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The revised 2022 European LeukemiaNet (ELN) AML risk stratification system requires validation in large, homogeneously treated cohorts. We studied 1118 newly diagnosed AML patients (median age, 58 years; range, 18–86 years) who received cytarabine-based induction chemotherapy between 1999 and 2012 and compared ELN-2022 to the previous ELN-2017 risk classification. Key findings were validated in a cohort of 1160 mostly younger patients. ELN-2022 reclassified 15% of patients, 3% into more favorable, and 12% into more adverse risk groups. This was mainly driven by patients reclassified from intermediate- to adverse-risk based on additional myelodysplasia-related mutations being included as adverse-risk markers. These patients (n = 79) had significantly better outcomes than patients with other adverse-risk genotypes (5-year OS, 26% vs. 12%) and resembled the remaining intermediate-risk group. Overall, time-dependent ROC curves and Harrel’s C-index controlling for age, sex, and AML type (de novo vs. sAML/tAML) show slightly worse prognostic discrimination of ELN-2022 compared to ELN-2017 for OS. Further refinement of ELN-2022 without including additional genetic markers is possible, in particular by recognizing TP53-mutated patients with complex karyotypes as “very adverse”. In summary, the ELN-2022 risk classification identifies a larger group of adverse-risk patients at the cost of slightly reduced prognostic accuracy compared to ELN-2017.

Key Points Prediction of induction failure in AML is possible using cytogenetic data and a gene expression–based classifier.Integration of PS29MRC in the clinical routine or trials may be facilitated by gene expression analysis with the NanoString platform., Visual Abstract, Prediction 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.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone–derived cells as healthy experimental controls.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR–based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR–based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Acute myeloid leukemia (AML) with initial hyperleukocytosis is associated with high early mortality and a poor prognosis. The aims of this study were to delineate the underlying molecular landscape in the largest cytogenetic risk group, cytogenetically normal acute myeloid leukemia (CN-AML), and to assess the prognostic relevance of recurrent mutations in the context of hyperleukocytosis and clinical risk factors.The authors performed a targeted sequencing of 49 recurrently mutated genes in 56 patients with newly diagnosed CN-AML and initial hyperleukocytosis of ≥100 G/L treated in the AMLCG99 study. The median number of mutated genes per patient was 5. The most common mutations occurred in FLT3 (73%), NPM1 (75%), and TET2 (45%).The predominant pathways affected by mutations were signaling (84% of patients), epigenetic modifiers (75% of patients), and nuclear transport (NPM1; 75%) of patients. AML with hyperleukocytosis was enriched for molecular subtypes that negatively affected the prognosis, including a high percentage of patients presenting with co-occurring mutations in signaling and epigenetic modifiers such as FLT3 internal tandem duplications and TET2 mutations.Despite these unique molecular features, clinical risk factors, including high white blood count, hemoglobin level, and lactate dehydrogenase level at baseline, remained the predictors for overall survival and relapse-free survival in hyperleukocytotic CN-AML.

Clonal hematopoiesis (CH) is characterized by somatic mutations in blood cells of individuals without hematologic disease. While the mutational landscape of CH in peripheral blood (PB) has been well characterized, detailed analyses addressing its spatial and cellular distribution in the bone marrow (BM) compartment are sparse. We studied CH driver mutations in healthy individuals (n = 261) across different anatomical and cellular compartments. Variant allele frequencies were higher in BM than PB and positively correlated with the number of driver variants, yet remained stable during a median of 12 months of follow-up. In CH carriers undergoing simultaneous bilateral hip replacement, we detected ASXL1-mutant clones in one anatomical location but not the contralateral side, indicating intra-patient spatial heterogeneity. Analyses of lineage involvement in ASXL1-mutated CH showed enriched clonality in BM stem and myeloid progenitor cells, while lymphocytes were particularly involved in individuals carrying the c.1934dupG variant, indicating different ASXL1 mutations may have distinct lineage distribution patterns. Patients with overt myeloid malignancies showed higher mutation numbers and allele frequencies and a shifting mutation landscape, notably characterized by increasing prevalence of DNMT3A codon R882 variants. Collectively, our data provide novel insights into the genetics, evolution, and spatial and lineage-specific BM involvement of CH.

Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are among the most frequent alterations in acute myeloid leukemia (AML) and can be found in ∼20% of patients at diagnosis. Among 4930 patients (median age, 56 years; interquartile range, 45-66) with newly diagnosed, intensively treated AML, we identified IDH1 mutations in 423 (8.6%) and IDH2 mutations in 575 (11.7%). Overall, there were no differences in response rates or survival for patients with mutations in IDH1 or IDH2 compared with patients without mutated IDH1/2. However, distinct clinical and comutational phenotypes of the most common subtypes of IDH1/2 mutations could be associated with differences in outcome. IDH1-R132C was associated with increased age, lower white blood cell (WBC) count, less frequent comutation of NPM1 and FLT3 internal tandem mutation (ITD) as well as with lower rate of complete remission and a trend toward reduced overall survival (OS) compared with other IDH1 mutation variants and wild-type (WT) IDH1/2. In our analysis, IDH2-R172K was associated with significantly lower WBC count, more karyotype abnormalities, and less frequent comutations of NPM1 and/or FLT3-ITD. Among patients within the European LeukemiaNet 2017 intermediate- and adverse-risk groups, relapse-free survival and OS were significantly better for those with IDH2-R172K compared with WT IDH, providing evidence that AML with IDH2-R172K could be a distinct entity with a specific comutation pattern and favorable outcome. In summary, the presented data from a large cohort of patients with IDH1/2 mutated AML indicate novel and clinically relevant findings for the most common IDH mutation subtypes.

Previous studies have demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies. Their clinical characteristics and aberrant splicing patterns have been explored in myelodysplasia, however, their functional consequences in acute myeloid leukaemia are largely unknown. The aim of this study was the comprehensive clinical and functional analysis of mutations in the three most commonly afflicted splicing factor genes: SRSF2 , U2AF1 and SF3B1 . To this end, we examined the prognostic role of splicing factor mutations in two large independent cohorts, encompassing a total of 2678 acute myeloid leukaemia patients treated with intensive chemotherapy. The clinical analysis was complemented by RNA-sequencing of 246 patients to identify targets of splicing dysregulation. Results were validated in an additional RNA-sequencing dataset of 177 patients. Patients with splicing factor mutations show inferior relapse-free and overall survival, however, these mutations do not represent independent prognostic markers. Differential isoform expression analysis revealed a characteristic expression profile for each splicing factor mutation with a strong dysregulation of several isoforms. Furthermore, by establishing a custom differential splice junction usage pipeline we accurately detected aberrant splicing in splicing factor mutated samples. Mutated samples were characterized by predominantly decreased splice junction utilization of a large number of genes. A large proportion of differentially used spliced junctions were novel. Targets of splicing dysregulation included several genes with a known role in acute myeloid leukaemia. In SRSF2 (P95H) mutants we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Taken together, our findings suggest that splicing factor mutations does not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of acute myeloid leukemia have been widely adopted, but have not yet been validated in large cohorts of AML patients. We studied 1116 newly diagnosed AML patients (age range, 18–86 years) who had received induction chemotherapy. Among 771 patients not selected by genetics, the ELN-2017 classification re-assigned 26.5% of patients into a more favorable or, more commonly, a more adverse-risk group compared with the ELN-2010 recommendations. Forty percent of the cohort, and 51% of patients ≥60 years, were classified as adverse-risk by ELN-2017. In 599 patients CEBPA mutations or inv(16) had particularly favorable outcomes, while patients with mutated TP53 and a complex karyotype had especially poor prognosis. DNMT3A mutations associated with inferior OS within each ELN-2017 risk group. Our results validate the prognostic significance of the revised ELN-2017 risk classification in AML patients receiving induction chemotherapy across a broad age range. Further refinement of the ELN-2017 risk classification is possible.

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of FLT3 are frequent mutations associated with unfavorable prognosis. At diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis, providing information about the length but not the position and sequence of the ITD. To overcome this limitation, we performed cDNA-based high-throughput amplicon sequencing (HTAS) in 250 FLT3-ITD positive AML patients, treated on German AML Cooperative Group (AMLCG) trials. FLT3-ITD status determined by routine diagnostics was confirmed by HTAS in 242 out of 250 patients (97%). The total number of ITDs detected by HTAS was higher than in routine diagnostics (n = 312 vs. n = 274). In particular, HTAS detected a higher number of ITDs per patient compared to fragment analysis, indicating higher sensitivity for subclonal ITDs. Patients with more than one ITD according to HTAS had a significantly shorter overall and relapse free survival. There was a close correlation between FLT3-ITD mRNA levels in fragment analysis and variant allele frequency in HTAS. However, the abundance of long ITDs (≥75nt) was underestimated by HTAS, as the size of the ITD affected the mappability of the corresponding sequence reads. In summary, this study demonstrates that HTAS is a feasible approach for FLT3-ITD detection in AML patients, delivering length, position, sequence and mutational burden of this alteration in a single assay with high sensitivity. Our findings provide insights into the clonal architecture of FLT3-ITD positive AML and have clinical implications.

