Cytogenetically Normal Acute Myeloid Leukemia Research Articles

[Gene Profile and Clinical Significance of Concomitant Mutations in CN-AML Patients with CEBPA Mutation].

To analyze the occurrence of concomitant gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients with CEBPA mutation and its impact on the clinical characteristics and prognosis of the patients. 151 newly diagnosed patients with CN-AML in the Second Hospital of Shanxi Medical University from June 2013 to June 2020 were analyzed retrospectively. 34 common genetic mutations associated with hematologic malignancies were detected by next-generation sequencing technology. The occurrence of concomitant gene mutations in patients with CEBPA positive and negative groups was compared, and the correlation between concomitant mutations in different functional groups and the clinical characteristics and prognosis of CN-AML patients with CEBPA mutation was analyzed. In 151 patients with CN-AML, 55 (36.42%) were positive for CEBPA mutation (including 36 cases of CEBPAdm and 19 cases of CEBPAsm), of which 41 (74.55%) had co-mutations with other genes. The main mutated genes were GATA2 (25.45%, 14/55), TET2 (21.82%, 12/55), FLT3 (20.00%, 11/55), NRAS (12.73%, 7/55) and WT1 (9.09%, 9/55), etc. Some cases had two or more concomitant gene mutations. Grouping the mutant genes according to their functions showed that CEBPA+ group had lower mutation rates of histone methylation (P =0.002) and chromatin modification genes (P =0.002, P =0.033), and higher mutation rates of transcription factors (P =0.037) than CEBPA- group. In 55 patients with CEBPA+ CN-AML, the platelet count at diagnosis in signaling pathway gene mutation-positive group was lower than that in the mutation-negative group (P =0.005), the proportion of bone marrow blasts in transcription factor mutation-positive group was higher than that in the mutation-negative group (P =0.003), and the onset age in DNA methylation gene mutation-positive group and chromatin modifier mutation-positive group was older than that in the mutation-negative group, respectively (P =0.002, P =0.008). DFS of CEBPA+ CN-AML patients in signaling pathway gene mutation group was shorter than that in signaling pathway gene mutation-negative group (median DFS: 12 months vs not reached) (P =0.034). Compared with DNA methylation gene mutation-negative group, CEBPA+ CN-AML patients with DNA methylation gene mutation had lower CR rate (P =0.025) significantly shorter OS and DFS (median OS: 20 months vs not reached, P =0.006; median DFS: 15 months vs not reached, P =0.049). OS in patients with histone methylation gene mutation was significantly shorter than that in the histone methylation gene mutation-negative group (median OS: 12 months vs 40 months) (P =0.008). Multivariate analysis of prognostic factors showed that the proportion of bone marrow blasts (P =0.046), concomitant DNA methylation gene mutation (P =0.006) and histone methylation gene mutation (P =0.036) were independent risk factors affecting the prognosis. CN-AML patients with CEBPA mutation have specific concomitant gene profile, and the concomitant mutations of different functional genes have a certain impact on the clinical characteristics and prognosis of the patients.

A new prognostic risk model for acute myeloid leukemia patients based on telomere-related genes

Telomere maintenance is critical to ensure unlimited cancer cell proliferation, but the role of telomere-related genes in acute myeloid leukemia (AML) has not yet been thoroughly discussed. This study aims to develop a new prognostic risk model based on telomere-related genes and analyze potential mechanisms and targets. Cox regression analyses were used to build the prognostic risk model. Kaplan-Meier (KM) survival analysis and receiver operating characteristic (ROC) curve were used to assess the model performance. At the same time, we analyzed the relationship between the risk score and chemotherapy and immunotherapy and preliminarily explored possible mechanisms of immune resistance. The real-time polymerase chain reaction (PCR) was used to detect the prognosis gene expression levels. Finally, a prognostic signature of six telomere-related genes (TGPS6) including ALDH2, CDK18, DNMT3B, FRAT2, LGALSL, and RBL2 was constructed. The TGPS6 score was confirmed as an independent prognostic factor (HR 2.74, CI [2.13–3.53], p < 0.001) in AML and the five-year area under the ROC curve (AUC) value of the score in the training and validation set reached 0.74, 0.81 respectively. In addition, the TGPS6 perfected the European LeukemiaNet (ELN) 2017 prognosis risk stratification and performed well in both AML and cytogenetically normal AML (CN-AML) cohorts. The TGPS6 score also provided a reference for chemotherapy and immunotherapy in patients with AML.

A novel 5-gene prognostic signature to improve risk stratification of cytogenetically normal acute myeloid leukemia.

