Enfortumab Vedotin in Metastatic Urothelial Carcinoma: Survival and Safety in a European Multicenter Real-world Patient Cohort.

Background: Treatment options for patients with urothelial cancer (UC) refractory to platinum and immunotherapy are limited and survival is short. Enfortumab vedotin (EV) is a monoclonal anti-NECTIN4 antibody conjugated to monomethyl auristatin. It was recently approved because of superior survival in comparison to standard-of-care (SOC) chemotherapy. Real-world patients, however, often have worse characteristics than patients included in clinical trials. Objective: To analyze the efficacy and safety of EV in a cohort of real-world patients. Design setting and participants: Retrospective data were collected from 23 hospitals and private practices for patients with metastatic and previously treated UC who received EV either when reimbursed by their insurance company before European Medicines Agency (EMA) approval, within a compassionate use program, or as SOC treatment after EMA approval. Imaging and therapy management were in accordance with local standards. Outcome measurements and statistical analysis: Adverse events (AEs) were reported according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 criteria. Objective responses were evaluated according to Response Evaluation Criteria in Solid Tumors version 1.1. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results and limitations: The median age for the 125 eligible patients was 66 yr (range 31-89). The Eastern Cooperative Oncology Group performance status (ECOG PS) was 0-1 for 76.0%, 2-4 for 13.6%, and unknown for 10.4% of patients. EV was administered in the fourth or later line for 44.8% of patients. The overall response rate was 41.6% (partial response 39.2%, complete response 2.4%). Median OS was 10.0 months (mo) (95% confidence interval 7.20-12.80) and median PFS was 5.0 mo (95% confidence interval 4.34-5.67). For patients with ECOG PS of 0-1, median OS was 14 mo. Any-grade AEs were observed in 67.2% and CTCAE grade ≥3 AEs in 30.4%. The most common AEs were peripheral sensory neuropathy and skin toxicity. Three fatal events (pneumonia, pneumonitis) occurred. Limitations include the retrospective design and short follow-up. Conclusions: Administration of EV for real-world patients was feasible with an acceptable toxicity profile. No new safety signals were reported. Antitumor activity in our cohort was comparable to data previously reported for trials. In summary, our results support the use of EV in patients with metastatic UC. Patient summary: Enfortumab vedotin is a medication that improved the survival of patients with bladder cancer in comparison to standard chemotherapy in clinical trials. However, patients included in clinical trials are highly selected and results for toxicities and improvements in survival do not always transfer to the real-world setting. We analyzed data for 125 patients who were treated with enfortumab vedotin. Our results are comparable to the outcomes from clinical trials regarding the safety and efficacy of this treatment.

Patients with indolent lymphomas are at high risk of infections: experience from a German outpatient clinic.

BACKGROUND: Patients with indolent B-cell non-Hodgkin lymphomas (B-NHLs) have an increased risk of infections which is caused by pathomechanisms of the diseases itself but also as a result of anti-tumor therapy. Especially the effects of anti-CD20 antibodies are well understood as these lead to decreased antibody production. Most studies regarding immunodeficiency in B-NHLs were conducted with multiple myeloma and chronic lymphocytic leukemia patients. As these studies not always represent the general population we collected and analyzed real world data from patients with indolent lymphomas and a control group (CG). RESULTS: Patients with B-NHLs undergoing therapy or who were regularly monitored in a watch and wait approach had, over the time of one year, an increased rate of infections compared to the CG of 145 healthy volunteers (mean: 11.66 vs. 7.13 infections per 1000 days). Consistent with this finding B-NHL patients received more antibiotic treatment (mean: 11.17 vs. 6.27 days) and were more often hospitalized than persons from the CG (mean: 5.19 vs. 0.99 days per 1000 days). Lymphoma patients without immunodeficiency had a lower infection rate than patients with non-symptomatic and symptomatic immunodeficiency (mean: 10.91 vs. 12.07 and 12.36 per 1000 days). The number of infections differed statistically significant for the subgroups and CG (7.13 per 1000 days). Patients with symptomatic immunodeficiency were mostly treated with regular immunoglobulin substitutions and infection rates were comparable to those of patients with asymptomatic immunodeficiency. CONCLUSIONS: Our data suggest the use of an approach with regular immune monitoring including the measurement of immunoglobulin levels and regular appointments for clinical assessment of all indolent lymphoma patients in order to identify patients with increased risk of infections. It also raises the question if patients with immunodeficiency should be treated more often with regular immunoglobulin substitution, but so far more studies are necessary to answer this question.

Curricular course for medical students at a hematology and oncology specialty practice, 2010-2022.

