Single-cell proteo-genomic reference maps of the hematopoietic system enable the purification and massive profiling of precisely defined cell states.

Single-cell genomics technology has transformed our understanding of complex cellular systems. However, excessive cost and a lack of strategies for the purification of newly identified cell types impede their functional characterization and large-scale profiling. Here, we have generated high-content single-cell proteo-genomic reference maps of human blood and bone marrow that quantitatively link the expression of up to 197 surface markers to cellular identities and biological processes across all main hematopoietic cell types in healthy aging and leukemia. These reference maps enable the automatic design of cost-effective high-throughput cytometry schemes that outperform state-of-the-art approaches, accurately reflect complex topologies of cellular systems and permit the purification of precisely defined cell states. The systematic integration of cytometry and proteo-genomic data enables the functional capacities of precisely mapped cell states to be measured at the single-cell level. Our study serves as an accessible resource and paves the way for a data-driven era in cytometry.

Treatment with the apoptosis inhibitor Asunercept reduces clone sizes in patients with lower risk Myelodysplastic Neoplasms.

In low-risk Myelodysplastic Neoplasms (MDS), increased activity of apoptosis-promoting factors such as tumor necrosis factor (TNFα) and pro-apoptotic Fas ligand (CD95L) have been described as possible pathomechanisms leading to impaired erythropoiesis. Asunercept (APG101) is a novel therapeutic fusion protein blocking CD95, which has previously shown partial efficacy in reducing transfusion requirement in a clinical phase I trial for low-risk MDS patients (NCT01736436; 2012-11-26). In the current study we aimed to evaluate the effect of Asunercept therapy on the clonal bone marrow composition to identify potential biomarkers to predict response. Bone marrow samples of n = 12 low-risk MDS patients from the above referenced clinical trial were analyzed by serial deep whole exome sequencing in a total of n = 58 time points. We could distinguish a mean of 3.5 molecularly defined subclones per patient (range 2-6). We observed a molecular response defined as reductions of dominant clone sizes by a variant allele frequency (VAF) decrease of at least 10% (mean 20%, range: 10.5-39.2%) in dependency of Asunercept treatment in 9 of 12 (75%) patients. Most of this decline in clonal populations was observed after completion of 12 weeks treatment. Particularly early and pronounced reductions of clone sizes were found in subclones driven by mutations in genes involved in regulation of methylation (n = 1 DNMT3A, n = 1 IDH2, n = 1 TET2). Our results suggest that APG101 could be efficacious in reducing clone sizes of mutated hematopoietic cells in the bone marrow of Myelodysplastic Neoplasms, which warrants further investigation.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

In Silico Pan-Cancer Analysis Reveals Prognostic Role of the Erythroferrone (ERFE) Gene in Human Malignancies.

The erythroferrone gene (ERFE), also termed CTRP15, belongs to the C1q tumor necrosis factor-related protein (CTRP) family. Despite multiple reports about the involvement of CTRPs in cancer, the role of ERFE in cancer progression is largely unknown. We previously found that ERFE was upregulated in erythroid progenitors in myelodysplastic syndromes and strongly predicted overall survival. To understand the potential molecular interactions and identify cues for further functional investigation and the prognostic impact of ERFE in other malignancies, we performed a pan-cancer in silico analysis utilizing the Cancer Genome Atlas datasets. Our analysis shows that the ERFE mRNA is significantly overexpressed in 22 tumors and affects the prognosis in 11 cancer types. In certain tumors such as breast cancer and adrenocortical carcinoma, ERFE overexpression has been associated with the presence of oncogenic mutations and a higher tumor mutational burden. The expression of ERFE is co-regulated with the factors and pathways involved in cancer progression and metastasis, including activated pathways of the cell cycle, extracellular matrix/tumor microenvironment, G protein-coupled receptor, NOTCH, WNT, and PI3 kinase-AKT. Moreover, ERFE expression influences intratumoral immune cell infiltration. Conclusively, ERFE is aberrantly expressed in pan-cancer and can potentially function as a prognostic biomarker based on its putative functions during tumorigenesis and tumor development.

