RUNX1 Mutations In Acute Myeloid Leukemia Research Articles

Maintenance Therapy with Chidamide Decreases the Relapse in Fusion Gene-Positive AML Patients with MRD-Positive or ELN-High Risk

Purpose: Acute myeloid leukemia (AML) patients with fusion gene-positive including core binding factor (CBF), mixed-lineage leukemia (MLL) gene rearrangement have a high incidence of relapse if they are continious measurable residual disease (MRD)-positive or in ELN-high risk and could not be bridged to allogenetic heamatopoitic stem cell transplantation (allo-HSCT). As known histone deacetylase (HDAC) is abnormally recruited in these fusion gene-positive AML, also RUNX1 mutated AML, and could be a therapy target. Aim of this study is to assess the effect and safety of HDAC inhibitor chidamide as maintemance therapy in these AML patients. Methods: A multi-center retrospective study was performed. Patients with MRD-positive CBF-AML, MLL rearrangement or RUNX1 mutation AML, without being bridged to allo-HSCT were included. Maintemance with chidamide was started one to three months after consolidation for at least six months, at a dose of 10mg/day or 30mg twice a week. Results: Twelve patients were enrolled, included 8 with AML1-ETO-positive, 3 with RUNX1 mutation, 1 with MLL rearrangement. The median age was 33(16-62) years old. The ratio of male to female was 6 to 5. They previously experienced median 6(4-8) cycles of chemotherapy, included one with allo-HSCT, one with autologous-HSCT. They were all maintemanced with chidamide, except one with chidamide plus sorafenib. Eleven patients were available for outcome assessment. With a median follow-up of 14(2-67) months, among the 9 patients with MRD-positive, 6(66.7%) achieved MRD-negative, 1 stable disease, 2 relapsed and died. The median time to achieved MRD-negative was 2.5(1-12) months. The other 2 patients was contenious MRD-negative. The cumulative incidence of relapse was 2/11(18.2%). The rate of MRD-nagetive was 8/11(72.7%). The mortality rate was 2/11(18.2%). During the maintemance, 4 patients underwent bone marrow suppression, including 2 with grade 3, 2 with grade 1/2. One patients had an anorexia of grade 2. Conclusion: Maintenance therapy with chidamide might decrease the relapse in fusion gene-positive AML patients with MRD-positive or ELN-high risk, who could not take allo-HSCT, and prolong their survival, with a well toleration. Key words: chidamide, maintenance, fusion gene, acute myeloid leukemia

Risk Stratification of Patients with RUNX1-mutated Acute Myeloid Leukemia

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.

Monitoring Minimal Residual Disease in RUNX1-Mutated Acute Myeloid Leukemia

Introduction: Mutated RUNX1 is considered a poor prognostic factor and usually is mutually exclusive with NPM1 mutations. Monitoring of molecular markers for minimal residual disease provides a powerful tool to assess remission and guide clinical decisions. Methods: Newly diagnosed RUNX1-mutated AML patients, designated to intensive chemotherapy-based treatment or nonintensive regimens, were monitored for mutated RUNX1 transcript levels by qPCR with patient-specific primers. Samples were obtained along the treatment course and follow-up. Results: A clear correlation was observed between mutated RUNX1 levels and response to treatment as observed by flow cytometry and STR-based assessment. Conclusion: We demonstrate the feasibility of RUNX1-based MRD to correlate with the clinicopathological status of leukemia. We further suggest how RUNX1 qPCR monitoring can influence clinical decision-making and contribute to improved personalized patient care.

Distinct gene expression pattern of RUNX1 mutations coordinated by target repression and promoter hypermethylation in acute myeloid leukemia.