Prognostic prediction is a challenging task in cytogenetically normal acute myeloid leukemia (CN-AML) patients. In this study, we aimed at developing a novel prognostic signature to predict and stratify the survival of CN-AML patients. Using a training dataset (GSE12417), 5-gene prognostic signature was established to predict survival of CN-AML patients. The prognostic performance of this prognostic signature was further validated in testing dataset (TCGA CN-AML cohort) and validation dataset (GSE6891 CN-AML cohort). In training, testing and validation datasets, the increased 5-gene risk score was significantly related with inferior overall survival (OS) of patients, and the area under the receiver operating characteristic curve (AUC) demonstrated that our prognostic signature had overall prediction accuracy. The excellent prognostic value of the 5-gene prognostic signature was also supported by the comparison with three previously proposed prognostic models. For the intermediate-risk CN-AML patients and the CN-AML patients with FLT3 or NPM1 mutation, our model could also well dichotomize them into two subgroups with distinct prognosis. Multivariate analysis demonstrated that 5-gene risk score was the only independent risk factor in TCGA CN-AML cohort. Nomogram including the 5-gene risk score performed well in predicting 1-year, 2-year and 3-year OS. In summary, our novel 5-gene prognostic signature facilitated the improvement in risk stratification of CN-AML patients.

Phe354Leu polymorphism of the liver kinase B1 gene as a prognostic factor in adult egyptian patients with acute myeloid leukemia

BackgroundThe human liver kinase B1 (LKB1) gene is a significant tumor suppressor widely expressed in all fetal and adult tissues. Despite its established role in solid tumors, the biological and clinical implications of LKB1 gene alterations in hematological malignancies have not been sufficiently recognized. AimThis study aimed to determine the frequency of the LKB1 Phe354Leu polymorphism in adult Egyptian patients with cytogenetically normal AML (CN-AML), evaluate its clinical prognostic significance, and investigate its effect on the therapeutic outcome and patient survival. MethodsDirect sequencing of amplified exon eight of the LKB1 gene was performed to detect the Phe354Leu polymorphism in 72 adult de novo CN-AML patients. ResultsThe LKB1 Phe354Leu polymorphism was detected in 16.7% of patients and associated with younger age and lower hemoglobin levels (p < 0.001). Patients in the mutated group had significantly higher total leukocytic count and bone marrow blasts (p = 0.001 and p < 0.001, respectively). The most common FAB subtypes in mutated patients were M4 and M2. The relapse rate was significantly higher in the mutated group (p = 0.004). There was a significant association between the FLT3-ITD polymorphism and LKB1 F354L (p < 0.001). The mutated group had shorter overall survival (p = 0.003). In multivariate analysis, the Phe354Leu polymorphism was a significant independent prognostic variable for the overall and disease-free survival of the studied patients (p = 0.049). ConclusionThe LKB1 Phe354Leu polymorphism was diagnosed at younger ages in Egyptian CN-AML patients and represented a poor independent prognostic factor in CN-AML. Patients who carried this polymorphism had shorter overall survival and more frequent relapses. Our findings may provide insight into the design of therapeutic targets, and molecular testing of the LKB1 gene is recommended for proper risk stratification of CN-AML patients.

Abstract A09: Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML

Abstract NPM1 mutations are among the most common mutations in CN-AML (Cytogenetically Normal-AML) and are known to enhance chemosensitivity in AML patients. Across multiple co-operative group studies, in the absence of deleterious mutations, patients with NPM1 mutations show superior overall and relapse-free survival. The FOXM1 regulatory network is a major predictor of poor prognosis in human cancers of different origin, including AML. Early AML cell line and patient sample data from our lab demonstrated that nuclear NPM colocalizes with nuclear FOXM1 while mutant NPM sequesters FOXM1 in the cytoplasm. Applying RNAseq differential expression results from published FOXM1 knockout experiments on KG-1 cells and MLL-AF9-transformed mice to Beat AML RNAseq data, we developed a ~200-gene FOXM1 signature that was significantly associated with inferior CR/CRi rate (OR=0.44 [95% CI: 0.21–0.95], p=0.037) in wild type NPM1, wild type FLT3 de novo AML patients. Notably, this FOXM1 signature was not significantly associated with CR/CRi in mutated NPM1, wild type FLT3 patients (OR=1.78 [95% CI: 0.15–21.51], p=0.651). These data suggest that inactivation of FOXM1 may underlie the chemosensitivity conferred by the highly prevalent NPM1 mutation in favorable risk AML. Using our novel network-centric approach, we found a first-in-class small molecule (STL427944) that affects a pathway that limits FOXM1 expression and significantly disrupts the associated regulatory network. To directly address the role of FOXM1 in chemosensitivity of AML cells, we tested two FOXM1-specific inhibitory compounds: STL427944 and its derivative, STL001 (their structures will be disclosed). From a medicinal chemistry standpoint, STL427944 has two main metabolic liabilities, a hydrolytically labile furane-carboxylic acid phenolic ester and a hydrazone. We applied several paths of structural activity relationship (SAR) optimization to overcome these issues from removal of liabilities to bio-isosteric replacement to cyclizations. The ring replacement is likely to have significantly improved the overall stability observed in STL001, resulting in at least !0-fold more active compound with better ‘drug-like’ properties and thus enhanced potency. We showed that 500nM of STL001 partially suppressed expression of FOXM1 protein in AML, while 5mM of STL001 (versus 50mM of STL427944) fully suppressed FOXM1 and sensitized AML cells to venetoclax and cytarabine treatment. Ex vivo treatment of AML patient samples with STL427944 and STL001 resulted in inhibition of nuclear FOXM1 expression according to IHC and downregulation of canonical FOXM1 transcriptional targets as indicated by real-time PCR. This treatment potentiated the efficacy of venetoclax in inhibiting colony forming activity in AML patient samples. Our data suggest that targeting FOXM1 in AML patients with wild type NPM1 may recapitulate NPM1 mutations and will have a robust therapeutic effect leading to more favorable outcome for AML patients. Citation Format: Irum Khan, Sandjeev Raghuwanshi, Andy Kaempf, Jeff Tyner, Alex Domling, Carlos Camacho, Andrei Gartel. Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A09.

