Although there is growing evidence that N-terminal pro-brain natriuretic peptide (NT-proBNP) can be used as a powerful tool in risk prediction in patients with non-ST-elevation acute coronary syndrome (NSTEACS), the dynamic variation of serum concentrations in time is poorly understood. To gain insight into the dynamics of NT-proBNP, a study was performed using serial serum samples in patients admitted with NSTEACS. METHODS: A total of 24 patients admitted with NSTEACS was included in this study. Serial samples were taken at baseline, 8 hours, 16 hours, 24 hours, and 36 hours after admittance. RESULTS: A highly dynamic pattern in serial measurements of NT-proBNP was observed. Although an increase in NT-proBNP serum levels already existed 8 hours after admittance, it did not reach significance as compared with baseline. The samples obtained 16, 24, and 36 hours after admission were all significantly increased as compared with the values at admission (P < .01), generally leading to a > 2-fold increase with peak values at 16 to 24 hours after admittance. Furthermore, considerable differences in NT-proBNP concentrations between patients were observed. CONCLUSIONS: It was shown that NT-proBNP is a highly dynamic cardiac peptide. Strategic sampling at 16 to 24 hours after admittance could prove representative regarding the assessment of risk prediction and subsequent clinical decision making.