Primary therapy resistance is a major problem in acute myeloid leukemia treatment. We set out to develop a powerful and robust predictor for therapy resistance for intensively treated adult patients. We used two large gene expression data sets (n=856) to develop a predictor of therapy resistance, which was validated in an independent cohort analyzed by RNA sequencing (n=250). In addition to gene expression markers, standard clinical and laboratory variables as well as the mutation status of 68 genes were considered during construction of the model. The final predictor (PS29MRC) consisted of 29 gene expression markers and a cytogenetic risk classification. A continuous predictor is calculated as a weighted linear sum of the individual variables. In addition, a cut off was defined to divide patients into a high-risk and a low-risk group for resistant disease. PS29MRC was highly significant in the validation set, both as a continuous score (OR=2.39, P=8.63·10−9, AUC=0.76) and as a dichotomous classifier (OR=8.03, P=4.29·10−9); accuracy was 77%. In multivariable models, only TP53 mutation, age and PS29MRC (continuous: OR=1.75, P=0.0011; dichotomous: OR=4.44, P=0.00021) were left as significant variables. PS29MRC dominated all models when compared with currently used predictors, and also predicted overall survival independently of established markers. When integrated into the European LeukemiaNet (ELN) 2017 genetic risk stratification, four groups (median survival of 8, 18, 41 months, and not reached) could be defined (P=4.01·10−10). PS29MRC will make it possible to design trials which stratify induction treatment according to the probability of response, and refines the ELN 2017 classification.

Introduction An increasing proportion of patients with Acute Myeloid Leukemia (AML) become long-term survivors. Somatic and psycho-social outcomes are therefore becoming increasingly important, but little is known about the long-term effects of the disease and its treatment. Methods We designed a comprehensive analysis of AML survivorship outcomes including psycho-social well-being and somatic health status and conducted a questionnaire-based study collecting data from AML long term survivors (AML-LTS) and their physicians. This report focuses on overall and health-related quality of life. Somatic, especially cardiovascular, morbidity in AML-LTS are reported separately (Moret et al.). The primary aim of this study was to compare quality of life (QoL, measured by the FACT-G questionnaire) and general and health-related life satisfaction (gLS/hLS, measured by the FLZ-M questionnaire) of AML-LTS with normative data of German adults who were not diagnosed with AML (Holzner et al. 2009; Daig et al. 2009). FLZ-M and FACT-G scores were standardized relative to the normal population mean and standard deviation, stratified by sex and age. These z-scores were then tested against the fixed value 0 (indicating no difference between AML-LTS and the general population) using Mann-Whitney U-tests. Our statistical design incorporated a sequentially rejective testing procedure to maintain the multiple testing significance level at 5%, using a graphical model as described by Bretz et al. (2009). Results 427 former AML patients who had been enrolled in AMLCG trials (AMLCG-1999, AMLCG-2004, AMLCG-2008) or the AMLCG patient registry, participated in this study between 5 and 18.6 years [y] after their initial AML diagnosis (median, 11.3y). Median age of AML-LTS was 61y (range 28y-93y), and 56% were female. Thirty-eight percent of participants had been treated with chemotherapy alone, while 62% received at least one allogeneic stem cell transplant (alloSCT). A relapse occurred in 24% of the participants. Unexpectedly, quality of life and general life satisfaction summary scores were significantly higher in AML-LTS (p Notably, a subgroup of participants (26%) reported poor physical well-being (PWB), indicated by a FACT PWB subscore more than one standard deviation (SD) below the age- and sex-matched general population value (Figure A). This resulted in poor overall QoL (i.e. >1 SD below normal) for 13% of the participants (Figure B). To identify factors potentially associated with poor overall QoL, we constructed a logistic regression model including pre-specified cofactors (age, sex, time since initial diagnosis, relapse and alloSCT) and additional covariables that associated with QoL in univariate analyses (Table C). We found that participants with younger age, male sex, lower educational level, shorter time since diagnosis and a altered financial situation reported significantly lower QoL. No influence was found for other characteristics including treatment (alloSCT vs. no alloSCT), previous relapse, or de novo vs. secondary or therapy-related AML. Discussion Unlike previous studies of AML survivorship, our large cohort included a diverse spectrum of patients regarding age, time since diagnosis, and treatment modalities, which allows for new insight into long-term quality of life. Our study establishes that overall QoL in AML long-term survivors is comparable to the general population. Improvement of QoL continues beyond five years post diagnosis. Importantly, disease- and treatment-related factors, such as prior relapse or status post allogeneic transplantation, are not associated with overall QoL. However, we were able to identify risk factors for worse QoL (younger age, male sex, alteration of the financial situation), delineating a subgroup of patients that may still have a need for targeted psycho-social interventions five or more years after an AML diagnosis. Figure 1 Figure 1. Disclosures Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hiddemann: Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Metzeler: AbbVie: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy; Celgene/BMS: Consultancy, Honoraria, Research Funding.