High trophinin-associated protein expression predicts good survival in acute myeloid leukemia with normal cytogenetics.

Nearly half of adult acute myeloid leukemia (AML) patients were classified into cytogenetic normal acute myeloid leukemia (CN-AML). The expression level of Trophinin associated protein (TROAP) was proven to be associated with the prognosis of several cancers, but it is still unclear in the prognosis of patients with CN-AML. We integrated CN-AML patients samples from 4 datasets to analyze the relationship between TROAP expression and the survival of CN-AML. In addition, we investigated 92 AML patients of The Cancer Genome Atlas (TCGA) database to analyze the relationship between TROAP expression and the survival of AML patients received chemotherapy. We investigated the relationship between the expression of TROAP and drug sensitivity in AML cell lines. CN-AML patients with high TROAP expression were related to good event-free survival (EFS) and overall survival (OS). In AML patients received chemotherapy, high TROAP expression was associated with good survival prognosis. Additionally, the expression of TROAP gene in leukemia stem cells (LSC) + group was lower. Among multiple drugs, the lower the expression of TROAP, the lower the IC50. TROAP could serve as an independent predictor of CN-AML patients and could act as a potential biomarker for the prognosis of CN-AML. TROAP expression levels were closely correlated with the drug sensitivity of multiple drugs.

The expression level of Neuronal Calcium Sensor 1 can predict the prognosis of cytogenetically normal AML.

Acute myeloid leukemia (AML) is malignant clonal expansion of myeloid blasts with high heterogeneity and numerous molecular biomarkers have been found to judge the prognosis in some specific classifications of AML. Furthermore, as for patients with cytogenetically normal acute myeloid leukemia (CN-AML), we need to find more new biomarkers to predict the patients' outcomes. Recently, the expression level of Neuronal Calcium Sensor 1 (NCS1) has been associated with the prognosis of breast cancer and hepatocellular carcinoma, but nothing related has been reported about hematological malignancies. Therefore, we make this study to explore the relationship between the NCS1 expression level and CN-AML. We analyzed the relation between survival and NCS1 RNA expression through 75 CN-AML patients from Cancer Genome Atlas (TCGA) database and 433 CN-AML patients (3 independent datasets) from Gene Expression Omnibus (GEO) database. Additionally, we compared the NCS1 RNA expression between 138 leukemia stem cells positive (LSCs+) samples and 89 leukemia stem cells negative (LSCs-) samples from 78 AML patients from GSE76004 dataset. In our study, CN-AML patients with high expression level of NCS1 have longer EFS or OS. In addition, the NCS1 expression level in leukemia stem cells was low (p = 0.00039). According to these findings, we concluded that the high expression of NCS1 can predict favorable prognosis in CN-AML patients. Furthermore, our work put forward that NCS1 expresses lower in LSCs+, which might be an important mechanism to explain the aggressiveness of AML.

The prognostic value of RASGEF1A RNA expression and DNA methylation in cytogenetically normal acute myeloid leukemia.