High erythroferrone expression in CD71+ erythroid progenitors predicts superior survival in myelodysplastic syndromes.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Genome-wide DNA methylation analysis pre- and post-lenalidomide treatment in patients with myelodysplastic syndrome with isolated deletion (5q).

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation.

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ acute lymphoblastic leukemia (ALL) treated with tyrosine kinase inhibitors (TKIs) and allogeneic stem cell transplantation (aSCT). Ninety-seven Ph+ ALL patients (median age 41 years; range 18-64 years) within the prospective multicenter German Multicenter ALL Study Group studies 06/99 (n = 8) and 07/2003 (n = 89) were analyzed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with single nucleotide polymorphism arrays and validated by multiplex ligation-dependent probe amplification. The frequencies of recurrently deleted genes were: IKZF1, 76%; CDKN2A/2B, 45%; PAX5, 43%; BTG1, 18%; EBF1, 13%; ETV6, 5%; RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (P = .023), disease-free survival (P = .012), and remission duration (P = .036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase 1 and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT.

Deep sequencing of bone marrow microenvironments of patients with del(5q) myelodysplastic syndrome reveals imprints of antigenic selection as well as generation of novel T-cell clusters as a response pattern to lenalidomide.

In myelodysplastic syndromes with a partial deletion of the long arm of chromosome 5, del(5q), lenalidomide is believed to reverse anergic T-cell immunity in the bone marrow resulting in suppression of the del(5q) clone. In this study we used next-generation sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements in bone marrow-residing and peripheral blood-circulating lymphocytes of patients with del(5q) myelodysplastic syndromes to assess the immune architecture and track adaptive immune responses during treatment with lenalidomide. The baseline bone marrow B-cell space in patients was comparable to that of age-matched healthy controls in terms of gene usage and IGH clonality, but showed a higher percentage of hypermutated IGH sequences, indicating an expanded number of antigen-experienced B lineage cells. Bone marrow B lineage clonality decreased significantly and hypermutated IGH clones normalized upon lenalidomide treatment, well in line with the proliferative effect on healthy antigen-inexperienced B-cell precursors previously described for this drug. The T-cell space in bone marrow of patients with del(5q) myelodysplastic syndromes showed higher TRB clonality compared to that of healthy controls. Upon lenalidomide treatment, myelodysplastic syndrome-specific T-cell clusters with low to medium spontaneous generation probabilities emerged; these clusters were shared across patients, indicating a common antigen-driven T-cell response pattern. Hence, we observed B lineage diversification and generation of new, antigen-dependent T-cell clusters, compatible with a model of adaptive immunity induced against the del(5q) clone by lenalidomide. Overall, this supports the concept that lenalidomide not only alters the functional T-cell state, but also the composition of the T- and B-cell repertoires in del(5q) myelodysplastic syndromes.

Mutational hierarchies in myelodysplastic syndromes dynamically adapt and evolve upon therapy response and failure.

Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.

Frontline therapy of acute promyelocytic leukemia: Randomized comparison of ATRA and intensified chemotherapy versus ATRA and anthracyclines.

OBJECTIVES: Randomized comparison of two treatment strategies in frontline therapy of acute promyelocytic leukemia (APL): all-trans retinoic acid (ATRA) and double induction intensified by high-dose cytosine arabinoside (HD ara-C) (German AMLCG) and therapy with ATRA and anthracyclines (Spanish PETHEMA, LPA99). PATIENTS AND RESULTS: Eighty of 87 adult patients with genetically confirmed APL of all risk groups were eligible. The outcome of both arms was similar: AMLCG vs PETHEMA: hematological complete remission 87% vs 83%, early death 13% vs 17% (P = .76), overall survival, event-free survival, leukemia-free survival, cumulative incidence of relapse at 6 years 75% vs 78% (P = .92); 75% vs 68% (P = .29); 86% vs 81% (P = .28); and 0% vs 12% (P = .04, no relapse vs four relapses), respectively. The median time to achieve molecular remission (RT-PCR negativity of PML-RARA) was 60 days in both arms (P = .12). The AMLCG regimen was associated with a longer duration of neutropenia (P = .02) and a higher rate of WHO grade ≥3 infections. CONCLUSIONS: The small number of patients limits the reliability of conclusions. With these restrictions, the outcomes of both approaches were similar and show the limitations of ATRA and chemotherapy. The HD ara-C-containing regimen was associated with a lower relapse rate in high-risk APL.

