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ETO2 is a nuclear co-repressor, which plays a critical role in the regulation of the cell cycle, self-renewal capacity, and differentiation of hematopoietic progenitor cells. We identified novel fusion transcripts involving ETO2 and CTCF by RNA-seq in a multiple relapsed AML case. The CTCF-ETO2 and ETO2-CTCF chimeric genes were validated by RT-PCR and Sanger sequencing. In addition, both transcripts apparently promoted cell proliferation via JAK/STAT3 pathway that is sensitive to STAT3 inhibitors. The novel fusions may have prognostic value and pathogenic mechanisms in acute myeloid leukemia.

Mixed lineage leukemia (MLL) T10 is a relatively rare partner for the KMT2A lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with KMT2A-MLLT10 remain unknown. In this study, 10 adult AML patients with KMT2A-MLLT10 fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with KMT2A-MLLT10 were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0–4). The most frequently mutated gene was NRAS (n = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (n = 7), a high rate of relapse, and biomarkers CD33 (n = 10), CD117 (n = 9), CD13 (n = 8), and CD64 (n = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.

Background Autosomal trisomy is a relatively rare abnormality observed in AML, occurring singly or as a secondary event in association with other karyotypic changes, and associated with prognosis. The molecular genetic and clinical characterizations of acute myeloid leukemia (AML) with isolated trisomy 4, 11, or 21 have been poorly investigated. Materials and methods Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for 41 chromosomal gene translocations/fusion genes, and next-generation sequencing (NGS) were performed on 29 AML patients with trisomy 4, 11, or 21 as the sole chromosomal anomaly. Results Of the 29 patients, one or more mutations were detected in 93.1% of patients. CEBPA had the highest mutation frequency, followed by TET2, NPM1, DNMT3A, and FLT3-ITD. The sole +11 AML patients exhibited more mutations in FLT3-ITD (P = .031) than the sole +21 AML patients, while CEBPA mutation was more frequently found in the sole +21 AML patients than that in the sole +11 AML patients(P = .07). The median overall survival (OS) and disease-free survival (DFS) for patients with +11 were shorter than those with +4(P = .015, 0.046) or +21 (0.057, 0.064), but no difference was found between +4 patients and +21 patients. In the whole cohort, only the FLT3-ITD mutation was significantly associated with inferior OS (18 vs. 35 months, P = .023) and DFS (12 months vs. NR, P = .046). There were no significant differences in OS and DFS according to the gene mutation status of CEBPA, TET2, NPM1, DNMT3A, and IDH1/2. Conclusion There was a significantly different mutation profile among the sole +4, +11, +21 AML patients. Our research provided new insight into the molecular characteristics of AML with isolated trisomy.

Objective Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) frequently emerge in acute myeloid leukemia (AML), but the clinical features and molecular characteristics of IDH mutational status and other coexisting mutations have not been investigated in a large extensively characterized AML series. The aim of this study was to gain insight into the mutational profile of IDH-mutated patients, such as the frequency and clinical characteristics of coexisting mutated genes. Materials and methods We investigated 485 newly diagnosed AML patients (range 18-81 years). DNA was extracted from bone marrow samples at the time of diagnosis. All samples were investigated with a panel of 49 mutational genes using next-generation sequencing (NGS). FLT3-ITD, NPM1, and CEBPA mutations were detected by Sanger PCR sequencing. Results We found 84 patients (17.3%) with IDH1 or IDH2 mutations. There were 40 IDH1R132 , 15 IDH2R140Q , 17 IDH2R172K , and 12 uncommon mutations. No patient was found to have both IDH1 and IDH2 mutations. Patients with IDH2R140Q mutations were more frequently older and presented with significantly lower average platelet counts, while IDH2R172K -mutated patients had significantly lower white blood cell (WBC) counts. On the background of IDH mutations, the presence of a normal karyotype showed a balanced distribution. The four most frequently coexisting mutated genes were NPM1, DNMT3A, TET2, and FLT3-ITD. The majority of coexisting mutated genes were involved in regulating transcription and DNA methylation. IDH mutation status had no effect on the CR rate, regardless of other molecular abnormalities. Conclusion Isocitrate dehydrogenases mutations are associated with a complex coexisting mutation cluster in AML. Future investigation is needed to reveal the association between IDH mutations and other genetic abnormalities, which may have an impact on the progression and prognosis of disease.

