Acute myeloid leukemia (AML) is a complex disease characterized by diverse molecular pathogenesis. Genetic alterations, including germline and somatic variants in the DEAD box helicase 41 gene ( DDX41) located on chromosome 5 play an increasingly recognized role. Recent reports indicate that 5% of intensively treated adult AML patients harbor DDX41 germline mutations ( DDX41MutGL), and their precise impact remains incompletely understood. These studies suggest that DDX41MutGL may define a distinct biological subgroup, associated with e.g. older age, male gender, low blast, and low white blood cell count (WBC). To further elucidate the role of DDX41 in AML, we performed a retrospective analysis of 906 unselected adult AML patients from the AML Cooperative Group (AMLCG) registry (2015-2022), by targeted sequencing. As DDX41MutGL are typically “null mutations”, leading to reduced DDX41 expression, we additionally investigated whether DDX41 gene expression correlates or resembles the observed germline phenotype. Our analysis encompassed >1000 independent gene expression profiles (GSE37642, GSE14468, and GSE106291) of intensively treated adult AML patients. We identified a sub-cohort with low DDX41 ( DDX41-low) expression and re-analyzed them with next-generation sequencing to detect DDX41MutGL. Additionally, we correlated gene expression data with 198 DNA methylation profiles (from patients who had undergone both analyses) to identify potential epigenetic mechanisms underlying DDX41-low expression. Among the 906 patients (median age 61 years; range 18-98 years), we identified 11 unrelated individuals with suspected DDX41MutGL (VAF > 40%). Notably, the overall frequency of DDX41MutGL in the German unselected AML patient population was merely 1%, considerably lower than previously reported (p<0.0001 in comparison to Duployez et al. 2022). Characteristically, DDX41MutGL patients in this cohort were mostly male (73%), with a median age of 67 years displaying low WBC (average 1,3G/l) and a normal karyotype (80%) at initial diagnosis. Remarkably, our analysis of independent large gene expression cohorts revealed a pattern of phenotypic association in patients with decreased DDX41 expression ( DDX41-low) closely resembling those with DDX41MutGL. These associations were consistent across different datasets and included e.g. older age, low WBC, and low blast count. Furthermore, DDX41-low patients had poor overall survival. The DDX41-low subgroup constituted almost 10% of all AML patients, surpassing the reported frequencies of DDX41MutGL. In an exploratory pilot study, we screened 48 patients exhibiting the lowest DDX41 gene expression by sequencing the coding regions for DDX41 mutations but found none, suggesting alternative mechanisms, such as copy number changes, non-coding alterations or aberrant DNA methylation patterns that may replicate the phenotypic effects associated with DDX41MutGL. To further elucidate this mechanism, we correlated the gene expression of DDX41 with all DNA methylation loci (CpG sites covered by the EPIC array) on chromosome 5 using Spearman correlation analysis (GSE106291). We plotted the correlations against the sorted chromosome length, identifying a peak at a specific location corresponding to the PCDH (Protocadherin) cluster (Figure). Thus, DDX41-low was associated with reduced methylation at the PCDH loci and linked to higher PCDH gene expression. Across several data sets, we confirmed the association between higher PCDH cluster gene expression and low DDX41 expression. In summary, our study reveals that the frequency of DDX41MutGL in an unselected population of German AML patients is considerably lower than previously reported. Additionally, we found that apart from DDX41MutGL, DDX41-low exhibits a comparable clinical profile and is linked to specific methylation and gene expression patterns, with a notable emphasis on the PCDH complex located on chromosome 5. Understanding the functional implications of PCDH genes and their interaction with DDX41 in AML may significantly advance our knowledge of AML pathogenesis. Gaining insights into DDX41 alterations may open avenues for personalized therapeutic approaches and further prognostic stratification for AML patients with distinct molecular characteristics.