Accurate quantification of chromosomal lesions via short tandem repeat analysis using minimal amounts of DNA.

BACKGROUND: Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings. METHODS: For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers. RESULTS: Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77-0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring. CONCLUSIONS: In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

Concomitant MDS with isolated 5q deletion and MGUS: case report and review of molecular aspects.

Patients with monoclonal gammopathy of undetermined significance (MGUS) have a higher risk for the development of concomitant primary cancers such as multiple myeloma (MM) and myelodysplastic syndrome (MDS). We report the case of patient initially suffering from MGUS of the IgG lambda subtype for more than 10 yr, which evolved to MM and MDS with deletion (5q) with severe pancytopenia. Due to pancytopenia, he received dose-reduced treatment with lenalidomide and dexamethasone. He achieved an ongoing transfusion independency after about 1 month of treatment. Bone marrow taken 14 months after start of treatment showed a complete cytogenetic response of the del(5q) clone and a plasma cell infiltration below 5%. In contrast to the development of MM in MGUS patients, the subsequent occurrence of MDS after diagnosis of MGUS is infrequent. Moreover, the biological association of MDS with MGUS is not sufficiently understood, but the non-treatment-related occurrence supports the pathogenetic role of pre-existing alterations of stem cells. Here, we summarize data on concomitant MDS and MGUS/MM with particular emphasis on molecular aspects.

Frequent pathway mutations of splicing machinery in myelodysplasia.

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3'-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

A comparison of two differing doses of promethazine for the treatment of postoperative nausea and vomiting.

PURPOSE: To compare the use of promethazine 6.25 mg intravenous (IV) (experimental group) with promethazine 12.5 mg IV (control group) among adult ambulatory surgery patients to control established postoperative nausea or vomiting (PONV). DESIGN/METHODS: In a double-blind, randomized controlled trial (n = 120), 59 subjects received promethazine 6.25 mg and 61 subjects received promethazine 12.5 mg to treat PONV. Study doses were administered postoperatively if the subject reported/exhibited nausea and/or vomiting. Outcomes for experimental and control groups were compared on the basis of relief of PONV and sedation levels. FINDINGS: Ninety-seven percent of subjects reported total relief of nausea with a single administration of promethazine at either dose. Sedation levels differed between groups at 30 minutes post-medication administration and at the time of discharge to home. CONCLUSIONS: Promethazine 6.25 mg is as effective in controlling PONV as promethazine 12.5 mg, while resulting in less sedation.

Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay.