Runt-related transcription factor 1 (RUNX1) is an essential regulator of normal hematopoiesis. Its dysfunction, caused by either fusions or mutations, is frequently reported in acute myeloid leukemia (AML). However, RUNX1 mutations have been largely under-explored compared with RUNX1 fusions mainly due to their elusive genetic characteristics. Here, based on 1741 patients with AML, we report a unique expression pattern associated with RUNX1 mutations in AML. This expression pattern was coordinated by target repression and promoter hypermethylation. We first reanalyzed a joint AML cohort that consisted of three public cohorts and found that RUNX1 mutations were mainly distributed in the Runt domain and almost mutually exclusive with NPM1 mutations. Then, based on RNA-seq data from The Cancer Genome Atlas AML cohort, we developed a 300-gene signature that significantly distinguished the patients with RUNX1 mutations from those with other AML subtypes. Furthermore, we explored the mechanisms underlying this signature from the transcriptional and epigenetic levels. Using chromatin immunoprecipitation sequencing data, we found that RUNX1 target genes tended to be repressed in patients with RUNX1 mutations. Through the integration of DNA methylation array data, we illustrated that hypermethylation on the promoter regions of RUNX1-regulated genes also contributed to dysregulation in RUNX1-mutated AML. This study revealed the distinct gene expression pattern of RUNX1 mutations and the underlying mechanisms in AML development.

Plasmacytoid dendritic cell expansion defines a distinct subset of RUNX1-mutated acute myeloid leukemia

AbstractPlasmacytoid dendritic cells (pDCs) are the principal natural type I interferon–producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.

Mutational landscape of patients with acute myeloid leukemia or myelodysplastic syndromes in the context of RUNX1 mutation

ABSTRACT Introduction: RUNX1 mutations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with distinct clinicopathologic features. However, the clinical and laboratory characteristics of the myeloid malignancies may be influenced by the presence of more concomitant mutations. The aim of this study is to provide a further understanding of mutational landscape in the context of RUNX1 mutation in AML/MDS. Methods: The present study screened for 49 mutations using next-generation sequencing (NGS). FLT3-ITD, NPM1, and CEBPA mutations were detected by PCR Sanger sequencing. Results: One or more co-mutations were detected in all AML and 92.3% MDS patients in the context of RUNX1 mutation. The most common co-mutation was DNMT3A, followed by NRAS, IDH1, and FLT3-ITD in AML. The four more frequently co-mutated genes were U2AF1, TET2, PTPN11, and ASXL1 in MDS. We also identified a significantly difference in co-mutational spectrums between RUNX1-mutatedAML and MDS patients, as reflected in incidence of DNMT3A (35.1% vs 7.7%), FLT3-ITD (16.2% vs 0%) and U2AF1 (10.8% vs 30.7%) mutations. RUNX1-mutated AML patients with 3, or ≥4 co-mutations showed much lower CR rate than that with 2 additional mutations (p = 0.0247, 0.00919). Conclusion: RUNX1-mutated AML and MDS are associated with a different complex co-mutation cluster. Some co-mutations have certain influence on the clinical feature and CR rate in the context of RUNX1 mutation.

Chemogenomic Approach Unveils the Increased Susceptibility of RUNX1-Mutated AML to Glucocorticoids

Abstract RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, highlighting the need for better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for patients with RUNX1mut AML. Toward this goal, we performed RNA-sequencing of a cohort of RUNX1mut primary AML specimens and used a chemogenomic approach combining mutational and gene expression analysis of these specimens with assessment of their drug sensitivity profile through chemical screening. Sequencing data analysis demonstrated that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Interestingly, these studies also unveiled the increased susceptibility of RUNX1-mutated specimens to Glucocorticoids (GCs) and revealed that RUNX1 allele dosage dictates sensitivity to these compounds in AML patient cells, unravelling a new role for RUNX1 in the Glucocorticoid Receptor (GR) pathway. GR is a nuclear receptor that modulates the expression of thousands of genes involved in several biological processes such as metabolism, immune function, skeletal growth, etc. GCs are commonly used to treat cancers of the lymphoid system, however their potential benefits for AML treatment have never been assessed formally and the mechanism of action of these drugs is not fully understood. Transcriptome analyses identified NR3C1 (encoding the GR) as one of the genes whose expression is determined by RUNX1 allele dosage, with increased expression in RUNX1mut specimens, indicating that RUNX1 inactivation could lead to GR upregulation, which might explain the increased sensitivity to GCs. We previously showed that RUNX1 silencing sensitizes human AML cell lines to GCs and that this acquired sensitivity is accompanied by the upregulation of the GR both at the transcript and protein levels. However, basal levels of GR could not explain GC sensitivity in all cases, indicating that other mechanisms are involved in the GC response. By performing co-immunoprecipitations (co-IP), we demonstrated that RUNX1 and GR physically interact in AML cells. Overexpression of FLAG-tagged RUNX1 mutants in HEK293 cells followed by co-IP identified the C-terminal inhibitory domain of RUNX1 as essential for the interaction with GR. Our results suggest that RUNX1 could be part of the GR transcriptional complex and could modulate the transcription of genes involved in the response to GCs. To identify regulators of the GC response, we are currently performing genome-wide CRISPR-Cas9-based genetic screens in AML cell lines. We generated RUNX1-deficient (GCs sensitive) AML cell lines that uniformly express Cas9 under a doxycycline-inducible promoter. These cell lines, along with parental cell lines (GC resistant) were used to conduct screens in GCs-supplemented media for the identification of genes that confer sensitivity or resistance to GCs. Mechanistic insights gained from these experiments will allow the design of additional therapeutic strategies to potentiate the effect of GCs on poor outcome AML. Disclosures No relevant conflicts of interest to declare.