Introduction: As outcomes of patients with acute myeloid leukemia (AML) have improved over the past decades, the fraction of patients surviving long-term is increasing. Information on long-term somatic and psycho-social health consequences of AML and its treatment is sparse. Previous studies suggested a higher prevalence of cardiovascular diseases in AML survivors, especially those treated with allogeneic stem cell transplantation (alloHSCT). The aim of our study was to perform a multi-dimensional analysis of health outcomes in AML long-term survivors (AML-LTS). This report focuses on somatic, especially cardiovascular, morbidity in AML-LTS. Overall and health-related quality of life are reported separately (Telzerow et al.). Methods: We conducted a cross-sectional study including AML survivors who had been enrolled in clinical trials or the patient registry of the AML-CG study group and were alive ≥5 years after initial diagnosis. Data concerning somatic health status were collected through patient questionnaires, assessment by the patients' physicians, and medical and laboratory reports. An age- and sex-matched control cohort was derived from German population-based health surveys (Robert Koch Institute, DEGS1 survey; n=6013; persons diagnosed with leukemia [n=11] were excluded). Results: 427 AML-LTS, aged 28 to 93 years, participated in this study. Data on somatic health status is available for 355 survivors, 5 to 19 years after their AML diagnosis. Thirty-eight percent of survivors were treated with chemotherapy with or without an autologous transplant (autoHSCT), whereas 62% had undergone alloHSCT. Focusing on cardiovascular diseases and risk factors, we found that that 49% of AML-LTS had hypertension, 33% had hypercholesterolemia, 15% had type 1/2 diabetes, 10% had congestive heart failure (CHF), and 9% had coronary artery disease (Figure A). The mean body-mass index (BMI) of AML-LTS was 26.7, similar to the DEGS1 cohort (mean BMI, 26.8). Next, we compared the prevalence of cardiovascular diseases and risk factors between AML-LTS and the general German population (represented by the DEGS1 sample), using multivariate models adjusting for age and sex (Table B). Compared to persons not diagnosed with leukemia, AML survivors had similar risks of hypertension, coronary heart disease and myocardial infarction. Prevalence of diagnosed hypercholesterolemia was higher in AML-LTS compared to non-AML controls. In addition, AML-LTS had a 2-fold higher risk of having type 1/2 diabetes, and a 3.5-fold increased risk of CHF compared to the general population. To identify factors associated with the increased risks of diabetes and CHF among AML-LTS, we constructed multivariate models incorporating patient- and treatment related covariables (age, sex, BMI, smoking, prior AML relapse, treatment [chemotherapy + autoHSCT vs. alloHSCT], and type of leukemia [de novo versus secondary / therapy-related]). We found an increased risk of CHF for AML-LTS who had had a relapse (OR, 3.16; 95% CI: 1.46 - 6.83; P=0.004) and, in trend, for patients with sAML or tAML (OR 2.19; 95%CI: 0.92 - 5.22, P=0.076). In addition, we found an increased risk of type 1/2 diabetes for AML-LTS who are smokers (OR: 3.43; 95% CI: 1.43 - 8.21; p: 0.006). Disease- or treatment-related factors did not significantly associate with any of the other comorbidities we studied. Conclusion: To the best of our knowledge, this is the largest analysis of somatic health outcomes in AML-LTS. Strengths of our study include the relatively large cohort representing a wide age range, the long follow-up period of 5 to nearly 20 years, and the heterogeneity regarding therapy regimens (chemotherapy + autoHSCT vs. alloHSCT). We found that, compared to the general population, AML-LTS have increased risks for CHF and diabetes, but not for hypertension or coronary artery disease. We identified AML relapse as a risk factor for the development of CHF, suggesting that cumulative chemotherapy exposure might be causally involved. On the other hand, we found no treatment- and disease-related risk factors that might explain the higher prevalence of diabetes in AML-LTS. Notably, AML-LTS who had undergone alloHSCT did not have increased risks of CHF, cardiovascular disease, hypertension or diabetes, compared to survivors treated with chemotherapy only. Our results may guide future recommendations for follow-up and inform personalized treatment decisions. Figure 1 Figure 1. Disclosures Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hiddemann: Janssen: Research Funding; F. Hoffmann-La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding. Metzeler: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; AbbVie: Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Pfizer: Consultancy; Astellas: Honoraria.