BACKGROUND: Investigation of X-chromosome inactivation patterns (XCIP) by determination of differential CpG-methylation has been widely applied for investigation of female cell clonality. Using this approach the clonal origin of various tumours has been corroborated. Controversially, strong age-related increase of peripheral blood (PB) cell clonality in haematologically healthy female subjects was reported. Recently, transcriptional XCIP ratio analysis challenged these results and questioned the suitability of methylation based clonality assays. METHODS: To reinvestigate XCIP-skewing in CD34, low-density mononuclear bone marrow (BM) as well as PB cells from healthy female subjects and patients with myelodysplastic syndromes (MDS), we established a transcriptional assay using pyrosequencing technique for quantification of single nucleotide polymorphism allele frequencies, representative for XCIP ratios. RESULTS: Our assay provides high sensitivity for XCIP ratio assessment as determined by standard curves, reproducibility, inter-marker correlation as well as correlation with the DNA-methylation based human androgen receptor (HUMARA) assay. Notably, in agreement with most studies investigating this issue, significant age-related increase of XCIP skewing in PB cells from healthy elderly female subjects was confirmed. Moreover, XCIP ratio analysis suggests even stronger clonal manifestation in BM and CD34 cells. In MDS, XCIP skewing levels were distinctively elevated as compared with controls of similar age and higher degrees were associated with poor clinical outcome. CONCLUSIONS: Transcriptional clonal profiling via pyrosequencing allows accurate assessment of XCIP ratios, confirms the validity of the DNA-methylation based HUMARA assay and reveals important insights into ageing healthy and myelodysplastic haematopoiesis.

Understanding iron homeostasis in MDS: the role of erythroferrone.

Myelodysplastic neoplasms (MDS) are a heterogenous group of clonal stem cell disorders characterized by dysplasia and cytopenia in one or more cell lineages. Anemia is a very common symptom that is often treated with blood transfusions and/or erythropoiesis stimulating factors. Iron overload results from a combination of these factors together with the disease-associated ineffective erythropoiesis, that is seen especially in MDS cases with SF3B1 mutations. A growing body of research has shown that erythroferrone is an important regulator of hepcidin, the master regulator of systemic iron homeostasis. Consequently, it is of interest to understand how this molecule contributes to regulating the iron balance in MDS patients. This short review evaluates our current understanding of erythroferrone in general, but more specifically in MDS and seeks to place in context how the current knowledge could be utilized for prognostication and therapy.

Cell-type-specific consequences of mosaic structural variants in hematopoietic stem and progenitor cells.

The functional impact and cellular context of mosaic structural variants (mSVs) in normal tissues is understudied. Utilizing Strand-seq, we sequenced 1,133 single-cell genomes from 19 human donors of increasing age, and discovered the heterogeneous mSV landscapes of hematopoietic stem and progenitor cells. While mSVs are continuously acquired throughout life, expanded subclones in our cohort are confined to individuals >60. Cells already harboring mSVs are more likely to acquire additional somatic structural variants, including megabase-scale segmental aneuploidies. Capitalizing on comprehensive single-cell micrococcal nuclease digestion with sequencing reference data, we conducted high-resolution cell-typing for eight hematopoietic stem and progenitor cells. Clonally expanded mSVs disrupt normal cellular function by dysregulating diverse cellular pathways, and enriching for myeloid progenitors. Our findings underscore the contribution of mSVs to the cellular and molecular phenotypes associated with the aging hematopoietic system, and establish a foundation for deciphering the molecular links between mSVs, aging and disease susceptibility in normal tissues.

High expression of the Ets-related gene (ERG) is an independent prognostic marker for relapse-free survival in patients with acute promyelocytic leukemia.

In acute promyelocytic leukemia (APL), relapse occurs in about 15 % of cases and is a major cause for death. Molecular markers identifying patients at high risk for relapse are not well established. High expression of the transcription factor Ets-related gene (ERG) is associated with inferior overall survival (OS) and disease-free survival in different types of hematologic malignancies. There are no data available about the impact of ERG expression in APL. ERG expression levels were analyzed in bone marrow samples of 86 APL patients at initial diagnosis. High ERG expression was significantly associated with an inferior OS in patients who had reached first complete remission. It was also significantly correlated with inferior relapse-free survival (RFS) and time to relapse (i.e., relapse-free interval, RFI). In multivariate analysis, high ERG expression had an independent negative impact on RFS and RFI. High ERG expression was significantly associated with inferior OS, RFS, and RFI. Moreover, in multivariate analysis, it maintained its value as an independent negative prognostic factor with regard to RFS and RFI. Therefore, ERG expression might serve as a molecular marker for risk stratification in APL and might identify patients who could benefit from intensified treatment regimens.