Identification of Key Genes and Pathways Associated with RUNX1 Mutations in Acute Myeloid Leukemia Using Bioinformatics Analysis.

BACKGROUND RUNXl plays a key regulatory role in the process of hematopoiesis and is a common target for multiple chromosomal translocations in human acute leukemia. Mutations of RUNX1 gene can lead to acute leukemia and affect the prognosis of AML patients. We aimed to identify pivotal genes and pathways involved in RUNX1-mutated patients of with acute myeloid leukemia (AML) and to explore possible molecular markers for novel therapeutic targets of the disease. MATERIAL AND METHODS The RNA sequencing datasets of 151 cases of AML were obtained from the Cancer Genome Atlas database. Differentially expressed genes (DEGs) were identified using edgeR of the R platform. PPI (protein-protein interaction) network clustering modules were analyzed with ClusterONE, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses for modules were performed. RESULTS A total of 379 genes were identified as DEGs. The KEGG enrichment analysis of DEGs showed significantly enriched pathways in cancer, extracellular matrix (ECM)-receptor interaction pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway. The top 10 genes ranked by degree were PRKACG, ANKRD7, RNFL7, ROPN11, TEX14, PRMT8, OTOA, CFAP99, NRXN1, and DMRT1, which were identified as hub genes from the protein-protein interaction network (PPI). Statistical analysis revealed that RUNX1-mutated patients with AML had a shorter median survival time (MST) with poor clinical outcome and an increased risk of death when compared with those without RUNX1 mutations. CONCLUSIONS DEGs and pathways identified in the present study will help understand the molecular mechanisms underlying RUNX1 mutations in AML and develop effective therapeutic strategies for RUNX1-mutation AML.

Identification of Key Genes and Pathways Associated with RUNX1 Mutations in Acute Myeloid Leukemia Using Bioinformatics Analysis

BackgroundRUNXl plays a key regulatory role in the process of hematopoiesis and is a common target for multiple chromosomal translocations in human acute leukemia. Mutations of RUNX1 gene can lead to acute leukemia and affect the prognosis of AML patients. We aimed to identify pivotal genes and pathways involved in RUNX1-mutated patients of with acute myeloid leukemia (AML) and to explore possible molecular markers for novel therapeutic targets of the disease.Material/MethodsThe RNA sequencing datasets of 151 cases of AML were obtained from the Cancer Genome Atlas database. Differentially expressed genes (DEGs) were identified using edgeR of the R platform. PPI (protein–protein interaction) network clustering modules were analyzed with ClusterONE, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses for modules were performed.ResultsA total of 379 genes were identified as DEGs. The KEGG enrichment analysis of DEGs showed significantly enriched pathways in cancer, extracellular matrix (ECM)-receptor interaction pathway, and cyclic adenosine monophosphate (cAMP) signaling pathway. The top 10 genes ranked by degree were PRKACG, ANKRD7, RNFL7, ROPN11, TEX14, PRMT8, OTOA, CFAP99, NRXN1, and DMRT1, which were identified as hub genes from the protein–protein interaction network (PPI). Statistical analysis revealed that RUNX1-mutated patients with AML had a shorter median survival time (MST) with poor clinical outcome and an increased risk of death when compared with those without RUNX1 mutations.ConclusionsDEGs and pathways identified in the present study will help understand the molecular mechanisms underlying RUNX1 mutations in AML and develop effective therapeutic strategies for RUNX1-mutation AML.