The outcome of patients with acute myeloid leukemia (AML) worsens with increasing age. Dichotomization into “younger” and “older” patients is clinically routine and often dictates treatment options. We aimed to delineate whether molecular genetic features and/or outcome measures support assorting patient populations by age, including division into “younger” and “older” groups. We analyzed 2823 adult AML patients enrolled onto frontline chemotherapy-based clinical protocols of two cooperative study groups from USA and Germany who were profiled molecularly via targeted sequencing platforms. Frequencies of gene mutations and cytogenetic findings were depicted in 5-year age increments. Clinical outcomes of 2756 AML patients were analyzed with respect to molecular features, genetic-risk groups and age. Age-associated distributions of gene mutations and cytogenetic abnormalities were similar in both cohorts. There was almost linear shortening of overall survival with increasing age among all patients (P < 0.001) and within 2022 European LeukemiaNet-defined genetic-risk groups, with survival decreasing as age increased (favorable-risk, P < 0.001; intermediate-risk, P < 0.001; adverse-risk, P < 0.001). Although mutational profiles and outcomes of the youngest patients differed from those of older patients, there was no age cut-off identifying “younger” and “older” patients. These findings support more age-associated flexibility for drug approval and trial eligibility.

Microvascular immunothrombotic dysregulation is a critical process in the pathogenesis of severe systemic inflammatory diseases. The mechanisms controlling immunothrombosis in inflamed microvessels, however, remain poorly understood. Here, we report that under systemic inflammatory conditions the matricellular glycoproteinvitronectin (VN) establishes an intravascular scaffold, supporting interactions of aggregating platelets with immune cells and the venular endothelium. Blockade of the VN receptor glycoprotein (GP)IIb/IIIa interfered with this multicellular interplay and effectively prevented microvascular clot formation. In line with these experimental data, particularly VN was found to be enriched in the pulmonary microvasculature of patients with non-infectious (pancreatitis-associated) or infectious (coronavirus disease 2019 (COVID-19)-associated) severe systemic inflammatory responses. Targeting the VN-GPIIb/IIIa axis hence appears as a promising, already feasible strategy to counteract microvascular immunothrombotic dysregulation in systemic inflammatory pathologies.

Growth factor independence 1 (GFI1) is a DNA-binding transcription factor and a key regulator of hematopoiesis. GFI1-36N is a germ line variant, causing a change of serine (S) to asparagine (N) at position 36. We previously reported that the GFI1-36N allele has a prevalence of 10% to 15% among patients with acute myeloid leukemia (AML) and 5% to 7% among healthy Caucasians and promotes the development of this disease. Using a multiomics approach, we show here that GFI1-36N expression is associated with increased frequencies of chromosomal aberrations, mutational burden, and mutational signatures in both murine and human AML and impedes homologous recombination (HR)-directed DNA repair in leukemic cells. GFI1-36N exhibits impaired binding to N-Myc downstream-regulated gene 1 (Ndrg1) regulatory elements, causing decreased NDRG1 levels, which leads to a reduction of O6-methylguanine-DNA-methyltransferase (MGMT) expression levels, as illustrated by both transcriptome and proteome analyses. Targeting MGMT via temozolomide, a DNA alkylating drug, and HR via olaparib, a poly-ADP ribose polymerase 1 inhibitor, caused synthetic lethality in human and murine AML samples expressing GFI1-36N, whereas the effects were insignificant in nonmalignant GFI1-36S or GFI1-36N cells. In addition, mice that received transplantation with 36N leukemic cells treated with a combination of temozolomide and olaparib had significantly longer AML-free vival than mice that received transplantation with GFI1-36S leukemic cells. This suggests that reduced expression leaves GFI1-36N leukemic cells particularly vulnerable to DNA damage initiating chemotherapeutics. data provide critical insights into novel options to treat patients with AML carrying the GFI1-36N variant.

Background: AML is a disease affecting predominantly older patients (pts), but does occur across the entire age spectrum; younger adults [age <60 years (y)] have better outcomes. Using 2 large datasets (from Germany and the US), we sought to identify whether mutational frequencies, cytogenetic aberrations or outcome measures would demonstrate unique patterns to independently assort populations by age.