Background: Clonal hematopoiesis (CH) describes the presence of genetic alterations and expansion of clonal cell populations in the peripheral blood (PB) of individuals without clinical manifestation of a hematologic malignancy. CH is a common, age-related state. However, individuals carrying CH are at greater risk for hematologic cancer. It has been shown that presence of TP53- and other high-risk mutations, variant allele frequency (VAF) >10% or multiple mutations in CH carriers may confer a higher risk of preleukemic development and transformation to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Nevertheless, further insights into the role of CH in clonal evolution towards leukemia and elaborating features that are predictive of leukemic progression are needed. Aims: We have recently shown that CH is common in individuals undergoing hip replacement surgery (Hecker, Hartmann et al., Blood 2021). Here, we compared mutational spectrum in these CH individuals to MDS and secondary AML (sAML) cohorts. Additionally, we analyzed spatial and lineage distribution of CH and longitudinally monitored CH and MDS clones over time. Methods: Samples from individuals without known hematologic disease undergoing hip replacement surgery (n=288 samples from 261 individuals), patients with MDS (n=92) or sAML (n=123) were screened for variants in 68 leukemia-associated genes using a targeted sequencing approach (VAF cut off, 1%). Follow-up (FU) PB samples were available for 21 individuals with CH and 16 untreated low-risk MDS patients, 6-24 months after screening. n=5 CH bone marrow (BM) samples carrying six ASXL1-mutations were sorted for seven different cell fractions. Results: At screening, variants were detected in 127/261 (49%) healthy individuals, 84/92 (91%) MDS and 117/123 (95%) sAML patients, with median VAFs of 2.7% (ranging from 1-44%), 18.8% (1.1-87%) and 37.1% (1-99%), respectively. Individuals with CH had a median number of 1 variant per individual, whereas median detected variants per patient increased with clonal evolution with 3 variants in the MDS and 4 variants per patient in the sAML cohort. CH, MDS and sAML showed entity-specific mutation profiles (Figure 1A). Most variants in CH affected epigenetic modifiers, while mutations in splicing factors, signaling pathways and transcription factors increased with clonal progression. During FU, untreated low-risk MDS patients more frequently gained additional mutations compared to CH individuals (7/16 vs 2/21, respectively; p=0.024). However, we did not observe significant changes in clone sizes over time. In 17 hip replacement individuals both femoral heads were removed simultaneously, allowing paired analysis of two different hematopoietic sites. CH prevalence in this subgroup was 70.6% (12/17). Ten individuals showed identical mutation patterns in BM obtained from the right and left femoral head, with little differences in VAFs (Table 1). In contrast, two other individuals showed significant differences in variant detection comparing one to the other side: ASXL1-mutations were only present in one hip sample, whereas all the other variants were detectable in both sides (p To further characterize ASXL1 mosaicism across hematopoietic lineages and differentiation stages, we sorted and sequenced n=5 CH BM samples carrying six ASXL1-mutations for seven different hematopoietic cell fractions. In all cases ASXL1-mutations were identified in CD34 +CD38 -CD90 + hematopoietic stem cells (HSC). VAFs of ASXL1-mutations were differentially distributed (p=0.0049, Kruskal Wallis test): detected VAFs were significantly higher in HSC and common myeloid progenitors (CMP) compared to VAFs in T-cells (p=0.045 and p=0.011, respectively; Dunn´s multiple comparison test; Figure 1B). Conclusions: CH, MDS and sAML show characteristic mutation profiles that remained stable over a 6-24-month period. Gains of additional variants and clonal expansion associated with disease progression. Cellular distribution analysis of ASXL1 CH variants revealed characteristic repartition patterns within the hematopoietic differentiation tree. Additionally, differences in variant detection between cellular BM compartments indicates spatial heterogeneity of CH clones warranting further investigation. J.S.H. and L.H. contributed equally. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Goetze: BMS/Celgene: Other: Advisory Board, Research Funding; Abbvie: Other: Advisory Board.

Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

Background: AML is a disease affecting predominantly older patients (pts), but does occur across the entire age spectrum; younger adults [age Methods: We analyzed the molecular profiles of 2,823 adult AML pts enrolled onto clinical frontline protocols of 2 large cooperative study groups from 2 continents [US, Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology (Alliance), n=1743; Germany, AML Cooperative Group [AMLCG], n=1080] between 1986 and 2016. Treatment of all pts included intensive induction therapy, whereas pts enrolled on CALGB/Alliance protocols precluded allogeneic transplantation in 1 st complete remission. Pts in both cohorts were profiled for molecular features via targeted sequencing platforms. Frequencies of mutations genes and selected cytogenetic findings were then calculated in both datasets for the group of pts aged 18-24 y and for older pts by 5-year intervals until the age of 74 y and for pts older than 75 y. We also analyzed survival outcomes of 1,669 AML pts younger than 60 y using the same age intervals up to age 59 y. Results: Our side-by-side analysis shows remarkable congruence of results between German and US pt populations. Selected AML-associated gene mutations (mutation frequency ≥4%) and recurrent cytogenetic abnormalities followed 3 basic distribution patterns across the age spectrum (Fig. 1A): group 1 with increasing frequency with increasing age [ASXL1, BCOR, IDH1/2, RUNX1, SRSF2, TET2, TP53; complex karyotype and cytogenetically normal AML (CN-AML)]; group 2 with decreasing frequency with increasing age (CEBPA, EZH2, FLT3-TKD, GATA2, KIT, KRAS, PTPN11, NRAS, WT1; inv(16), t(8;21) and 11q23/KMT2A rearrangements) and group 3 with non-linear frequency distribution, which included the 3 most common AML-associated gene mutations (NPM1, DNMT3A, FLT3-ITD), SF3B1 and mutations in the cohesin complex genes (RAD21, SMC1A, SMC3, STAG2) (Fig. 1A). Notably, within the first 2 distribution groups, there seem to be no obvious age that could serve as a cut point separating age groups that are markedly different with regard to their molecular patterns. Particularly, this includes an age group that is commonly used for pt cohort definitions such as pts aged 18-39 y referred to as adolescent and young adults (AYA) or even treatment decisions and eligibility (eg, ages 60 or 65 and older for consideration as elderly AML). With respect to pt outcomes, expectedly, there was almost linear shortening of overall survival (OS) as age increased (p Conclusions: To our knowledge, this is the first large scale depiction of mutational patterns in AML inclusive of the entire adult age spectrum. Our international study demonstrates that patterns of individual mutations based on age are remarkably consistent between countries, and defy assortment based on typical age conventions. Given the continuous distribution of either increasing or decreasing frequency of many mutations, there are distinctly different mutational profiles for the youngest pts compared with older pts, however choosing a precise cut-off, such as age 39 for AYA pts or 59 for consideration as "younger AML", does not seem to be supported by our analyses. This observation supports a more personalized approach that also considers molecular subgroups in clinical practice instead of the age rigidity set in many clinical trials. *shared first: M.C.,K.L.; #last: T.H.,AK.E. Figure 1 Figure 1. Disclosures Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hiddemann: F. Hoffmann-La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Mims: Glycomemetics: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Aptevo: Research Funding; Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Xencor: Research Funding; Kartos Pharmaceuticals: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Blum: Celyad Oncology: Research Funding; Forma Therapeutics: Research Funding; Xencor: Research Funding; Nkarta: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie: Honoraria; AmerisourceBergen: Honoraria; Syndax: Honoraria. Larson: Epizyme: Consultancy; Astellas: Consultancy, Research Funding; Gilead: Research Funding; CVS/Caremark: Consultancy; Takeda: Research Funding; Novartis: Research Funding; Rafael Pharmaceuticals: Research Funding; Cellectis: Research Funding. Stone: Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Onconova: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Innate: Consultancy; GlaxoSmithKline: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Boston Pharmaceuticals: Consultancy; Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Arog: Consultancy, Research Funding; Aprea: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Macrogenics: Consultancy. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Metzeler: Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy; Daiichi Sankyo: Honoraria; Astellas: Honoraria; AbbVie: Honoraria; Pfizer: Consultancy; Celgene/BMS: Consultancy, Honoraria, Research Funding. Eisfeld: Karyopharm (spouse): Current Employment.