Acute myeloid leukemia (AML) is a significantly heterogeneous malignancy of the blood. Cytogenetic abnormalities are crucial for the prognosis of AML. However, since more than half of patients with AML are cytogenetically normal AML (CN-AML), predictive prognostic indicators need to be further refined. In recent years, gene abnormalities are considered to be strong prognostic factors of CN-AML, already having clinical significance for treatment. In addition, the relationship of methylation in some genes and AML prognosis predicting has been discovered. RASGEF1A is a guanine nucleotide exchange factors of Ras and widely expressed in brain tissue, bone marrow and 17 other tissues. RASGEF1A has been reported to be associated with a variety of malignant tumors, examples include Hirschsprung disease, renal cell carcinoma, breast cancer, diffuse large B cell lymphoma, intrahepatic cholangiocarcinoma and so on [1, 2]. However, the relationship between the RASGEF1A gene and CN-AML has not been reported. By integrating the Cancer Genome Atlas (TCGA) database 75 patients with CN-AML and 240 Gene Expression Omnibus (GEO) database CN-AML samples, we examined the association between RASGEF1A's RNA expression level and DNA methylation of and AML patients' prognosis. Then, we investigated the RASGEF1A RNA expression and DNA methylation's prognostic value in 77 patients with AML after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) as well as 101 AML patients after chemotherapy respectively. We investigated the association between sensitivity to Crenolanib and expression level of RASGED1A in patients by integrating 191 CN-AML patients from BeatAML dadataset. We integrated the expression and methylation of RASGEF1A to predict the CN-AML patients' prognosis and investigated the relationship between prognostic of AML patients with different risk classification and expression levels or methylation levels of RASGEF1A. We found that RASGEF1A gene high expression group predicted poorer event-free survival (EFS) (P< 0.0001) as well as overall survival (OS) (P< 0.0001) in CN-AML samples, and the identical results were found in AML patients receiving chemotherapy (P< 0.0001) and Allo-HSCT (P< 0.0001). RASGEF1A RNA expression level is an CN-AML patients' independent prognostic factor (EFS: HR = 5.5534, 95% CI: 1.2982-23.756, P= 0.0208; OS: HR = 5.3615, 95% CI: 1.1014-26.099, P= 0.0376). The IC50 (half maximal inhibitory concentration) of Crenolanib of CN-AML samples with RASGEF1A high expression level is lower. In addition, patients with high RASGEF1A methylation level had significant favorable prognosis (EPS: P< 0.0001, OS: P< 0.0001). Furthermore, the integrative analysis of expression and methylation of RASGEF1A could classify CN-AML patients into subgroups with different prognosis (EFS: P= 0.034, OS: P= 0.0024). Expression levels or methylation levels of RASGEF1A help to improve risk classification of 2010 European Leukemia Net. Higher RASGEF1A RNA expression and lower DNA methylation predicts CN-AML patients' poorer prognosis. The RASGEF1A high expression level from patients with CN-AML have better sensitivity to Crenolanib. The integrative analysis of RASGEF1A RNA expression and DNA methylation can provide a more accurate classification for prognosis. Lower RASGEF1A expression is a favorable prognostic factor for AML patients receiving chemotherapy or Allo-HSCT. 2010 European Leukemia Net's risk classification can be improved by RASGEF1A expression levels or methylation levels.

Transgenic IDH2R172K and IDH2R140Q zebrafish models recapitulated features of human acute myeloid leukemia

Isocitrate dehydrogenase 2 (IDH2) mutations occur in more than 15% of cytogenetically normal acute myeloid leukemia (CN-AML) but comparative studies of their roles in leukemogenesis have been scarce. We generated zebrafish models of IDH2R172K and IDH2R140Q AML and reported their pathologic, functional and transcriptomic features and therapeutic responses to target therapies. Transgenic embryos co-expressing FLT3ITD and IDH2 mutations showed accentuation of myelopoiesis. As these embryos were raised to adulthood, full-blown leukemia ensued with multi-lineage dysplasia, increase in myeloblasts and marrow cellularity and splenomegaly. The leukemia cells were transplantable into primary and secondary recipients and resulted in more aggressive disease. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in T-cell development at embryonic and adult stages. Single-cell transcriptomic analysis revealed increased myeloid skewing, differentiation blockade and enrichment of leukemia-associated gene signatures in both zebrafish models. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in interferon signals at the adult stage. Leukemic phenotypes in both zebrafish could be ameliorated by quizartinib and enasidenib. In conclusion, the zebrafish models of IDH2 mutated AML recapitulated the morphologic, clinical, functional and transcriptomic characteristics of human diseases, and provided the prototype for developing zebrafish leukemia models of other genotypes that would become a platform for high throughput drug screening.

The Role of Mutations Genes NPM1, FLT3, DNMT3A, CEBPA, IDH1&amp; IDH2 in Cytogenetically Normal Acute Myeloid Leukemia

Cytogenetically normal acute myeloid leukemia (CN-AML) is a type of hematopoietic tissue (bone marrow) cancer called acute myeloid leukemia. In normal bone marrow, early blood cells called hematopoietic stem cells develop into several types of blood cells: white blood cells (leukocytes) that protect the body against infection, red blood cells (erythrocytes) that carry oxygen and platelets (thrombocytes) play a role in blood coagulation, in acute myeloid leukemia, the bone marrow produces a large number of abnormal and immature white blood cells called myeloid blasts. Instead of becoming normal white blood cells, myeloid blasts turn into blood cancer cells.

