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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.

We evaluated the mutational landscape of chronic myelomonocytic leukemia (CMML) and its potential clinical significance. We analyzed 47 samples with a panel of 112 genes using next-generation sequencing. Forty-five of the 47 patients (95.74%) had at least one mutation identified, with an average of 3.7 (range 0-9) per patient. The most common mutation was NRAS, followed by ASXL1, TET2, SRSF2, RUNX1, KRAS, and SETBP1. Patients 60 years and older more frequently had mutations in TET2 (56% vs. 9.09%, P = 0.001) and ASXL1 (48% vs. 18.18%, P = 0.031) than patients younger than 60 years. Median overall survival (OS) in patients with CMML was 22.0 months (95% CI 19.7-24.3 months). ASXL1 (18 vs. 22 months, P = 0.012), RUNX1 (17 vs. 22 months, P = 0.001), and SETBP1 (20 vs. 27 months, P = 0.032) mutations predicted inferior OS. However, only RUNX1 mutation was significantly associated with inferior acute myeloid leukemia (AML)-free survival. Our data showed that mutation profile differed significantly between CMML patients aged 60 years and older versus those younger than 60 years, and some of these mutations impact the progression and prognosis of the disease to a certain extent.

The aim of the study was to determine molecular genetic and clinical characterization of acute myeloid leukemia (AML) with trisomy 8 as the sole chromosome abnormality, a recurrent but rare chromosomal abnormality in AML. Interphase fluorescence in situ hybridization, reverse transcriptase-quantitative polymerase chain reaction for gene rearrangement and next-generation sequencing (NGS) were performed on sole trisomy 8 AML patients. A total of 35 AML patients with trisomy 8 as the sole chromosome abnormality were screened. The most frequently mutated genes were DNMT3A(37.1%), RUNX1(28.6%), FLT3-ITD(28.6%), IDH2(22.9%), NPM1(17.1%), and ASXL1 (14.3%). The sole +8 AML patients exhibited more mutations in RUNX1 (28.6% vs. 4.8%, P = 0.001) and ASXL1 (14.3% vs. 4.8%, P = 0.039) by comparing with normal karyotype AML (NK AML) patients(n = 63). The sole +8 AML patients(n = 35) with RUNX1 or IDH2 mutations showed significantly lower WBC counts, while FLT3-ITD showed higher white blood cell (WBC) counts as compared to the corresponding wild-type groups. Total of 45.7% patients achieved complete remission (CR) after the first induction therapy. The CR rate of patients with FLT3-ITD or IDH1 mutation was significantly lower than that in the corresponding wild-type cases (P = 0.047, 0.005, respectively). The median overall survival (OS) and disease-free survival (PFS) were 18.0 (95% CI: 10.8–25.2) and 10 (95% CI: 6.7–13.3) months, respectively. FLT3-ITD mutations and allogeneic hematopoietic stem cell transplantation (allo-HSCT) were independent prognostic markers for OS in multivariable analysis. The results suggest a possible association between trisomy 8 and additional mutations that may influence clinical feature and prognosis.

BACKGROUND: Chordoma is a rare malignant bone tumor with high recurrence and metastasis rates. Little is known about the mutational process of this incurable disease. The aim of our research was to explore the potential driver genes and signal pathways in the pathogenesis of chordoma and provide a new idea for the study of molecular biological therapy of chordoma. METHODS: We performed whole-exome-sequencing (WES) on 8 sacrum chordoma tissue samples (matched to peripheral blood samples that had been drawn from patients before surgery) to identify genetic alterations in Chinese patients. We analyzed the sequencing data from known driver genes, pathway enrichment analysis and significantly mutated genes (SMGs) after quality control of sequencing, comparison of reference genomes, analysis of mutations and identification of somatic mutations. Immunohistochemistry staining, Sanger sequencing and GeneChip were used to verify the related genes obtained from the analysis of sequencing data. RESULTS: The driver genes Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA), Phosphoinositide-3-Kinase Regulatory Subunit 1 (PIK3R1), and Phosphatase And Tensin Homolog (PTEN) were enriched in the Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway and could be potential therapeutic targets for the treatment of sacrum chordoma. The significantly mutated gene Claudin 9 (CLDN9) may play a critical role in the development and progression of sacrum chordoma. CONCLUSIONS: Collectively, our results identified the genetic signature of sacrum chordoma and could be used to develop a potential promising therapeutic strategy for the treatment of sacrum chordoma in Chinese 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.