Although acute myeloid leukemia (AML) is caused by somatically acquired gene mutations, germline variants can also modulate disease characteristics such as sensitivity to treatment and are therefore associated with patient outcomes. Several single-nucleotide polymorphisms (SNPs) have been iden-tified in the past decade to be significantly associated with patient characteristics such as survival, therapy response, and other outcome variables in AML.(1-3) Furthermore, susceptibility loci are increasingly recognized as being associated with the risk to develop leukemia.(4-6) Although these led to some intriguing hypotheses, SNPs are currently not included in routine risk assessment.7 Guidelines provided by the European LeukaemiaNet (ELN) in 2017 and 2022 improved risk stratification of patients with AML; however, recent publications suggest that further improvement beyond ELN2017 is possible.(7,8) In this context, SNPs present attractive candidates because they can easily be implemented in current routine panel sequencing analysis. Multiple studies have reported associations between germline polymorphisms and disease character-istics in AML.(1,9-14) For example, SNP rs12036333 (G>A, LINC01139;CHRM3) has been associated with inferior overall survival (OS) and relapse-free survival (RFS).(9,10) Furthermore, many studies reported associations of SNPs with clinical characteristics (supplemental Table 1). However, these associations are often derived from small cohorts and have not been systematically validated so far. In addition, to our knowledge, there has not yet been a comprehensive analysis of the prognostic/clinical relevance of common SNPs in genes linked to AML pathogenesis. The aim of our study, therefore, was to validate previously identified SNPs that were associated with AML outcomes in prior publications in a large and uniformly treated patient cohort. As a secondary goal, we investigated the potential prog-nostic role of SNPs in genes commonly affected by somatic mutations in AML, which are included in most routinely used diagnostic panels, because such polymorphisms might allow any easily imple-mentable refinement of risk classifications. Local ethic committees of participating institutions approved all protocols, and patients were treated according to the Declaration of Helsinki.

Supplementary Figure 3, related to main Figure 5.

Mutations in RUNX1 ( RUNX1mut) occur in ~15% of intensively treated AML cases. RUNX1mut have no specific hotspot and various types of alteration are observed. The European LeukemiaNet (ELN) risk stratification assigns adverse prognosis to RUNX1mut if they do not co-occur with favorable-risk genotypes. Considering the biological complexity of RUNX1 it seems implausible that all alterations have similar consequences. Using clinical and genetic variables, we developed a prognostic risk stratification model for ELN adverse-risk RUNX1mut AML patients. We combined data from five groups, totaling 609 patients with intensively treated RUNX1mut AML, to develop the model. Our training set included 448 patients treated on trials of the AML Cooperative Group (AMLCG; Herold et al, Leukemia, 2020; (n=178)), AML Study Group (Gerstung et al, NEJM, 2016; (n=116)) and Study Alliance Leukemia (n=154). Patients from the Munich Leukemia Laboratory (MLL; (n=107)) and of the Alliance group (trials NCT00048958, NCT00899223, NCT00900224; Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org; (n=54)) served as independent validation cohorts. Additionally, 955 patients without RUNX1mut treated on AMLCG trials served as controls. Patients with t(15;17), prior treatment, or RUNX1mut with co-occurring favorable-risk genotypes according to ELN 2017 were excluded. Differences between RUNX1mut patients and controls were investigated using univariate logistic regression. Testing was performed using likelihood ratio tests and adjusted for study group. Univariate analyses were adjusted for multiple testing using the Benjamini-Hochberg procedure. We obtained risk prediction models using multivariate Cox regression. Missing values were imputed using the missForest approach. Model selection was performed using forward selection based on the Bayesian information criterion. Cut-offs were based on the 25 th- 50 th- and 75 th-percentile score values obtained in the training data. Performance was evaluated using Kaplan-Meier curves. For internal validation Harrell's C index was estimated using cross-study validation. For external validation, the final risk prediction models were separately applied to the external datasets. RUNX1mut were more common in older, male patients and sAML (table). White blood cells, lactate dehydrogenase and bone marrow blasts were lower in RUNX1mut patients. Mutations in several myelodysplasia-related genes were enriched in RUNX1mut patients ( ASXL1, BCOR, BCORL1, EZH2, KMT2A, PHF6, and STAG2, SF3B1, SRSF2, and U2AF1), whereas DNMT3A, NPM1 and FLT3 were more frequently altered in controls. A strong association with mutations of the splicing factor complex was identified (49% vs. 13%, p<0.0001). Contradicting previous reports, we found no association with IDH mutations. The risk prediction score we obtained for OS is as follows: 0.03054 x age (y) + 0.74996 x adverse MRC + 0.43779 x FLT3-ITD + 0.00317 x WBC count (10^9/L) - 0.00158 x platelet count (10^9/L) + 0.37401 x NRAS-mutation, where the obtained cut-off values are: <1.592 (low risk); 1.592 to 2.303 (moderate risk) >2.303 (high risk). Binary variables are coded as 0 or 1. Harrell's C index estimated using internal validation was 0.6. Kaplan-Meier curves estimated using the training set suggest strong differences in survival between the risk categories. Median overall survival (OS) was 2.5, 1.0 and 0.6 years for low-, intermediate-, and high-risk. External validation using the Alliance cohort shows similar results (Figure). Results obtained for the MLL cohort show smaller differences. However, we still observe clear separation of risk groups with a significant difference between low- and high-risk groups (adjusted p-value: 0.0266). Scores for relapse-free survival (RFS) as well as for OS and RFS censored for allogeneic transplant show similar results. We analyzed a large collection of intensively treated RUNX1mut AML patients and observed heterogenous outcomes that could be predicted by applying few variables (age, MRC-score, FLT3/NRAS mutation-status, and WBC/platelet count). The OS of RUNX1mut high-risk patients is discouraging, highlighting the unmet need of these patients. In addition, our work demonstrates that ELN risk groups can be further stratified and that integrated approaches using routinely available variables can further advance risk prediction.