Background: Mutations in the protein tyrosine phosphatase gene PTPN11 (also known as SHP2) are found in approximately 10% of adult patients with acute myeloid leukemia (AML). A recent study reported that mutated PTPN11 associates with inferior response rates and shorter survival among intensively treated AML patients, independently of the ELN prognostic groups (Alfayez et al., Leukemia 2020). Earlier analyses of the genomic landscape of AML did not uncover a similar prognostic relevance of PTPN11 mutations. Therefore, our aim was to clarify the prognostic relevance of mutated PTPN11 variants in AML patients receiving intensive front-line therapy. Patients and Methods: We studied 1116 AML patients enrolled on two subsequent multicenter phase III trials of the German AML Cooperative Group (AML-CG 1999, NCT00266136; and AML-CG 2008, NCT01382147) who were genetically characterized by amplicon-based targeted next-generation sequencing (Herold et al., Leukemia 2020). All patients had received induction chemotherapy containing cytarabine and daunorubicin or mitoxantrone. Results: We identified 146 PTPN11 mutations in 114 of 1116 patients (10%). Mutations clustered in two hotspot regions (5': codons 52-79; n=108 and 3': codons 491-512, n=38) as previously reported. Associations of PTPN11 mutations with baseline clinical and genetic patient characteristics are shown in Figure A. PTPN11 mutations were most frequent in the European LeukemiaNet (ELN) "favorable" genetic risk group, and associated with higher leukocyte counts. Patients with mutated PTPN11more commonly had mutated NPM1, IDH1 and DNMT3A, and less frequently had FLT3-ITD, IDH2 and TP53 mutations, compared to patients with wild-type PTPN11. With regard to treatment outcomes, the rate of complete remission was similar among patients with mutated and wild-type PTPN11 (65% vs. 59%, P=.25). In univariate analyses, PTPN11-mutated patients had significantly longer relapse-free survival (RFS; 5-year estimate, 55% vs 33% for PTPN11-wild type patients; P=.001; Figure B) and tended to have longer overall survival (OS; 5-year estimate, 43% vs 32%; P=.06; Figure C). However, in multivariable models adjusting for age, sex, leukocyte count, AML type (de novo/sAML/tAML) and ELN-2017 genetic risk group, mutated PTPN11 no longer associated with RFS (hazard ratio [HR], 0.89, 95% confidence interval [CI], 0.63 - 1.27; P=0.53) or OS (HR, 1.03; 95% CI, 0.80 - 1.33; P=.79). Moreover, PTPN11 mutations did not significantly associate with RFS or OS within any of the ELN genetic risk groups. Finally, we detected no significant differences in baseline characteristics or outcomes between patients with PTPN11 mutations affecting the 5' hotspot region (n=82), the 3' hotspot region (n=21), or mutations at both hotspots (n=11). Conclusion: In our cohort of newly diagnosed and intensively treated AML patients, mutations in PTPN11 occurred in 10% and associated with prognostically favorable genetic characteristics such as mutated NPM1 and absence of FLT3-ITD and TP53mutations. Consequently, PTPN11 mutations were most commonly found within the ELN-2017 favorable risk category. While patients with PTPN11 mutations had relatively favorable survival outcomes, multivariable models suggest this observation is confounded by the frequent co-occurrence of known favorable genetic markers. Our data are in disagreement with a recently published study on 880 newly diagnosed patients that found an unfavourable prognostic impact of mutated PTPN11, particularly among the 410 patients who received intensive treatment. Possible explanations for these discrepant results include differences in treatment regimens between the two cohorts, as well as the play of chance when studying a relatively rare gene mutation in medium-sized cohorts. In summary, our data do not support a role of PTPN11 mutations as an adverse prognostic biomarker in newly diagnosed, intensively treated adult AML patients. Figure Disclosures Metzeler: Daiichi Sankyo: Honoraria; Otsuka Pharma: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy; Astellas: Honoraria. Subklewe:AMGEN: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Morphosys: Research Funding; Seattle Genetics: Research Funding; Roche AG: Consultancy, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding.

The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.

Key Points We report longitudinal mutational analyses of 2 patients with high-risk MDS and AML experiencing spontaneous disease remissions. Both patients had persistent clonal hematopoiesis during remission, harboring all but 1 of the mutations from the initial diagnostic sample.

Calcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRLhigh AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR- Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML.

Resistance against chemotherapy remains a major obstacle in treating patients with acute myeloid leukemia (AML). Novel therapeutic concepts are especially desired to target and eliminate resistant AML stem cells. Here we show that AML stem cells harbor the plasticity to switch from a low-cycling, chemotherapy resistant state into an actively proliferating state associated with treatment response. We used patient-derived xenograft (PDX) cells from patients with high risk or relapsed AML, which were lentivirally transduced for marker expression, stained with the proliferation-sensitive dye Carboxyfluorescein succinimidyl ester (CFSE), and re-transplanted into next-recipient mice. A rare subpopulation of AML cells displayed reduced proliferation in vivo, associated with increased treatment resistance. The proportion of AML cells with stem cell potential was identical in both, the highly and lowly proliferative sub-fraction. In re-transplantation experiments, proliferation behavior proved reversible, and AML stem cells were able to switch between a high and low proliferation state. Our data indicate that AML stem cells display functional plasticity in vivo, which might be exploited for therapeutic purposes, to prevent AML relapse and ultimately improve the prognosis of patients with AML.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.