Introduction: Several studies have confirmed that mutations in the Wilms tumor 1 (WT1) gene occur in adult acute myeloid leukemia (AML). However, few data are available regarding the incidence of WT1 mutations in CEBPAmut AML and their impact. Methods: We retrospectively analyzed the frequency and clinical impact of WT1 mutations in 220 newly diagnosed AML patients with CEBPA mutations. Chromosome karyotype analysis was performed by R or G banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT-PCR). Mutations were detected with a panel of 112 mutational genes using next-generation sequencing (NGS). FLT3-ITD, NPM1 and CEBPA mutation were detected by PCR Sanger sequencing.Results: Overall, 30 WT1 mutations were detected in 29 of the 220 patients (13.18%) screened. These mutations clustered overwhelmingly in exon 7 (16 mutations in 15 patients), but they were also detected in exon 8 (n=6) and exon 9 (n=8). WT1-mutated (WT1mut) patients presented with lower platelet counts (P=0.0481), higher WT1 expression (P=0.0371), and a negative trend with the M5 subtype (P=0.07) compared to WT wild-type (WTwt) patients. WT1 mutations were found to be significantly more frequent in AML patients with double-mutated CEBPA (CEBPAdm) than in AML patients with single-mutated CEBPA (P=0.043). Of note, the concomitant mutations in epigenetic regulators were inversely correlated with WT1 mutations (P=0.003). Patients with newly diagnosed WT1mut AML had inferior relapse-free survival, event-free survival and overall survival compared with patients with WT1wt AML (P=0.002, 0.004, and 0.010, respectively). Conclusion: Our data showed that WT1 mutations are frequently identified in CEBPAmut AML, especially in CEBPAdm AML. Patients with WT1 mutations show distinct clinical features, a spectrum of comutations, and poor prognosis compared to WT1 wild-type patients.

More than 90% of NPM1-mutated acute myeloid leukaemia (NPM1

Objectives: The 8p11 myeloproliferative syndrome (EMS) is an extremely rare, generally aggressive haematologic malignancies. This study provided the clinical outcomes and therapeutic strategies for...

Introduction: RUNX1 mutations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with distinct clinicopathologic features. However, the clinical and laboratory chara...

Background Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive and poor prognostic malignant tumor along with high-level infection of Epstein-Barr virus (EBV). Gemcitabine (Gem) and Thymosin alpha 1 (Tα1) exert an anti-tumor effect in various cancers. However, the effect of the combination of Gem and Tα1 in NNKTL remains unknown. Methods SNK6 cells were treated with Gem, Tα1 and Gem plus Tα1 for 48 h. The expression levels of EBV and inflammatory factors were measured by qRT-PCR assay. The effect of Gem and Tα1 on cell viability, proliferation, apoptosis, autophagy was detected by CCK-8, colony formation, flow cytometry, autophagic flux measurement, respectively. Western blot was used to evaluate the expression of proteins related to epithelial-mesenchymal transition (EMT), apoptosis and autophagy. In vivo xenograft models were used to further verify the roles of Gem and Tα1. Tumors were removed for weight measurement, H&E and IHC staining. Results We identified that the half maximal inhibitory concentration (IC50) of Gem and Tα1 was 116.5 μmol/ml and 1.334 μmol/ml. Alone or combined administration of Gem and Tα1 dramatically attenuated the EBV viral load and promoted inflammatory factors expression in SNK6 cells, among which the combination of Gem and Tα1 treatment showed the most significant effect. Besides, combination treatment with Gem and Tα1 markedly inhibited cell growth and EMT progress, and enhanced apoptosis and autophagy. Similarly, Gem combined with Tα1 suppressed tumor growth, promoted apoptosis and autophagy in vivo. Additionally, combination treatment with Gem and Tα1 inhibited PI3K/AKT/mTOR pathway. Conclusion In summary, combination administration of Gem and Tα1 suppressed the progression of NNKTL in vivo and in vitro. Our study provided an effective therapeutic strategy potentially for the clinical treatment of NNKTL.

INTRODUCTION Cytogenetics and molecular testings for disease classifying and prognosis estimation are becoming routine in clinical practice. However, the molecular characteristics of acute monocytic leukemia (AML-M5) remain unclear. The aim of this study was to investigate the association between karyotypes and gene mutations, especially in AML-M5 patients with 11q23/KMT2A (MLL) rearrangement and normal karyotype. METHODS A total of 126 de novo AML-M5 patients were screened for mutations in the 51 genes known or suspected to have a role in myeloid malignancies or in monocytic differentiation using next-generation sequencing (NGS). Chromosome karyotype analysis was performed by R-banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT-PCR). RESULTS Of the 126 patients, one or more mutations were detected in 83.3% patients. FLT3-ITD and NRAS had the highest mutation frequency, followed by NPM1, DNMT3A, TET2, KRAS, and RUNX1. We also identified a significant difference in mutational spectrums between KMT2A-rearranged (KMT2Ar) patients and normal karyotype (NK) patients, as reflected in the average number of gene mutations per patient (1.66 vs 2.46), and in the frequencies of commonly mutated genes (FLT3-ITD: 6% vs 43.5%; NPM1: 0% vs 43.5%; RUNX1: 2.0% vs 15.2%; DNMT3A: 4% vs 26.1%; KRAS: 24.0% vs 4.35%). Patients harboring ≥3 mutations showed much lower complete remission rate than that with double mutations (P = 0.043) in high-risk group. CONCLUSION There was a significantly different mutation profile between KMT2Ar-patients and NK patients. Our research provided new insight into the molecular characteristics of AML-M5.

Summary Introduction: Gene mutations play an important role in acute myeloid leukemia (AML) pathogenesis. Several genes have been identified in AML, such as FLT3, KIT, NPM1, and JAK2. This study investigated the frequency of novel mutations in IDH1 (amino acid R132) and IDH2 (R140 and R172) and analyzed their impact on disease biology and interaction with other mutations in Chinese patients with de novo AML. Methods: A total of 195 patients were screened for mutations in the IDH1, IDH2, JAK2 V617F, NPM1, FLT3, and KIT genes, using polymerase chain reaction (PCR)-based and direct sequencing assays. Results: IDH mutations occurred at a considerable frequency of 15.89% in Chinese AML cases; IDH2 R140Q was the most frequent genetic alteration and was associated with older age, normal karyotype, and French-American-British classification M2 at diagnosis. There was a strong association of IDH2 mutation with NPM1 mutations and a trend with FLT3-internal-tandem duplication. Conclusion: IDH mutations may be a novel genetic marker in cytogenetically normal AML and may cooperate in leukemogenesis.

Abstract 4647 Introduction: Myelodysplastic syndromes (MDSs) comprise a heterogeneous group of stem cell disorders characterized by ineffective hematopoiesis presenting with peripheral cytopenias together with hyperplastic bone marrow.Patients with MDS show an increased but variable risk of progression to acute myeloid leukemia (AML).Although, cytogenetic abnormalities are known as a major role in pathogenetic mechanisms, only 30% to 50% of patients with de novo MDS have abnormal karyotype,An further understanding of the molecular pathogenesis of MDS is needed, however,littleis known about the genetic events and their potential value for predicting progression to AML. We aimed at studying the potential significance of IDH1,IDH2, JAK2V617F, NPM1, FLT3-ITD,c-KIT genes mutations in MDS and analyzed the interaction with other mutations in Chinese patients with MDS. Methods: We examined Chinese patients with de novo MDS for mutations in the IDH1,IDH2, JAK2, NPM1, FLT3-ITD,C-KIT genes.Female/male ratio was 39/49 and age ranged from 28.0–79.0 years (median, 58.0years).Genomic DNA was extracted from diagnostic marrow specimens,FLT3 internaltandem duplication(FLT3 –ITD) and JAK2V617F mutation was screened using polymerase chain reaction (PCR),c-KIT, NPM1 and IDH genes were assessed by PCR followed by direct sequencing, according to previously described protocols. Results: F Overall, in 7 patients (7.95%) IDH mutations were detected. None of them had IDH1 mutation,6 missense mutations (6.8%) in IDH2 (R140Q n=6) and 1 synonymous substitution (c.519G>A) in IDH2. Molecular genetic analysis of NPM1 and JAK2V617F revealed the presence of NPM1 exon 12 (2.27% 2/88) and JAK2V617F(3.4% 3/88 ) mutations. FLT3-ITD,c-KIT mutations in exon 8 and 17 were not detected at MDS stage and concurrent mutations were not found. 4 of 6 cases with IDH2 missense mutations presented normal karyotype. IDH2 mutations were restricted to 3 morphologic categories:refractory cytopenia with multilineage dysplasia (RCMD n=2),refractory anemia with excess blasts (RAEB-2 n=3) and refractory anemia with ringed sideroblasts(RARS n=1), Patients with IDH2 mutations were older (P60years),but mutation status that is or not potentially useful for predicting progression to AML need further studying. Conclusions: F IDH2 mutations are frequent in Chinese patients with de vono MDS, and associated with older age, lower WBC count at diagnosis.JAK2V617F and NPM1 mutations are rare in MDS,but both of them correlated with morphologic categories.IDH1,c-KIT,FLT3-ITD were detected in our cohort,suggesting that these mutations may be not a key role in pathogenetic mechanisms of MDS. Disclosures: No relevant conflicts of interest to declare.