Aim: For several years now, medical students have also been taught general practice at academic medical teaching practices. Specialty practices have not yet been included in the curricular education. Since 1998, we have conducted a block seminar in hematology twice per semester for eighth-semester medical students. This block seminar was offered from 1998-2001 to students at the Philipps University in Marburg and since 2001 to students at the Johannes Gutenberg University in Mainz. Since 2010 our block seminar has been part of the curriculum at the Johannes Gutenberg University. Method: Standardized course evaluation by students who had attended our block seminar between January 2010 and March 2022. Courses that were held virtually due to corona were not included in the analysis. The questionnaire used to evaluate courses in the medical degree program at the Johannes Gutenberg University served as the evaluation instrument. Results: Since 1998 more than 1,000 students have attended our seminar. The systematic evaluation of the course by 500 students who participated in the curricular, classroom-based seminar sessions since 2010 shows that the highest ratings possible are given for practical relevance, learning atmosphere, teaching and effectiveness. Conclusion: High quality in teaching curricular courses to medical students at a specialty practice is possible. Insights into the possibilities connected with working in the outpatient setting at a medical practice broadens students' experience. This teaching format facilitates external university instructors in terms of teaching and, at the same time, relieves the university in terms of staff and financial budget.

Outcome of relapsed or refractory acute B-lymphoblastic leukemia patients and BCR-ABL-positive blast cell crisis of B-lymphoid lineage with extramedullary disease receiving inotuzumab ozogamicin.

Acute lymphoblastic leukemia (ALL) can relapse in the extramedullary compartment, with or without medullary involvement. Response to treatment may be individual. We evaluated response to inotuzumab ozogamicin in 31 patients with relapsed/refractory B-ALL with extramedullary disease. Median age was 31 years (range, 19-81). All patients were heavily pretreated, including allogeneic hematopoietic stem cell transplantation (HSCT; n=18). Overall response rate after two cycles of inotuzumab ozogamicin was 84% (complete remission, 55%; partial remission, 29%; resistant disease, 13%; early death, 3%). The median follow-up was 29 months and median overall survival was 12.8 months. One-year and 2-year overall survival rates were 53% (95% CI: 37-76%) and 18% (95% CI: 8-43%), respectively. Age had no impact on overall survival when assessed as a continuous variable or dichotomized at 60 years. Twelve patients proceeded to allogeneic HSCT (complete remission, n=6; partial remission, n=3; resistant disease, n=3). Prior to allogeneic HSCT, eight patients received two or fewer cycles and four patients received three or four cycles of inotuzumab ozogamicin. Sinusoidal obstruction syndrome was reported in three patients, including one after transplantation. Allogeneic HSCT, evaluated as a time-dependent variable, had no impact on overall survival. Inotuzumab ozogamicin seems to be effective as a debulking strategy in relapsed/refractory ALL with extramedullary disease. However, inotuzumab ozogamicin followed by allogeneic HSCT seems not to be effective in maintaining long-term disease control.

Do Privacy Concerns About Social Robots Affect Use Intentions? Evidence From an Experimental Vignette Study.

While the privacy implications of social robots have been increasingly discussed and privacy-sensitive robotics is becoming a research field within human-robot interaction, little empirical research has investigated privacy concerns about robots and the effect they have on behavioral intentions. To address this gap, we present the results of an experimental vignette study that includes antecedents from the privacy, robotics, technology adoption, and trust literature. Using linear regression analysis, with the privacy-invasiveness of a fictional but realistic robot as the key manipulation, we show that privacy concerns affect use intention significantly and negatively. Compared with earlier work done through a survey, where we found a robot privacy paradox, the experimental vignette approach allows for a more realistic and tangible assessment of respondents' concerns and behavioral intentions, showing how potential robot users take into account privacy as consideration for future behavior. We contextualize our findings within broader debates on privacy and data protection with smart technologies.

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics.

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

The JmjC-domain protein NO66/RIOX-1 affects the balance between proliferation and maturation in acute myeloid leukemia.

As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34+/CD38- phenotype and is regarded as a "stem cell-like" model system. Similarly, NO66 is not detectable in CD34+/CD38- cells purified from healthy donors but is clearly expressed in the more committed CD34+/CD38+ cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.

Analysis of nonleukemic cellular subcompartments reconstructs clonal evolution of acute myeloid leukemia and identifies therapy-resistant preleukemic clones.

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (<1.9% ALDH+ cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure.

Towards Transparency by Design for Artificial Intelligence.