Alterations of RUNX1 in acute myeloid leukemia (AML) are associated with either a more favorable outcome in the case of the RUNX1/RUNX1T1 fusion or unfavorable prognosis in the case of point mutations. In this project we aimed to identify genes responsible for the observed differences in outcome that are common to both RUNX1 alterations. Analyzing four AML gene expression data sets (n = 1514), a total of 80 patients with RUNX1/RUNX1T1 and 156 patients with point mutations in RUNX1 were compared. Using the statistical tool of mediation analysis we identified the genes CD109, HOPX, and KIAA0125 as candidates for mediator genes. In an analysis of an independent validation cohort, KIAA0125 again showed a significant influence with respect to the impact of the RUNX1/RUNX1T1 fusion. While there were no significant results for the other two genes in this smaller validation cohort, the observed relations linked with mediation effects (i.e., those between alterations, gene expression and survival) were almost without exception as strong as in the main analysis. Our analysis demonstrates that mediation analysis is a powerful tool in the identification of regulative networks in AML subgroups and could be further used to characterize the influence of genetic alterations.

Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ≥1 mutation during remission at a VAF of ≥2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (p

Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML. In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells. To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1. To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity. In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. Disclosures Hiddemann: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Background: Mutations in the NPM1 gene are among the most common genetic alterations in patients with acute myeloid leukemia (AML). NPM1 mutations predominantly occur in patients with normal or intermediate-risk abnormal cytogenetics, and define a distinct subgroup of AML patients recognized in the 2016 WHO classification. Overall, mutated NPM1 associates with favorable response to induction chemotherapy and relatively favorable overall survival. However, this prognostic impact is modulated by the presence of other gene mutations including FLT3 internal tandem duplications (ITD) and DNMT3A mutations. Recently, Patel and colleagues reported that a high variant allele frequency (VAF) of mutated NPM1 at the time of initial diagnosis associates with unfavorable outcomes in de novo AML (Blood 131:2816-25). This interesting and unexpected observation prompted us to investigate the association between NPM1 VAF and outcomes in a large AML patient cohort. Patients and Methods: We studied NPM1 mutated AML patients who had been enrolled on two successive multicenter phase III trials of the German AML Cooperative Group (AML-CG 1999, NCT00266136; AML-CG 2008, NCT01382147) and genetically characterized by amplicon-based targeted next-generation sequencing (NGS, Agilent Haloplex; Metzeler et al., Blood 128:686-98 and unpublished data). All patients had received induction chemotherapy containing cytarabine with either daunorubicin plus thioguanine or mitoxantrone. The minimum VAF for calling of insertion/deletion variants was 0.05, and samples with NPM1 coverage Results: We identified 417 NPM1-mutated patients (type A mutations, 316; type B, 28; type D, 35; and other types, 38). Median patient age was 56 years (range, 19 - 86 years), and 31/414 patients (7.5%) with cytogenetic data had abnormal karyotypes. The median NPM1 VAF was 0.43 (range, 0.05 to 1.0). Type A NPM1 mutations had significantly higher VAFs than non-type A mutations (median, 0.43 vs. 0.41; P=.0002), while type B mutations had lower VAFs than non-type B mutations (median, 0.34 vs. 0.43; P=.0025) (Figure A). Age or karyotype did not associate with NPM1 VAF. NPM1 VAF, as a continuous variable, did not associate with response to induction chemotherapy (P=.6) or relapse-free survival (P=.22). A higher NPM1 VAF did, however, associate with shorter OS (hazard ratio for an increase in NPM1 VAF equal to the interquartile range, 1.14; 95% confidence interval, 1.00-1.30; P=.049). In particular, patients in the lowest quartile of NPM1 allele burden ('low NPM1 VAF') had longer OS than patients with allele burdens above the 25th percentile ('high NPM1 VAF') (median OS, 63.7 vs. 27.0 months; 5-year OS, 51% vs 42%; P=.05; Figure B). Patients with high NPM1 VAF had higher leukocyte counts (median, 46000/µl vs. 9300/µl; P Conclusion: Our study confirms the recent report that adult AML patients with high NPM1 mutant allele burden have shorter survival. In our cohort, however, higher NPM1 VAF also associated with higher leukocyte counts and marrow blast percentage, and with prognostically adverse FLT3-ITD and DNMT3A mutations. After adjusting for these confounders, NPM1 allelic burden did not independently predict survival in our analysis. We therefore suspect that high NPM1 VAF may be a surrogate marker of highly proliferative AML subsets, for example those with high allelic ratio FLT3-ITD, rather than a novel independent prognostic factor. Figure. Figure. Disclosures Prassek: Jannsen: Other: Travel support; Celgene: Other: Travel support. Subklewe:Roche: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy.

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.