Supplementary Methods - revised, plus Supplementary Tables 1-6

Supplementary Figure 1, related to main Figure 1

Supplementary Figure 2, related to main Figure 3

Zusammenfassung Ziel der Studie Dieser Beitrag visualisiert regionale und zeitliche Heterogenität der SARS-CoV-2 assoziierten Mortalität in Bayern und untersucht den Einfluss regionaler Faktoren wie Inzidenzrate, Hospitalisierungen, sozio-ökonomischer Status und Pflegeheime auf die SARS-CoV-2 assoziierte Mortalität. Methodik Die offiziellen bayerischen Meldedaten zu SARS-CoV-2 wurden für drei Altersgruppen (50–64, 65–74,>74 Jahre) zwischen März 2020 und April 2021 betrachtet. Karten mit regionalen standardisierten Mortalitätsraten wurden mit einem Bayesianischen hierarchischen Modell räumlich geglättet. Ergebnisse Das Bild der regionalen Mortalität ist heterogen mit einem steigenden Gradienten nach Nord-Osten. Die Adjustierung nach standardisierten Inzidenzraten, Hospitalisierungen der infizierten Personen und Verfügbarkeit von Heimplätzen für ältere Menschen nivelliert die Heterogenität. Schlussfolgerung Der Nord-Ost Gradient in den bayerischen SARS-CoV-2 spezifischen Sterberaten wird deutlich durch den vergleichbaren Gradienten der regionalen Inzidenzraten erklärt. Andere regionale Faktoren zeigen einen weniger deutlichen Einfluss.

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

OBJECTIVE:The aim of this study was to investigate the influence of regional factors such as incidence rate, hospitalizations, socio-economic status and nursing homes on the regional and temporal heterogeneity of SARS-CoV-2-associated mortality in Bavaria.METHODOLOGY:Official Bavarian SARS-CoV-2 reporting data were considered for three age groups (50-64, 65-74,>74 years) between March 2020 and April 2021. Maps of regional standardized mortality rates were spatially smoothed using a Bayesian hierarchical model.RESULTS:The picture of regional mortality was heterogeneous with an increasing gradient toward the northeast. Adjustment for standardized incidence rates, hospitalizations of infected persons, and availability of care homes for the elderly levelled the heterogeneity.CONCLUSION:The north-east gradient in Bavarian SARS-CoV-2-specific mortality rates is clearly explained by the comparable gradient in regional incidence rates. Other regional factors show a less clear influence.