In this article, we develop the concept of Transparency by Design that serves as practical guidance in helping promote the beneficial functions of transparency while mitigating its challenges in automated-decision making (ADM) environments. With the rise of artificial intelligence (AI) and the ability of AI systems to make automated and self-learned decisions, a call for transparency of how such systems reach decisions has echoed within academic and policy circles. The term transparency, however, relates to multiple concepts, fulfills many functions, and holds different promises that struggle to be realized in concrete applications. Indeed, the complexity of transparency for ADM shows tension between transparency as a normative ideal and its translation to practical application. To address this tension, we first conduct a review of transparency, analyzing its challenges and limitations concerning automated decision-making practices. We then look at the lessons learned from the development of Privacy by Design, as a basis for developing the Transparency by Design principles. Finally, we propose a set of nine principles to cover relevant contextual, technical, informational, and stakeholder-sensitive considerations. Transparency by Design is a model that helps organizations design transparent AI systems, by integrating these principles in a step-by-step manner and as an ex-ante value, not as an afterthought.

Mathematical Modeling Provides Evidence for Niche Competition in Human AML and Serves as a Tool to Improve Risk Stratification.

Acute myeloid leukemia (AML) is a stem cell-driven malignant disease. There is evidence that leukemic stem cells (LSC) interact with stem cell niches and outcompete hematopoietic stem cells (HSC). The impact of this interaction on the clinical course of the disease remains poorly understood. We developed and validated a mathematical model of stem cell competition in the human HSC niche. Model simulations predicted how processes in the stem cell niche affect the speed of disease progression. Combining the mathematical model with data of individual patients, we quantified the selective pressure LSCs exert on HSCs and demonstrated the model's prognostic significance. A novel model-based risk-stratification approach allowed extraction of prognostic information from counts of healthy and malignant cells at the time of diagnosis. This model's feasibility was demonstrable based on a cohort of patients with ALDH-rare AML and shows that the model-based risk stratification is an independent predictor of disease-free and overall survival. This proof-of-concept study shows how model-based interpretation of patient data can improve prognostic scoring and contribute to personalized medicine. SIGNIFICANCE: Combining a novel mathematical model of the human hematopoietic stem cell niche with individual patient data enables quantification of properties of leukemic stem cells and improves risk stratification in acute myeloid leukemia.

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.

Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.

The molecular signature of AML with increased ALDH activity suggests a stem cell origin.

Enrichment of leukemic blasts with a stem cell phenotype correlates with poor survival in acute myeloid leukemia (AML). In this context, measurement of the stem cell marker aldehyde-dehydrogenase (ALDH) activity can distinguish poor prognosis cases with increased fractions of ALDH-positive cells (ALDH-numerous AML) and favorable outcome cases with low percentages (ALDH-rare AML). It has been shown that ALDH-numerous AML favor leukemic engraftment in xenotransplantation assays which suggests increased leukemic stem cell (LSC) potential. To test if this reflects an immature cell of origin, comparative gene-expression studies of CD34+ leukemic blasts were performed. This analysis revealed increased expression of LSC and HSC signatures in ALDH-numerous AML, whereas ALDH-rare AML were enriched for a progenitor signature. The enrichment of stemness-associated transcriptional programs suggests that ALDH-numerous AML derive from immature hematopoietic progenitors and offers an explanation for the poor prognosis and therapy resistance of this subgroup which is likely caused by inherited stem cell properties.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Mesenchymal stromal cells contribute to quiescence of therapy-resistant leukemic cells in acute myeloid leukemia.

OBJECTIVE: Persistence of leukemic cells after induction therapy has been shown to correlate with poor survival in acute myeloid leukemia (AML). In this study, we tested if human mesenchymal stromal cells (hMSCs) have protective effects on leukemic cells undergoing chemotherapy. METHODS: Persistent disease was used as marker to identify cases with therapy-resistant leukemic cells in 95 patients with AML. Immunophenotyping, cell cycle, and apoptosis assays were assessed by flow cytometry. AML coculture studies were performed with hMSC of healthy donors. RESULTS: Samples from patients with persistent disease had increased fractions of CD34+ CD38- and quiescent leukemic cells. Comparison of sample series collected at time points of diagnosis and blast persistence showed a relative therapy resistance of quiescent leukemic cells. Consistent with these observations, relapsed disease always displayed higher proportions of quiescent cells compared to samples of first diagnosis suggesting that quiescence is an important therapy escape mechanism of resistant cells. Co-culture studies demonstrated that hMSC protect leukemic cells from the effect of AraC treatment by enriching for quiescent cells, mimicking the effects observed in patients. This effect was even detectable when no direct stromal contact was established. CONCLUSIONS: Our data suggest that hMSC contribute to quiescence and therapy resistance of persistent AML cells.

Reduced hematopoietic stem cell frequency predicts outcome in acute myeloid leukemia.

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse.

Human haematopoietic stem cell lineage commitment is a continuous process.

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.