Prognostic Value of RUNX1 Mutations in AML: A Meta-Analysis

The RUNX1 (AML1) gene is a relatively infrequent mutational target in cases of acute myeloid leukemia (AML). Previous work indicated that RUNX1 mutations can have pathological and prognostic implications. To evaluate prognostic value, we conducted a meta-analysis of 4 previous published works with data for survival according to RUNX1 mutation status. Pooled hazard ratios for overall survival and disease-free survival were 1.55 (95% confidence interval (CI) = 1.11–2.15; p-value = 0.01) and 1.76 (95% CI = 1.24–2.52; p-value = 0.002), respectively, for cases positive for RUNX1 mutations. This evidence supports clinical implications of RUNX1 mutations in the development and progression of AML cases and points to the possibility of a distinct category within the newer WHO classification. Though it must be kept in mind that the present work was based on data extracted from observational studies, the findings suggest that the RUNX1 status can contribute to risk-stratification and decision-making in management of AML.

Number of RUNX1 mutations, wild-type allele loss and additional mutations impact on prognosis in adult RUNX1-mutated AML.

RUNX1-mutated acute myeloid leukemia (AML) show a distinct pattern of genetic abnormalities and an adverse prognosis. We analyzed the impact of multiple RUNX1 mutations and RUNX1 wild-type (WT) loss in 467 AML with RUNX1 mutations (mut): (1) RUNX1 WT loss (n=53), (2) >1 RUNX1mut (n=94) and (3) 1 RUNX1mut (n=323). In 1 RUNX1mut, +8 was most frequent, whereas in WT loss +13 was the most abundant trisomy (+8: 66% vs 31%, P=0.022; +13: 15% vs 62%, P<0.001). Analyses of 28 genes in 163 selected cases revealed SRSF2 (39%), ASXL1 (36%), DNMT3A (19%), IDH2 (17%) and SF3B1 (17%) as most frequently mutated genes. RUNX1 WT loss showed a higher frequency of ASXL1mut compared with the other cases (50% vs 29%, P=0.009). Median overall survival (OS) in the total cohort was 14 months. WT loss (OS: 5 months) and >1 RUNX1mut (14 months) showed an adverse impact on prognosis compared with 1 RUNX1mut (22 months; P=0.002 and 0.048, respectively). Mutations in ASXL1 and ⩾2 additional mutations correlated with shorter OS (10 vs 18 months, P=0.028; 12 vs 20 months, P=0.017). Thus, the number of RUNX1mut, RUNX1 WT loss and the number and type of additional mutations is biologically and clinically relevant.

Clinical Outcomes and Co-Occurring Mutations in Patients with RUNX1-Mutated Acute Myeloid Leukemia.

(1) Runt-related transcription factor 1 (RUNX1) mutations in acute myeloid leukemia (AML) are often associated with worse prognosis. We assessed co-occurring mutations, response to therapy, and clinical outcomes in patients with and without mutant RUNX1 (mRUNX1); (2) We analyzed 328 AML patients, including 177 patients younger than 65 years who received intensive chemotherapy and 151 patients >65 years who received hypomethylating agents. RUNX1 and co-existing mutations were identified using next-generation sequencing; (3) RUNX1 mutations were identified in 5.1% of younger patients and 15.9% of older patients, and were significantly associated with increasing age (p = 0.01) as well as intermediate-risk cytogenetics including normal karyotype (p = 0.02) in the elderly cohort, and with lower lactate dehydrogenase (LDH; p = 0.02) and higher platelet count (p = 0.012) overall. Identified co-occurring mutations were primarily ASXL1 mutations in older patients and RAS mutations in younger patients; FLT3-ITD and IDH1/2 co-mutations were also frequent. Younger mRUNX1 AML patients treated with intensive chemotherapy experienced inferior treatment outcomes. In older patients with AML treated with hypomethylating agent (HMA) therapy, response and survival was independent of RUNX1 status. Older mRUNX1 patients with prior myelodysplastic syndrome or myeloproliferative neoplasms (MDS/MPN) had particularly dismal outcome. Future studies should focus on the prognostic implications of RUNX1 mutations relative to other co-occurring mutations, and the potential role of hypomethylating agents for this molecularly-defined group.