Expression levels of long non-coding RNA (lncRNA) have been shown to associate with clinical outcome of patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, the frequency and clinical significance of genetic variants in the nucleotide sequences of lncRNA in AML patients is unknown. Herein, we analyzed total RNA sequencing data of 377 younger adults (aged <60 years) with CN-AML, who were comprehensively characterized with regard to clinical outcome. We used available genomic databases and stringent filters to annotate genetic variants unequivocally located in the non-coding transcriptome of AML patients. We detected 981 variants, which are recurrently present in lncRNA that are expressed in leukemic blasts. Among these variants, we identified a cytosine-to-thymidine variant in the lncRNA RP5-1074L1.4 and a cytosine-to-thymidine variant in the lncRNA SNHG15, which independently associated with longer survival of CN-AML patients. The presence of the SNHG15 cytosine-to-thymidine variant was also found to associate with better outcome in an independent dataset of CN-AML patients, despite differences in treatment protocols and RNA sequencing techniques. In order to gain biological insights, we cloned and overexpressed both wild-type and variant versions of the SNHG15 lncRNA. In keeping with its negative prognostic impact, overexpression of the wild-type SNHG15 associated with higher proliferation rate of leukemic blasts when compared with the cytosine-to-thymidine variant. We conclude that recurrent genetic variants of lncRNA that are expressed in the leukemic blasts of CN-AML patients have prognostic and potential biological significance.

Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.

AbstractPrediction of resistant disease at initial diagnosis of acute myeloid leukemia (AML) can be achieved with high accuracy using cytogenetic data and 29 gene expression markers (Predictive Score 29 Medical Research Council; PS29MRC). Our aim was to establish PS29MRC as a clinically usable assay by using the widely implemented NanoString platform and further validate the classifier in a more recently treated patient cohort. Analyses were performed on 351 patients with newly diagnosed AML intensively treated within the German AML Cooperative Group registry. As a continuous variable, PS29MRC performed best in predicting induction failure in comparison with previously published risk models. The classifier was strongly associated with overall survival. We were able to establish a previously defined cutoff that allows classifier dichotomization (PS29MRCdic). PS29MRCdic significantly identified induction failure with 59% sensitivity, 77% specificity, and 72% overall accuracy (odds ratio, 4.81; P = 4.15 × 10−10). PS29MRCdic was able to improve the European Leukemia Network 2017 (ELN-2017) risk classification within every category. The median overall survival with high PS29MRCdic was 1.8 years compared with 4.3 years for low-risk patients. In multivariate analysis including ELN-2017 and clinical and genetic markers, only age and PS29MRCdic were independent predictors of refractory disease. In patients aged ≥60 years, only PS29MRCdic remained as a significant variable. In summary, we confirmed PS29MRC as a valuable classifier to identify high-risk patients with AML. Risk classification can still be refined beyond ELN-2017, and predictive classifiers might facilitate clinical trials focusing on these high-risk patients with AML.

In an effort to identify target genes in acute myeloid leukemia (AML), we compared gene expression profiles between normal and AML cells from various publicly available datasets. We identified CD99, a gene that is up-regulated in AML patients. In 186 patients from The Cancer Genome Atlas AML dataset, CD99 was over-expressed in patients with FLT3-ITD and was down-regulated in patients with TP53 mutations. CD99 is a trans-membrane protein expressed on leukocytes and plays a role in cell adhesion, trans-endothelial migration, and T-cell differentiation. The CD99 gene encodes two isoforms with distinct expression and functional profiles in both normal and malignant tissues. Here we report that, although the CD99 long isoform initially induces an increase in cell proliferation, it also induces higher levels of reactive oxygen species, DNA damage, apoptosis and a subsequent decrease in cell viability. In several leukemia murine models, the CD99 long isoform delayed disease progression and resulted in lower leukemia engraftment in the bone marrow. Furthermore, the CD99 monoclonal antibody reduced cell viability, colony formation, and cell migration, and induced cell differentiation and apoptosis in leukemia cell lines and primary blasts. Mechanistically, CD99 long isoform resulted in transient induction followed by a dramatic decrease in both ERK and SRC phosphorylation. Altogether, our study provides new insights into the role of CD99 isoforms in AML that could potentially be relevant for the preclinical development of CD99 targeted therapy.

AbstractPrevious studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukaemia remain largely unexplored. In this study we characterized mutations inSRSF2, U2AF1andSF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline we accurately detected aberrant splicing in splicing factor mutated samples. Mutated samples were characterized predominantly by decreased junction usage. A large proportion of differentially used junctions were novel. Targets of splicing dysregulation included several genes with a known role in leukaemia. InSRSF2(P95H) mutants we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.