Stratification and therapeutic potential of ELL in cytogenetic normal acute myeloid leukemia

Optimizing prognostic stratification of patients with cytogenetic normal acute myeloid leukemia (CN-AML), a highly heterogeneous subgroup in AML, appears to be important to improve its treatment and clinical outcome. Here, we report a potential role of ELL, a gene associated with leukemogenesis in AML, in prognostic stratification of CN-AML patients. By analyzing public available databases, we found that ELL was highly expressed in AML patients compared with healthy donors. Kaplan-Meier analysis revealed that ELL expression markedly correlated with short overall survival (OS) of CN-AML patients. In COX multivariable regression analysis, higher ELL expression was an independent prognostic factor for OS in CN-AML. Knockdown of ELL by shRNAs sensitized KG-1α cells to anti-leukemic agents such as idarubicin (IDA) and chidamide (CS055), supporting its role in therapeutic response and outcome in AML. To understand its function in CN-AML, we further analyzed the ELL-driving gene signature. ELL-related genes were particularly enriched in cell adhesion molecules, cell differentiation, pathways in cancer, sequence-specific DNA binding, and extracellular matrix (ECM)-receptor interaction. Analysis of the PPI network identified 25 hub genes, including the stem cell gene BMP4. While BMP4 expression was significantly associated with ELL in CN-AML, knockdown of ELL markedly down-regulated BMP4 expression, suggesting that ELL might function via regulating BMP4 in AML. Together, these observations suggest a novel mechanism underlying pro-leukemogenic role of ELL via BMP4 up-regulation in AML and its potential value to serve as a predictive biomarker for therapeutic response and outcome of CN-AML patients.

Clinical Implication of DNMT3A and TET2 Genes Mutations in Cytogenetically Normal Acute Myeloid Leukemia

Refining risk stratification of cytogenetically normal AML (CN-AML) cases is important for decision making and tailoring of therapy. In this context genetic and epigenetic mutations was considered. Among these epigenetic regulators are DNMT3A & TET2 genes. Therefore, the aim of this study was to determine the prevalence of DNMT3A and TET2 genes mutations and their impact on the outcome of adult AML patients. The present study is cross sectional study which was conducted on 39 adult CN-AML patients at diagnosis. For all included patients sanger sequencing was done for DNMT3A exon 23 and TET2 exon 3 genes. DNMT3A mutations were detected in 8 of 39 patients (20.5%), and in 5 of 39 patients(12.8%) in TET gene. Two CN-AML patients had combined mutations in both genes. All of the mutations detected were missense and only one was frame shift. Mutated TET2 or DNMT3A genes were significantly associated with failure of complete remission (CR) (p <0.001), higher mortality rate, shorter OS (mean=16 versus 22.7 months) and shorter DFS (mean= 9.5 versus 21.4 months) when compared to non-mutated ones. Mutated TET2 and DNMT3A detection define a subgroup of CN-AML patients with poor outcome.

Comprehensive analysis of ID genes reveals the clinical and prognostic value of ID3 expression in acute myeloid leukemia using bioinformatics identification and experimental validation

BackgroundDysregulation of inhibitor of differentiation/DNA binding (ID) genes is linked to cancer growth, angiogenesis, invasiveness, metastasis and patient survival. Nevertheless, few investigations have systematically determined the expression and prognostic value of ID genes in acute myeloid leukemia (AML).MethodsThe expression and clinical prognostic value of ID genes in AML were first identified by public databases and further validated by our research cohort.ResultsUsing public data, the expression of ID1/ID3 was markedly downregulated in AML, and the expression of ID2 was greatly upregulated in AML, whereas ID4 showed no significant difference. Among the ID genes, only ID3 expression may be the most valuable prognostic biomarker in both total AML and cytogenetically normal AML (CN-AML) and especially in CN-AML. Clinically, reduced ID3 expression was greatly associated with higher white blood cell counts, peripheral blood/bone marrow blasts, normal karyotypes and intermediate cytogenetic risk. In addition, low ID3 expression was markedly related to FLT3 and NPM1 mutations as well as wild-type TP53. Despite these associations, multivariate Cox regression analysis revealed that ID3 expression was an independent risk factor affecting overall survival (OS) and disease free survival (DFS) in CN-AML patients. Biologically, a total of 839 mRNAs/lncRNAs and 72 microRNAs were found to be associated with ID3 expression in AML. Importantly, the expression of ID3 with discriminative value in AML was further confirmed in our research cohort.ConclusionThe bioinformatics analysis and experimental verification demonstrate that low ID3 expression independently affects OS and DFS in patients with CN-AML, which might be seen as a potential prognostic indicator in CN-AML.