Abstract 1509: RUNX1 dosage dictates gene signature and in vitro response to glucocorticoids in acute myeloid leukemia

Abstract Background: Poor prognosis subgroups of Acute Myeloid Leukemia (AML), such as RUNX1-mutated (RUNX1mut) AML, would greatly benefit from more efficacious therapies that target leukemic stem cells (LSC) and improve patient outcome. To understand factors that predict sensitivity to drugs, we used a chemogenomic approach to interrogate primary AML specimens. Methods: We performed RNA sequencing of 415 primary AML specimens comprising various cytogenetic subgroups. Using culture conditions that support LSC activity ex vivo, we carried out a viability screen including 20 primary AML specimens and ~5,100 low molecular weight compounds. RUNX1mut specimens showed increased sensitivity to glucocorticoid compounds (GCs). Validation screens were done in 248 primary AML samples and 32 AML cell lines treated with selected GCs in a dose-response manner. The effect of RUNX1 dosage in the in vitro response to drugs was assessed by shRNA gene knockdown. GCs target, the glucocorticoid receptor (GR), was validated by chemical blockage and shRNA silencing. Results: RUNX1mut specimens were associated with older age, French-American-British (FAB) M0 morphology, intermediate-risk cytogenetics with abnormal karyotype and poor patient survival. RUNX1mut gene expression signature showed the overexpression of previously described genes such as DNTT, BAALC and CD34, as well as novel genes such as PROM1 and EGFEM1P. Most interestingly, the expression levels of these genes was influenced by the nature of the RUNX1 mutations, with levels progressively increasing with mutations corresponding to decreased levels of functional RUNX1. Chemical screens comprising 33 RUNX1mut specimens confirmed that RUNX1mut are more sensitive to GCs than RUNX1 wild-type samples. In agreement with the RUNX1mut gene signature, the anti-proliferative effect of GCs anti-correlated with levels of functional RUNX1. Specimens harboring loss-of-function and dominant-negative RUNX1 mutations showed increased sensitivity to GCs when compared to samples carrying missense mutations expected to have little impact on RUNX1 function. Mutations in other genes, such as CEBPA and SRSF2, had an additive effect on GC sensitivity when combined with RUNX1 mutations. In accordance with our hypothesis, the downregulation of RUNX1 could reverse GC-resistance in AML cell lines, and the sensitivity to compounds was proportional to knockdown levels. Treatment of cell lines with the GR antagonist RU486 blocked the inhibitory response induced by GCs and GR silencing completely abrogated the anti-proliferative effects of GCs in GC-sensitive cells, confirming that GC operate through the GR in AML. Conclusion: Altogether, these findings highlight the impact of RUNX1 dosage on gene expression and GCs sensitivity in AML cells in vitro. Further studies should investigate the benefits of repositioning GCs in the treatment of RUNX1mut AML patients. Citation Format: Laura Simon, Vincent-Philippe Lavallée, Marie-Eve Bordeleau, Jana Krosl, Irène Baccelli, Geneviève Boucher, Bernhard Lehnertz, Tara MacRae, Réjean Ruel, Sébastien Lemieux, Anne Marinier, Josée Hébert, Guy Sauvageau. RUNX1 dosage dictates gene signature and in vitro response to glucocorticoids in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2017-1509

RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features.