We report the co-mutations in AML with CEBPAWe retrospectively sequenced 112 genes in 302 patients with CEBPA① The average number of mutations in CEBPACEBPA

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...

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.

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

To detect ASXL1 gene variants among patients with myelodysplastic syndrome (MDS) and explore their correlation with variants of other genes and clinical features of patients.For 149 patients with MDS, genomic DNA was amplified by PCR and subject to direct sequencing to identify variants of ASXL1, U2AF1, SF3B1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT genes.ASXL1 variants were found among 37 patients (24.8%). Other commonly mutated genes included U2AF1 (22.8%), TET2 (11.4%), DNMT3A (9.4%), NPM1 (8.1%) and SF3B1 (6.0%). The frequency of concurrent U2AF1 and TET2 variants among patients with ASXL1 variants was slightly higher than that of wild-type patients. No significant difference was found in median age, MDS subtype, karyotype, peripheral leukocytes, hemoglobin, platelet levels, and bone marrow blast counts between the ASXL1-variant and the wild-type groups (P0.05). Twenty-nine patients harboring ASXL1 variants were followed up, 37.9% progressed to acute myeloid leukemia (AML). The rate of transformation in ASXL1-variant group was significantly higher than the wild-type group (37.9% vs. 14.1%, P0.01).ASXL1 showed a high frequency of variant among MDS patients, which was frequently accompanied with U2AF1 and TET2 variants. Compared with the wild type group, patients with ASXL1 variants were more likely to progress to AML.

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.

Highlights l Next generation sequencing (NGS) applied to establish clonality in a patient with hypereosinophilia. l The first study reported the clonal evolution in a long-term follow-up patient from CEL-NOS stage to MDS and s-AML. l Disease progression was associated with clonal evolution., The natural history of primary hypereosinophilia remains poorly defined, given the underlying disease heterogeneity. Recently, targeted NGS helps to establish clonality in a subset of patients with hypereosinophilia. We first reported the clonal evolution in a long-term follow-up patient with hypereosinophilia. This case initially presented with chronic eosinophilic leukemia, not otherwise specified (CEL-NOS), successively transformed to myelodysplastic syndromes (MDS) and acute myeloid leukemia(s-AML). We identified three mutations at CEL-NOS phase, five and seven mutations at MDS and s-AML stages, respectively. Our data illustrate the clonal dynamic process associated with disease evolution from CEL-NOS to s-AML.

To carry out mutation analysis for patients with myelodysplastic syndromes (MDS) and a normal karyotype.Targeted capture and next-generation sequencing (NGS) was carried out using a customized 49-gene panel. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutations were detected by PCR and Sanger sequencing.Sixty-two patients (80.5%) were found to harbor at least one mutation. Each patient has carried 2.21 mutations in average. Coexistence of ≥ 3 mutations was common (43.7%). The most commonly mutated genes were RUNX1 (23.4%, 18/77), ASXL1 (18.2%, 14/77), NPM1 (15.6%, 12/77), U2AF1 (15.6%, 12/77), DNMT3A (11.7%, 9/77). Patients with SF3B1 mutations were significantly older than those with ASXL1 mutations (P=0.023). Mutations of the DNMT3A gene were significantly associated with the blood platelet level compared with BCOR mutations (P=0.02). No significant difference was found in the number and rate of mutations between those under or above 60-year-old. Among 67 patients with clinical follow-up, 20 (29.8%) has transformed to acute myeloid leukemia, and the time of transformation has ranged from 1 to 44 months, with a average of 5.3 months. RUNX1, U2AF1 and FLT3 mutations are associated with leukemic transformation.Coexistence of ≥ 3 mutations are frequent among patients with normal-karyotype MDS. Certain mutations are associated with age and leukemic transformation.

To characterize the mutational profile of patients with core-binding factor acute myeloid leukemia (CBF-AML).A total of 81 acute myeloid leukemia patients were recruited, which included 36 cases of CBF-AML and 45 cases of cytogenetically normal acute myeloid leukemia (CN-AML) . Mutations of FLT3-ITD, FLT3-TKD, NPM1, c-KIT, NRAS, KRAS, TET2, IDH1/2, RUNX1, DNMT3A, GATA2, ASjXL1, TP53, PTPN11, JAK2V617F, SETBP1 and CEBPA genes were simultaneously detected by DNA-based PCR and Sanger sequencing.Over all, mutations were detected in 68 patients (83.9%), with the most common ones including double CEBPA mutations (n=17), followed by NPM1 (n=15), c-KIT (n=11), NRAS (n=10), TET2 (n=9), FLT3-TKD (n=9), FLT3-ITD (n=8), IDH1 (n=7), RUNX1 (n=7), KRAS (n=7), DNMT3A (n=6), IDH2 (n=4), and GATA2 (n=4) mutations. AML1-ETO and CBFβ-MYH11 fusions were present in 21 and 15 patients, respectively. Coexistence of ≥2 mutations was more common in CN-AML comparing with CBF-AML. The mutation rate of NPM1, FLT3-ITD, DNMT3A, IDH1 and CEBPA double mutations were higher in patients with CN-AML. NRAS, c-KIT and KRAS mutations were identified more frequently in patients with CBF-AML (P0.05). Based on the function, aberration of genes involved in DNA methylation, NPM1 proteins and transcription predominated in CN-AML, while tyrosine kinase receptor signaling and RAS pathways have predominated in CBF-AML.The genomic landscape of CBF-AML patients has differed from that of CN-AML patients. Synergy of fusion genes with particular mutations may impact the clinical phenotype and prognosis of patients.

To characterize the molecular genetics of 81 patients with acute monocytic leukemia (AML).Fluorescence in situ hybridization (FISH) was employed to detect MLL gene rearrangements. Combined mutations of 17 genes were detected by DNA-based PCR and Sanger sequencing.Sixty seven patients were found to harbor at least one mutation. The most commonly mutated gene was NPM1 (n=18), which was followed by FLT3-ITD (n=16), NRAS (n=16), DNMT3A (n=15), TET2 (n=12), RUNX1 (n=11) and KRAS (n=9). Based on the functions of mutated genes, the most frequently involved genes were those involved in DNA methylation (38.27%), tyrosine kinase receptor signaling (32.1%), transcription regulation (28.4%), and RAS pathway (24.7%). Single gene mutation predominated in patient with cytogenetic abnormalities, while coexistence of 2 mutations have predominated in patient with normal cytogenetic findings. Stratified by cytogenetic findings, patients with single gene mutations (intermediate-risk group) had significantly higher complete remission (CR) rates than those with ≥2 gene mutations (unfavorable-risk group) (91.7% vs. 57.6% , 87.5% vs. 25.0%, P =0.0319, 0.0117, respectively).Over 80% of AML patients were found to harbor at least one mutation. Their clinical phenotype and prognosis may be impacted by the synergy of MLL gene rearrangement and multiple mutations. For patients under the same risk stratification, the number of mutations is reversely correlated with the CR rate.

To study the correlation of hematomorphology, bone marrow cytogenetics and clinical biochemical parameters with the prognosis of non-Hodgkin's lymphoma with bone marrow invasion.Morphological analysis of bone marrow cells was performed by routine bone marrow puncture.Chromosome samples were prepared by short-term bone marrow culture. Karyotype analysis was carried out by R-banding in 28 patients. P53 gene was detected by fluorescence in situ hybridization (FISH). Serum lactate dehydrogenase (LDH) of all patients was determined and compared.In all patients, bone marrow morphology showed invasion of lymphoma. Chromosome analysis revealed abnormal karyotypes in 19 cases, which yielded an incidence of 67.85%. The proportion of lymphoma cells in bone marrow among those with an abnormal karyotype was much higher than those with a normal karyotype (60.2% vs. 33.5%, P0.05). FISH assay showed that 9 (32.14%) patients had P53 gene deletion. And the deletion was much more common among those with an abnormal karyotype (42.11% vs. 11.11%, P0.05). The serum LDH level in patients with an abnormal karyotype was significantly higher compared with whose with a normal karyotype (1464.37 U/L vs. 294.33 U/L, P0.05).Patients with abnormal karyotypes have a higher rate of P53 gene deletion, and their LDH level is significantly higher than those with a normal karyotype, which predicted a relatively poor prognosis.

To delineate the clinical and molecular characteristics of a patient with myeloid neoplasm and co-existence of t(7;11)(p15;p15) and t(5;12)(q33;p13) translocations.Clinical data of the patient was collected. Conventional karyotyping, reverse transcriptase (RT)-PCR and next generation sequencing (NGS) were carried out to delineate its genetic features.The patient has featured recurrent rash, fatigue, loss of appetite and splenomegaly. Laboratory test suggested hyperleukocytosis of FAB-M2-subtype. Neither eosinophilia nor basophilia was presented. NUP98/HOXA9 and ETV6/PDGFRB fusion genes were detected by RT-PCR. NGS and DNA-PCR showed the co-existence of WT1 p.C423Y, KRAS p.G12D and DNMT3A p.R882C mutations. The patient achieved morphological remission after imatinib plus coventional chemotherapy (standard IAC regimen). However, the disease has relapsed shortly after. Treatment was switched to HHT-Ara-C-Acla regimen, no hematological response was observed. The ETV6/PDGFRB fusion gene was undetectable in bone marrow sample, though strong expression of NUP98/HOXA9 was detectable throughout the whole course.Acute myeloid leukemia in association with the co-existence of NUP98/HOXA9 and ETV6/PDGFRB fusion genes have unique clinical and genetic features. Imatinib seems to have no impact on the overall survival in such cases.

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.

Todelineate the clinical and genetic features of a patient with myeloproliferative neoplasm (MPN) in association with PDGFRA and EVI1 genes rearrangements.Clinical data of the patient was collected. Conventional cytogenetics, fluorescence in situ hybridization (FISH) and nested PCR were carried out for the patient.The patient has featured recurrent rash, joint pain, and intermittent fever. Laboratory tests showed hyperleukocytosis and marked eosinophilia. Physical examination revealed splenomegaly. His karyotype was 46,XY,t(3;5)(q26;q15)[6]/46,XY[10]. FISH assay showed that both PDGFRA and EVI1 genes were rearranged. Molecular studies of the mRNA suggested that there was a in-frame fusion between exon 12 of the PDGFRA gene and exon 9 of the FIP1L1 gene. Imatinib was initiated at a dosage of 200 mg, and after 10 months, the signal of the FIP1L1-PDGFRA fusion gene was undetectable in bone marrow sample. However, the expression of EVI1 mRNA was stable, with no significant difference found between the patient and 10 healthy controls.MPN in association with PDGFRA and EVI1 genes rearrangements have unique clinical and genetic features. Genetic testing is helpful for early diagnosis. Imatinib may be effective for the treatment.

To assess the correlation of cytogenetic changes with serum vascular endothelial growth factor (VEGF) and serum tartrate resistant acid phosphatase (TRacp-5b) levels among elderly patients with multiple myeloma (MM).Chromosomal changes were analyzed with a modified culturing method in the presence of IL-6. Serum levels of VEGF and TRacp-5b were determined with enzyme-linked immunosorbent assays (ELISA).Among the 60 MM patients, chromosomal abnormalities were found in 27 cases, including 22 with numerical abnormalities and 15 with structural abnormalities. Many patients had both numerical and structural abnormalities. For 33 patients with a normal karyotype, the levels of VEGF and TRacp-5b were 117.35 ± 55.26 pg/mL and 4.15 ± 2.15 U/L, respectively, while for 27 patients with an abnormal karyotype, the levels of VEGF and TRacp-5b were 190.26 ± 85.74 pg/ml and 5.96 ± 2.24 U/L, respectively. The difference between the two groups was significant (P0.05).Compared with MM patients with a normal karyotype, the levels of VEGF and TRacp-5b are higher in those with cytogenetic abnormalities.

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.

An as-cast Mg–Al–Y–Zn alloy was successfully processed by equal channel angular extrusion (ECAE) in the temperature range of 225–400 °C, and the influences of processing temperature on the microstructure and mechanical properties were investigated. The use of back pressure during one-pass ECAE of Mg–Al–Y–Zn alloy was favorable for eliminating the undeformed area in the billet. At the processing temperature below 250 °C, the microstructures were characterized by unrecrystallised structure and the precipitated phase Mg 17 Al 12 was elongated along the extrusion direction. With increasing processing temperature to 350 °C, a large number of recrystallised grains were obtained. Increasing processing temperature promoted workability but led to decrease in the strength of Mg–Al–Y–Zn alloy. Then billets of as-cast Mg–Al–Y–Zn alloy were extruded at different numbers of ECAE passes. It was found that the microstructure was effectively refined by ECAE and mechanical properties were improved with numbers of ECAE passes increasing from one-pass to four passes. However, strengths decreased slightly after five passes though the grain size decreased considerably.

Semi-solid processing of magnesium alloys is generally based on conventional magnesium-based casting alloys such as Mg–Al series. However, these casting alloys do not give such high mechanical properties as the alloys that are conventionally wrought such as Mg–Zn series. In this paper, a ZK60 magnesium alloy with the addition of Y was thixoforged. The semi-solid thermal transformation (SSTT) route and the recrystallisation and partial melting (RAP) route were used to obtain the semi-solid feedstocks for thixoforging. Microstructural evolution during partial remelting was studied at temperatures for times. Tensile mechanical properties of thixoforged components at room temperature were examined. Results show that a fine spheroidal microstructure can be obtained by the RAP route. Compared to the RAP route, the SSTT alloy shows coarsened solid grains with a relatively high proportion of intragranular liquid droplets. With prolonged holding time, the solid grain sizes of the SSTT alloy and the RAP alloy increased. Coalescence was dominant in the SSTT alloy and Ostwald ripening was dominant in the RAP alloy. Thixoforging for the SSTT alloy and the RAP alloy resulted in successful filling of the die. The tensile properties of the thixoforged RAP alloy were satisfactory and exceeded those of the thixoforged SSTT alloy. However, the mechanical properties of both the thixoforged SSTT alloy and the thixoforged RAP alloy decreased with prolonged holding time.

AZ31 magnesium alloy sheets with a thickness of about 2mm were prepared by using a unique hot rolling process which was featured by heating the rolls during rolling. Two different rolling routes were used to achieve the final thickness through 6 passes of rolling. The major rolling parameters were chosen as the same for both rolling routes except that the roll temperature was set to be 400°C for route A and the 1st to 4th passes of route B, but lowered to 350°C for the 5th and 6th passes of route B. The microstructure and mechanical properties of the alloy sheets were comparatively investigated. The results showed that dynamic recrystallization occurred during hot rolling, and by choosing the processing parameters appropriately the grain size could be refined steadily with increasing rolling passes. The final alloy sheet prepared by 6 passes of rolling via the rolling route B was featured by a very fine microstructure, with the grain size being 5μm in average. Correspondingly, it presented very high strength and tensile elongation, with its yield strength and tensile elongation achieving 206MPa and 26.4% in the transverse, and 196MPa and 27.6% in the rolling direction, respectively.

Ultrafine-grain size of ~1µm was achieved in an AZ31B Mg alloy wires with high strength prepared by cold drawing and subsequent annealing in the present study. Effects of cold-drawn area reduction (CAR) on strain hardening, recrystallized grain refinement and annealing temperature were investigated. The results showed that the maximum cold area reduction as high as 65.1% could be reached at room temperature, which resulted in the notable strain hardening, grain refinement and the decrease of annealing temperature.

To explore the mechanism of NK cell dysfunction in patients with multiple myeloma (MM).The expression of inhibitory receptors (CD158a and CD158b) and activating receptors NKG2D and NCRs (NKp30, NKp44 and NKp46) on CD3-CD56+NK cell of 13 MM patients and 30 healthy controls were analyzed by flow cytometry. The concentration of soluble NKG2D ligands (MICA, MICB, ULBP1, ULBP2 and ULBP3) in serum was detected by enzyme- linked immunosorbent assay (ELISA), and the cytotoxicity of NK cell against MM cell line by flow cytometry.There are no significant differences of percentage and absolute number of NK cells, and the expression level of CD158a and CD158b between MM patients and healthy individuals (P0.05). No NKp44 expression was detected on fresh isolated NK cells from both groups. There is no difference in inhibitor receptors expression between MM patients and healthy individuals but the expression of NKG2D, NKp30 and NKp46 on NK cells were higher in MM patients as compared with that in healthy individuals. The concentration of soluble NKG2D ligands in serum was higher in MM patients as compared with that in healthy individuals (P0.05). Cultured healthy individual's NK cells with MM patient's serum could significantly decrease its cytotoxicity against MM cell line U266 cells [(38.5 ± 6.5) % vs (25.4 ± 5.9)%, P=0.044］.The higher level of soluble NKG2D ligands in serum may be the mechanism of NK cell dysfunction in MM patient.

OBJECTIVE To explore the clinical and laboratory features of a patient with 8p11 myeloproliferative syndrome (EMS) and CEP110-FGFR1 fusion. METHODS Combined bone marrow cytology, fluorescence in situ hybridization, fusion gene detection was used to analyze the patient. RESULTS Clinically, the patient had many features similar to those with chronic myelomonocytic leukemia, which included hyperleukocytosis, marked eosinophilia, monocytosis, myeloid hyperplasia and hyperplasia. Fluorescence in situ hybridization analysis for FGFR1 gene rearrangement was positive. Further study of the mRNA also confirmed an in-frame fusion between exon 38 of the CEP110 gene and exon 9 of FGFR1 gene. CONCLUSION EMS with CEP110-FGFR1 fusion is a very rare and distinct myeloproliferative neoplasm. FISH and molecular studies may improve its diagnosis.

To improve the understanding of patients with 8p11 myeloproliferative syndrome (EMS) harboring ins(13;8)(q12;p11p23)/ZNF198 -FGFR1.We reported here a 8p11 EMS case and provided more details on the clinical and molecular features of ins(13;8)(q12;p11p23)/ZNF198-FGFR1，full length ZNF198-FGFR1 was cloned by overlap extension PCR method，and the literatures on this topic were reviewed.Clinically, the case with ins(13;8)(q12;p11p23)/ZNF198-FGFR1 had distinct hematological and clinical characteristics: hyperleukocytosis, myeloid hyperplasia，widespread adenopathy and lymphoma; Fluorescence in situ hybridization (FISH) disclosed the positive FGFR1 gene rearrangement; Further molecular studies confirmed a mRNA in-frame fusion between exon 17 of the ZNF198 gene and exon 9 of FGFR1 gene ，the full length ZNF198-FGFR1 was composed of a NH2 terminus of ZNF198 including the ZNF and proline-rich domains, whereas the COOH terminus of FGFR1 included 2 tyrosine kinase domains.EMS with ins(13;8)(q12;p11p23)/ZNF198 -FGFR1 was a very rare， distinct myeloproliferative neoplasm, the fusion gene and chimeric protein with constitutive activation of the FGFR1 tyrosine kinase.

A previously healthy 47-year-old Chinese man presented in January 2009 with extreme fatigue, fever, and night sweats. Hepatomegaly (palpable, 2 cm in the right hypochondrium) and splenomegaly (palpable, 2 cm in the left hypochondrium) were identified, and axillary lymph nodes were swollen. A laboratory investigation showed a white blood cell (WBC) count of 39.8 × 109/L with eosinophils 52% (absolute eosinophil count of 20.7 × 109/L), hemoglobin level of 14.9 g/dl and platelet count of 50.0 × 109/L. Bone marrow (BM) aspirate revealed hypercellular and occupied by approximately 22.0% blasts; eosinophils comprised 23.5% of total cellularity [Figure 1a]. Flow cytometry of these blast cells demonstrated expression of CD13, CD33, CD34, CD117, CD14, CD15, and human leukocyte antigen-DR indicating that myeloblasts were presented. Cytogenetic studies on BM showed a 46, XY, t (8;13)(p11.2;q12.1)[15]/46, idem,-7, der (14), +Mar[5] karyotype [Figure 1c]. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect gene rearrangements of BCR/ABL, FIPIL1/PDGFRA, TEL/PDGFRB were all negative as well as c-KIT and JAK2V617F mutations. Fluorescence in situ hybridization using a dual-fusion probe confirmed FGFR1 gene rearrangement [Figure 1b], the ZNF198-FGFR1 fusion test was done by RT-PCR using ZNF198-and FGFR1-specific primers (5′-TCCCTGTGCCTGTG TATATCCC-3′ and 5′-CGGGAAGCTCATACTCAGAGAC-3′, respectively) and showed a specific amplification product of 195 bp [Figure 1d]. Sequencing identified an in-frame fusion of exon 17 of ZNF198 to exon 9 of FGFR1 [Figure 1e], which was indicative of 8p11 myeloproliferative syndrome (EMS, World Health Organization [WHO] 2008, myeloid and lymphoid neoplasms with FGFR1 abnormalities[1]). Treatment was started with imatinib (400 mg once daily) plus urbason (40 mg once daily) at day 5, about 2 weeks after initiating treatment, WBC count (30.43 × 109/L) did not achieve a significant decrease and the level of hemoglobin and platelet count gradually reduced to 8.6 g/dl and 10.0 × 109/L, respectively. Therefore, imatinib and urbason therapy was stopped. Subsequently, the patient received AA (aclacinomycin plus cytarabine) regimen and had a marked initial response to the first cycle of chemotherapy: The WBC and platelet count returned to normal. However, 25 days later, the patient again developed worse, his WBC rose to 52.7 × 109/L. BM study revealed inadequate aspirate due to dry tap. At the time, a bone biopsy was performed and revealed a hypercellular with marked hypereosinophilia. High-dose cytarabine therapy was recommended and completed. Nonetheless, the patient failed to respond to aggressive chemotherapy and supportive treatment. He died in May 2009, 5 months after the initial diagnosis. Figure 1 (a) Morphologic changes of bone marrow (BM) smear (Wrighte Giemsa, 1 000) showed markedly increased abnormal myeloblasts with eosinophils. (b) Fluorescence in situ hybridization analysis of BM cells with an FGFR1 break-apart DNA probe revealing FGFR1 ...

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.

Abstract 4638 Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of aggressive disease with complex process,gene mutations play an important role in AML pathogenesis. Several genes have been identified in AML,such as FLT3, c-KIT, NPM1 and JAK2. Indeed, some mutations have revealed prognosis subgroups and modified the therapeutic management of these leukemias. Recently,novel mutations in IDH1(amino acid R132)and IDH2 (R140 and R172)have been found in patients with AMLs,but the frequency and impact on biological and prognostic features in Chinese patients with AMLs remain unknown. We aimed at studying the potential significance of IDH1 and IDH2 mutations in AML and analyzed their interaction with other mutations in Chinese patients with de novo AMLs. Methods: We searched 163 Chinese patients with de novo AML for mutations in the IDH1,IDH2, JAK2, NPM1, FLT3-ITD,C-KIT genes.Female/male ratio was 98/65 and age ranged from 17.0–74.0years (median, 41.0years).Genomic DNA was extracted from diagnostic marrow specimens,FLT3 internaltandem duplication(FLT3 –ITD) and JAK2V617F mutations were 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: Overall, IDH mutations were found in 25 patients (15.3%) —IDH1 in 7 patients (4.29%) and IDH2 in 18 patients (11.04%). A total of 4 types of IDH1 mutations were identified(c.395G>A, p.R132H,n=4 Gc.394C>A, p.R132S,n=1 Gc.394C>G, p.R132G,n=1 Gc.315C>T, n=1),six out of the seven patients with the missense mutations and the overall frequency of missense mutations in IDH1 was 3.68% (6/163). Both synonymous substitution (c.315C>T; rs11554137) in IDH1 and IDH2R140 mutation occurred in one patient. No mutated cases had both IDH1 and IDH2 missense mutations, suggesting that the mutations are mutually exclusive. IDH2 mutations caused changes of R140 (c.419G>A,p.R140Q,n=18), R172 mutation was not found in our study. The NPM1 mutation in exon 12 was present in 18.35% (n=29 29/158), 25 cases had FLT3-ITD(15.8% 25/158),One case had frame insertion/deletions of c-KIT in exon 8,7 cases had the substitution of a single amino acid in exon 17(4.57%,7/153),none of them had JAK2V617F mutation. IDH- mutated cases showed a higher frequency of concurrent NPM1 mutation compared with wildtype cases (48.3% 14/29 vs 10.8% 11/129), IDH mutations were also more frequent in FLT3-ITD mutation vs FLT3-ITD wildtype (44.0% 11/25 vs 10.5%14/133).10 patients had both FLT3-ITD and NPM1 mutations, concurrent mutations in NPM1, FLT-ITD were detected in 5 cases with the IDH mutation. None of all cases had both IDH and C-KIT mutations. IDH mutations were more often in cytogenetically normal AML cases(20.5% 15/73 vs5.8% 3/52. Patients with IDH mutations were older (51:40 P=0.003), whereas, there were no differences in sex,WBC and platelet count for IDHmut and IDHwt. Conclusion: Our results show that IDH mutations, especially IDHR140 were frequent genetic alterations in Chinese patients with de novo AMLs, and associated with older age, normal karyotype at diagnosis.There was strong association of IDH mutations with NPM1 and FLT3-ITD mutations, suggesting that IDH mutations may act cooperatively in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.