A Novel Risk Stratification Model for Cytogenetically Normal Acute Myeloid Leukemia with FLT3-ITD Mutation

Introduction Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are observed in about 30 % of patients with newly diagnosed cytogenetically normal acute myeloid leukemia (CN-AML) and has now been acknowledged to be associated with a poor survival. However, long-term follow-up revealed a high degree of heterogeneity in the clinical prognosis of these patients. Further risk stratification to identify high-risk patients is urgently needed. Methods A total of 106 patients with CN-AML and FLT3-ITD mutations who visited the People's Hospital of Peking University from 2007-2019 were included, and target region sequencing was performed by 266 gene panel. Patients were randomly divided into training and validation cohort according to the ratio of 7:3. Molecular and clinical data in training cohort were incorporated to construct a nomogram model. Cumulative incidence of relapse (CIR), Relapse-free survival (RFS) and survival by allogenic hematopoietic stem cell transplantation (allo-HSCT) and consolidation chemotherapy were compared. Results Clinical variables, genes, and genetic groups with mutations occurring in more than 10 patients were enrolled for univariate analysis of RFS (allo-HSCT was recorded as a censored event). And to avoid bias patients with RFS ≤ 4.57 months (median interval from complete remission to allo-HSCT) would be excluded from the subsequent analyses unless receiving an allo-HSCT was treated as a censored event. Three variables were eventually entered into the nomogram analysis: DNMT3A mutation (+ vs. -), minimal residual disease detected by multiparameter flow cytometry (MPFC-MRD) at the end of the 2nd course of consolidation chemotherapy (CON2) (+ vs. -, represents ≥ vs. ＜0.01%), typical ITDs (+ vs. -, represents ITDs have insertion sequences that exactly match wild-type FLT3 gene vs. ITDs have insertion sequences containing other exogenous nucleotides). And the model demonstrated good discrimination in both the training and validation cohort with C-index value of 0.82 (95% confidence interval, 0.7-0.86), 0.86 (95% confidence interval, 0.76-0.95), respectively. The Nomogram model was constructed as: Score = 1.44×DNMT3A mutation+2.16×MFPC-MRD-0.94×typical ITDs (Figure 1, A). According to the variables in nomogram model, patients with risk scores ≤ 0 were assigned to the low-risk subgroup (N = 39) and the rest were assigned to the high-risk subgroup (N = 67). The 5-year RFS was significantly higher in the low-risk group than in the high-risk group (training cohort, HR 3.68 [1.16-11.66], P = 0.002; validation group, HR 4.45 [1.52-13.00], P = 0.03) (Figure 1, B-C). We then interrogated the effect of consolidation chemotherapy and allo-HSCT as post-remission therapies in the two risk subgroups. Allo-HSCT did not further improve the prognosis of patients in the low-risk group (allo-HSCT vs. chemotherapy, 5-year CIR, HR 0.46 [0.11-1.93], P = 0.29; 5-year RFS, HR 0.41 [0.09-1.86], P = 0.24; 5-year survival, HR 0.91 [0.18-4.68], P = 0.91) (Figure 2, A-D). In contrast, in the high-risk group of patients, transplantation showed a significant survival benefit (5-year CIR, HR 0.21 [0.06-0.74], P = 0.01; 5-year RFS, HR 0.23 [0.06-0.80], P = 0.01; 5-year survival, HR 0.25 [0.06-1.11], P = 0.05) (Figure 2, E-H). Conclusion Our study suggests that risk stratification by our novel nomogram scoring system can identify patients at high risk of relapse in CN-AML with FLT3-ITD mutations and allo-HSCT further benefits their prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acute Myeloid Leukemia with Isolated Trisomy 4 Is Characterized By Frequent Mutations of TET2 and a Distinct DNA Methylation Signature