We evaluated the frequency, genetic architecture, clinico-pathologic features and prognostic impact of RUNX1 mutations in 2439 adult patients with newly-diagnosed acute myeloid leukemia (AML). RUNX1 mutations were found in 245 of 2439 (10%) patients; were almost mutually exclusive of AML with recurrent genetic abnormalities; and they co-occurred with a complex pattern of gene mutations, frequently involving mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components of the spliceosome complex (SRSF2, SF3B1) and STAG2, PHF6, BCOR. RUNX1 mutations were associated with older age (16-59 years: 8.5%; ⩾60 years: 15.1%), male gender, more immature morphology and secondary AML evolving from myelodysplastic syndrome. In univariable analyses, RUNX1 mutations were associated with inferior event-free (EFS, P<0.0001), relapse-free (RFS, P=0.0007) and overall survival (OS, P<0.0001) in all patients, remaining significant when age was considered. In multivariable analysis, RUNX1 mutations predicted for inferior EFS (P=0.01). The effect of co-mutation varied by partner gene, where patients with the secondary genotypes RUNX1mut/ASXL1mut (OS, P=0.004), RUNX1mut/SRSF2mut (OS, P=0.007) and RUNX1mut/PHF6mut (OS, P=0.03) did significantly worse, whereas patients with the genotype RUNX1mut/IDH2mut (OS, P=0.04) had a better outcome. In conclusion, RUNX1-mutated AML is associated with a complex mutation cluster and is correlated with distinct clinico-pathologic features and inferior prognosis.

Frequency, Characteristics and Prognostic Significance of RUNX1 mutations in Patients with Acute Myeloid Leukemia, Not Otherwise Specified

Background: Somatic mutations in RUNX1 gene have been identified in a substantial proportion of patients with de novo acute myeloid leukemia (AML). It is suggested as a new candidate molecular marker and, therefore, is suggested to be routinely performed at the diagnostic stage of AML. Despite its clinical importance, however, previous cohorts have been heterogeneous in terms of cytogenetic and molecular subtypes of AML. Here, the aim of this study was to evaluate the frequency, biologic characteristics, and prognostic significance of RUNX1 mutations focusing on patients with AML, not otherwise specified (NOS).Methods: Diagnostic samples from 202 patients with AML were analyzed for RUNX1 mutations. We excluded AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, and therapy-related AML because these entities have prognostic relevances of their own. RUNX1 mutations were detected using standard PCR techniques and direct sequencing.Results:RUNX1 mutations were found in 27 (13.4%) patients. The mutations were clustered in Runt homology domain (13, 48.1%) and transactivation domain (9, 33.3%). Frameshift mutations were most common (52.9%), followed by missense mutations (35.3%) and nonsense mutations (11.8%). As shown in Table 1, patients with RUNX1 mutations had a lower platelet count (P = 0.03), a higher rate of trisomy 8 (P = 0.02) and trisomy 13 (P = 0.039), and a trend toward older age (P = 0.063) than patients without mutations. Presence of RUNX1 mutations and NPM1 or CEBPA mutations were mutually exclusive. At the median follow-up of 12.1 months, RUNX1 mutations predicted for shorter overall survival (OS; P = 0.007) and relapse-free survival (RFS; P = 0.003). In the multivariate analysis, RUNX1 mutation was a significant marker for inferior OS (hazard ratio, 3.037; P = 0.014) and RFS (hazard ratio, 5.699; P = 0.001).Conclusion: The findings of our study further strengthen the previous data about RUNX1 mutations in AML. Furthermore, AML NOS with RUNX1 mutations is characterized by distinct biology and is associated with adverse clinical outcome. Our study supports the notion that RUNX1 mutational status would be integrated into diagnostic workup of AML, particularly for AML, NOS subgroup.Table 1Clinical and biologic features of the cohort by RUNX1 mutationsRUNX1 mutationsP -valueMutated, n (%)Wild type, n (%)Number27 (13.4)175 (86.6)Male sex17 (63.0)94 (53.7)0.489Median age, years (range)63 (14 - 80)55 (1 - 83)0.063WBC count, ¡¿109/L (median, range)7.9 (1.1 - 133.3)14.0 (0.8 - 231.3)0.636Hemoglobin, g/dL (median, range)8.6 (5.0 - 10.6)8.8 (4.1 - 17.3)0.376Platelet count, ¡¿109/L (median, range)35 (14 - 230)59 (9 - 900)0.03Blood blasts, % (median, range)29 (0 - 94)38.5 (0 - 93)0.312FAB subtypesM03 (11.1)11 (6.3)0.609M1, M221 (77.8)129 (73.7)0.831M4, M53 (11.1)27 (15.4)0.767M6, M708 (4.6)0.546Cytogenetic abnormalitiesNormal karyotype (%)11 (40.7)104 (59.4)0.106Trisomy 8 (%)5 (18.5)8 (4.6)0.02Trisomy 11 (%)1 (3.7)4 (2.3)0.823Trisomy 13 (%)3 (11.1)3 (1.7)0.039Trisomy 21 (%)1 (3.7)2 (1.1)0.866Distribution of other mutationsFLT3 -ITD5 (18.5)50 (28.6)0.39FLT3 -TKD1 (3.7)4 (2.3)0.823NPM1055 (31.4)0.002CEBPA017 (9.7)0.187MLL -PTD1 (3.7)14 (8.0)0.691 DisclosuresNo relevant conflicts of interest to declare.

