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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.

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.

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.

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...

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 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.

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.

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.