Background: Trisomy 4 (+4) is a rare cytogenetic abnormality found in less than 1% of acute myeloid leukemia (AML) either as sole abnormality (isolated AML+4, iAML+4) or associated with other genetic lesions. However, iAML+4 is inadequately studied due to its rarity and little is known about its molecular profile. Aim: The aim of this study was to characterize the molecular profile of iAML+4, including gene mutations, gene expression and DNA methylation changes. Methods: For the molecular characterization, we have performed targeted amplicon sequencing (n=68 target genes), gene expression profiling (SCRB-seq) and DNA methylation profiling (Illumina Methylation EPIC arrays) on 11 iAML+4 patients from German AML study groups (AML-CG 1999 and AML-CG 2008) and 105 patients with cytogenetically normal AML (CN-AML). Results: By targeted sequencing, 35 mutations were detected in 11 genes with a median of 3 mutations per patient (range 1-7, Figure 1A). Recurrently mutated genes were TET2 (n=9 patients), NPM1 (n=5) and FLT3-ITD (n=4), DNMT3A (n=2), and WT1 (n=2). Of note, in 8/9 patients with TET2 mutations, a truncating mutation has been detected. Thus, the frequency of truncating TET2 mutations was exceptionally high (8/11; 73%) in iAML+4, as compared to CN-AML with 12/105 patients mutated (11%, P=2.4e-5, Fisher exact test), which is in line with previous findings in the literature. Moreover, 3 patients harbored multiple TET2 mutations. We have further performed targeted amplicon sequencing in 8 AML patients with non-isolated +4, where trisomy 4 was present along with other cytogenetic abnormalities. In non-isolated +4 AML, TET2 mutations were less frequently found, as compared to iAML+4 (2/8, 25%, P=0.07). Gene expression profiling revealed a distinct gene expression pattern in iAML+4 as compared to CN-AML patients. Across all chromosomes, differentially expressed genes were detected; with the highest numbers of overexpressed genes located on chromosome 4, followed by chromosomes 2, 12, 17 and 18, as well as a substantial number of downregulated genes, particularly on chromosomes 11 and 19, indicating a trans-regulatory effect of +4 (Figure 1B). DNA methylation analysis revealed a specific hypermethylation signature among iAML+4 patients, as compared to CN-AML (Figure 1C). A global increase of CpG methylation levels in iAML+4 may be likely due to the high frequency of truncating TET2 mutations, as TET2 is known to be involved in hydroxymethylation of the DNA. However, the hypermethylation signature was confirmed even when comparing iAML+4 to CN-AML within TET2 mutated patient samples, indicating DNA hypermethylation beyond the effect of truncating TET2 mutations. Hierarchical clustering of iAML+4 and CN-AML samples resulted in a separate cluster with a distinct hypermethylation signature, which is strongly enriched for iAML+4 patients. Of note, besides TET2 truncating variants, no additional mutation was found to be enriched in that cluster, indicating that +4 is the major (cyto) genetic driver mediating this pattern. Conclusion: Our comprehensive multi-omics analysis revealed a homogenous molecular profile of iAML+4 with high frequency of truncating TET2 mutations indicating oncogenic cooperativity, a global pattern of differential gene expression as well as a uniform DNA hypermethylation signature, which is generally not associated with truncating TET2 mutations but a specific feature of trisomy 4 in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Expression of BCAT1 Affects DNA Damage Response By Inhibiting α-KG-Dependent Histone Demethylase

Branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1) correlates with AML aggression, not only that, it has been reported that overexpression of BCAT1 in leukemia cells decreases intracellular αKG and impairs the activities of αKG-dependent dioxygenase. In this study, we firstly analyzed the prognostic significance of BCAT1 expression in Cytogenetically normal AML(CN-AML) patients. We found that patients with high levels of BCAT1 had poor prognosis, the median overall survival time is shorter in BCAT1 high group (356 days versus 570 days,p=0.004)(Figure A).Next, we performed gene knockout in THP-1 and MV4-11 cells and gene overexpressing in OCI-AML3 cells. In summary, BCAT1 suppression of BCAT1 markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B,C),whereas overexpression contributed to colony-forming and cell growth(Figure D,E). In vivo experiments confirmed that BCAT1 knockout can reduce the tumor burden of xenograft mice, while BCAT1 overexpression remarkably increased tumor burden (Figure F).Survival analysis showed that the BCAT1 OE group significantly shortened survival time(22.5 days vs 26.0 days), and the median survival time of the knockout group was 2.5 days longer than control(26.0 days vs 28.5 day ).(Figure G). To further clarify the mechanism by which BCAT1 affects the proliferation of AML cells, we collected specimens from 87 AML patients and performed RNA-Seq analysis. Through Gene Set Enrichment Analysis, we found that the DNA Genes down-regulated in response to ultraviolet (UV) radiation pathway was enriched in the high expression group of BCAT1(Figure H). Therefore, we further examined the effect of BCAT1 expression level on DNA damage repair in vitro.DNA damage foci studies were performed in control and BCAT1 OE cells. More γ-H2AX focis in BCAT1 overexpressed cells were observed. About 48.20% of BCAT1 OE cell were obeseved ＞5 γ-H2AX foci, which was much higher than the control group(13.6%). And significant regression of γ-H2AX levels in BCAT1 overexpressing cells after the addition of DM-αKG(21%)(Figure I).We used western blot to detect DSB repair-related proteins and found that ATM was also down-regulated in BCAT1 overexpressing cells, along with an upregulation γ-H2AX and the changes of these proteins can also be rescued by exogenous supplementation of DM-aKG(Figure J). High expression of BCAT1 reduces the level of intracellular α-KG and inhibits the activity of α-KG-dependent dioxygenases. Histone demethylase KDM2-7 are αKG-dependent dioxygenase, so we speculated that the decrease in a-KG caused by high BCAT1 expression would limit the activity of histone demethylase, thereby affecting the cellular histone methylation levels. We detected several classic histone methylation markers after BCAT1 overexpression, results showed that the levels of H3K9me3 were significantly up-regulated after BCAT1 overexpression, which could be reversed by supplementation of DM-α-KG(Figure K). Therefore, we believe that the regulation of cellular histone methylation levels by BCAT1 is affected by α-KG levels. H3K9me3 is an epigenetic suppressive marker. IDH1mut cells inhibited ATM expression by upregulating histone H3K9 methylation levels and impede cellular DNA damage repair. The elevated DNA damage level in BCAT1 overexpression condition may be therapeutically exploited using therapies that induce or augment DNA damage, such as chemotherapy and PARP inhibitor, Next, we tried to compare the responses of BCAT1 overexpression and control AML cells to the PARP inhibitor BMN673(Talazoparib). After treatment with BMN673, the survival fraction of BCAT1 overexpressing AML cells was lower than that of negative control in MTS assay. Vice versa, The IC50 of BMN673 in BCAT1 knockout AML cells is much larger than that of the control(Figure L). In summary, this study verified BCAT1 high expression is an independent prognostic predictor of CN-AML.BCAT1 reduces intracellular αKG levels, inhibits αKG-dependent histone demethylation enzymes to inhibit H3K9me3 demethylation, thereby suppressing ATM expression and inhibiting DNA damage response in AML cells. Therefore, BCAT1 high expressing AML cells have higher sensitivity to PARP inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular profiling of patients with cytogenetically normal acute myeloid leukemia and hyperleukocytosis.

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 ≥100G/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.

Clinical implications and genetical insights of SOX6 expression in acute myeloid leukemia.

Transcription factor SOX6 belongs to Sry-related high-mobility-group box (SOX) family, has been reported to be downregulated and acts as a tumor-suppressor gene in various solid tumors, but in acute myeloid leukemia (AML) is incompletely understood. The SOX6 expression was analyzed between AML patients and normal controls from public data and our research cohort. Correlations between SOX6 expression and clinical, genetic features together with survival were further analyzed. In both public and our present datasets, we demonstrated that SOX6 expression is notably downregulated in AML patients compared with normal controls. Moreover, the expression level of SOX6 was dynamic, along withthediseasestatus. SOX6 was significantly decreased in relapsed/refractory AML compared with complete remission AML. Clinically, SOX6 underexpression was significantly correlated with bone marrow blasts, and WBC counts. Furthermore, decreased expression of SOX6 was more common in core binding factor AML (CBF-AML), rarely found in complex karyotype AML (CK-AML), and correlated with FLT3 mutations. By survival analyses, low-expression of SOX6 was associated with shorter overall survival (OS) and event-free survival (EFS) among cytogenetic normal AML (CN-AML) patients. Moreover, both univariate and multivariate analyses showed that low SOX6 expression was an independent unfavorable prognostic biomarker for CN-AML. Our findings indicated that SOX6 underexpression, as a frequent event in AML, was associated with genetic abnormalities and prognosis in AML. SOX6 might be a valuable biomarker for risk stratification, predictingprognosisand relapse of AML.

Overexpression of Prohibitin 2 Protein is Associated with Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia.

Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40%–50% of all AML cases. Despite advances in understanding the molecular pathophysiology of CN-AML, its clinical outcome remains unsatisfactory and unpredictable. To investigate its clinical implication in CN-AML, we measured the expression of prohibitin 2 (PHB2) using immunohistochemical staining (IHCS) of paraffin-embedded bone marrow sections from 134 CN-AML patients. IHCS results were semi-quantitatively scored. Clinical outcome was analyzed in comparison with other prognostic markers, including NPM1 polymorphism and FLT3 internal tandem duplication, and WT1 and BAALC mRNA expression. Except for BAALC mRNA expression, the known molecular markers showed no prognostic effect in the CN-AML patients. PHB2 protein overexpression was significantly associated with adverse prognosis in CN-AML patients. The PHB2 protein expression status may serve as an independent prognostic indicator in CN-AML.

A confirmation of ASXL1 as biological marker of normal cytogenetic acute myeloid leukemia with myelodysplasia-related changes (AML-MRC): A case successfully treated with CPX-351

We here report the case of a 56 yr old patient affected by acute myeloid leukemia with myelodisplasia related changes (AML‐MRC), ASXL1, successfully transplanted after CPX-351 treatment. Prats Martin et al.