The Landscape of RUNX1 Mutations in Acute Myeloid Leukemia: Investigations On Stability of Mutations At Relapse and Utility As a Marker for Minimal Residual Disease

AbstractAbstract 657 Introduction:RUNX1 mutations constitute disease-defining aberrations in acute myeloid leukemia (AML) and were demonstrated to be particularly frequent in secondary and de novo AML with normal karyotype or non-complex alterations and to confer an unfavorable prognosis. Monitoring minimal residual disease (MRD) in AML has been shown to provide prognostic information and is increasingly used for treatment decisions. A variety of molecular markers has been identified suitable for MRD assessment, yet there still is a lack of such markers in a significant number of patients. The use of RUNX1 mutations may bridge a gap. Aims:1.Evaluate the stability of RUNX1 mutations between diagnosis and relapse.2.Test the utility and clinical relevance of RUNX1 mutations as MRD marker. Patients and Methods:RUNX1 mutation screening was prospectively performed in 814 patients with AML at diagnosis (645 de novo, 109 s-AML, and 60 t-AML). The median age of the patients was 69.6 years (range: 1 – 93 years), including 375 female and 439 male patients, respectively. 50.5% (411/814) of cases presented with a normal karyotype, 38.8% (316/814) with non-complex cytogenetic alterations, 9.6% (78/814) with a complex aberrant karyotype, and 1.1% (9/814) with prognostically favorable cytogenetics. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT). Moreover, in a subset of 44 AML patients and additional 59 retrospectively analyzed cases the prognostic impact of MRD levels of RUNX1 mutations was studied at a second time point after completion of intensive induction therapy (median sampling interval: 128 days after diagnosis; range 60 – 180 days). In these follow-up samples the RUNX1 mutations already detected at diagnosis were investigated with a higher coverage (835-fold median coverage) as compared to the diagnostic assessment (759-fold median coverage) resulting in a sensitivity level of 1%. Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed to assess the stability of RUNX1 mutations. Results:211/814 patients (25.9%) were detected to carry RUNX1 mutations. The median clone size was 39% and revealed a significant heterogeneity ranging from 2% to 96%. 73.9% (156/211) of mutated patients carried one mutation only, whereas 26.1% (55/211) harbored two (n=46) or more (n=9) mutations. In detail, the 211 patients harbored a total number of 275 alterations in RUNX1: 42.5% (117/275) frame-shift mutations, 34.9% (96/275) missense, 14.2% (39/275) nonsense, 4.4% (12/275) exon-skipping/splicing, and 4.0% (11/275) in-frame insertion/deletion alterations, respectively. Regarding MRD assessment, patients were separated according to the median MRD level (3.92%; range 0.03% - 48.00%) into “good responders” (n=78) with MRD levels below 3.92% and “poor responders” (n=25) with MRD levels above 3.92%. This resulted in significant differences in both event-free survival (median 21.4 vs 5.7 months, p<0.001) and overall survival (73.3% vs 66.1% at 2 years, p=0.016). Moreover, in 57 cases the stability of individual RUNX1 mutations was studied at the time of relapse. In 46/57 (80.7%) cases the same alterations detected at diagnosis were present at relapse, whilst in 2/57 (3.5%) cases the RUNX1 mutation from the diagnostic sample was no longer detectable at relapse. Importantly, in 7/57 (12.3%) patients novel RUNX1 mutations were detected in regions different from those affected at diagnosis. Conclusion:Next-generation deep-sequencing accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. Thus, the measurement of mutation load by next-generation sequencing may contribute to refine the assignment into distinct risk categories in AML. Analysis of RUNX1 mutations should be considered for the complete coding region at relapse to detect new RUNX1 mutations. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia